Compositions and methods for treatment of cerebral amyloid angiopathies (CAAS)

Abstract

Compositions and methods for the treatment of cerebral amyloid angiopathies, e.g., hereditary cerebral hemorrhage with amyloidosis (HCHWA) and Alzheimer's disease, are disclosed. In some embodiments, it includes methods for reducing cerebral vascular basement membrane remodeling in a subject in need thereof are provided. In certain embodiments, the methods comprise administering to said subject, an effective amount of a composition comprising N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier, thereby reducing cerebral vascular basement membrane remodeling in the subject.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATMENT OF CEREBRAL AMYLOID ANGIOPATHIES (CAAS)

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the priority of U. S. Provisional Application No. 63 / 744,699, filed January 13. 2025, the entire contents of which is incorporated herein by reference.

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the fields of cerebral amyloid angiopathies (CAAs), and other brain disorders associated with pathogenic fibril formation. More specifically the invention provides compositions and methods useful for the treatment and management of diseases associated with aberrant fibril formation, particularly hereditary cystatin C amyloid angiopathy (HCCAA) like diseases, such as hereditary cerebral hemorrhage with amyloidosis (HCHWA), Familial British dementia (FBD) and Alzheimer's Disease (AD), as over 80% of AD patients develop CAA that progresses during their disease course.

[0005] BACKGROUND OF THE INVENTION

[0006] Several publications and patent documents are cited through the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

[0007] The phrase “cerebral amyloid angiopathy (CAA)” encompasses a group of diseases in which amyloid deposits accumulate in the walls of cerebral blood vessels, which ultimately leads to structure degradation in the vessel walls causing them to rupture, often resulting in cerebral hemorrhages, ranging from microbleeds to major hemorrhages. CAA usually happens sporadically in elderly people. Notably, almost all Alzheimer's disease patients have different levels of pathological amyloid angiopathy changes in the brain that are identical to other CAA. However, the hereditary familial forms of CAA allow for better studies of the disease-causing mechanism as these usually lead to more rapid and more serious illness in younger people. CAA adversely affects the blood vessels of the leptomeningeal space, the most common form being Ap -CAA, associated with Ap deposits into cerebral blood vessels. This is the same amyloid forming protein which causes Alzheimer’s disease (AD), where deposits build up within braintissue and the blood vessel walls within the brain. Nearly all patients affected by AD have symptoms of Ap-CAA.

[0008] Currently, no known effective permanent treatments for CAA are available which reduce disease symptoms or slow progression of this devastating disease. Accordingly, there is a clear need for improved methods and compositions for treating CAAs.

[0009] SUMMARY OF THE INVENTION

[0010] In a first aspect, methods of treating a cerebral amyloid angiopathy (CAA) in a subject in need thereof are provided. An exemplary method comprises administering to said subject, an effective amount of a composition comprising an agent that disrupts both hydrogen and disulfide bonds between amyloid dimers and / or oligomers, thereby disrupting amyloid deposition in the brain and / or the walls of blood vessels. In certain embodiments, the blood vessels are cerebral blood vessels. In certain embodiments the treatment ameliorates symptoms of the CAA in the patient.

[0011] In another aspect of the invention, methods for reducing cerebral vascular basement membrane remodeling in a subject in need thereof are provided. In certain embodiments, the methods comprise administering to said subject, an effective amount of a composition comprising N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier, thereby reducing cerebral vascular basement membrane remodeling in the subject. In certain embodiments, the composition further reduces extracellular matrix accumulation of the one or more CAA extracellular matrix proteins. In certain embodiments, the subject has a cerebral amyloid angiopathy. In certain embodiments, the treatment ameliorates symptoms of the CAA in the subject.

[0012] In another aspect of the invention, method for reducing extracellular matrix accumulation of one or more CAA extracellular matrix proteins in a subject in need thereof are provided. In certain embodiments, the methods comprise administering to said subject, an effective amount of a composition comprising N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier, thereby reducing extracellular matrix accumulation of the one or more CAA extracellular matrix proteins. In certain embodiments, the composition further reduces cerebral vascular basement membrane remodeling. In certain embodiments, the subjecthas a cerebral amyloid angiopathy. Tn certain embodiments, the treatment ameliorates symptoms of the CAA in the subject.

[0013] In certain embodiments, the one or more CAA extracellular matrix proteins is selected from fibronectin (FN1) and collagen IV (COL IV). In certain embodiments, the methods disclosed herein reduces accumulation of collagen and / or fibronectin within cerebral vessel walls. In certain embodiments, the composition attenuates vascular fibrosis associated signaling. In certain embodiments, the vascular fibrosis associated signaling is selected from TGF-P signaling-associated vascular fibrosis and / or WNT signaling-associated vascular fibrosis. In certain embodiments, the composition preserves vascular smooth muscle cell integrity and / or prevents phenotypic switching of vascular smooth muscle cells to fibroblast-like states.

[0014] In certain embodiments, the composition improves intramural periarterial drainage (IPAD) or reduces protein elimination failure angiopathy (PEFA). In certain embodiments, the subject exhibits increased levels vascular collagen IV or fibronectin prior to administration of the composition. In certain embodiments, the composition reduces extracellular matrix expansion within one or more layers of a cerebral vessel wall and / or attenuates profibrotic signaling in cerebral vasculature. In certain embodiments, the profibrotic signaling comprises transforming growth factor-β (TGF-β) signaling and / or WNT signaling. In certain embodiments, administration of the composition reduces activation of SMAD2 / 3, phosphorylated SMAD2 / 3, WNT-1, or combinations thereof and / or preserves vascular smooth muscle cell integrity.

[0015] In certain embodiments, the composition inhibits phenotypic switching of vascular smooth muscle cells to fibroblast-like or myofibroblast-like cells. In certain embodiments, the phenotypic switching is associated with expression of a-smooth muscle actin and vimentin. In certain embodiments, the composition improves perivascular or intramural periarterial drainage of proteins from the brain. In certain embodiments, the composition reduces protein elimination failure angiopathy (PEFA) and / or the composition reduces vascular retention of amyloidogenic proteins. In certain embodiments, the amyloidogenic proteins comprise one or more of amyloid-P, cystatin C. or medin. In certain embodiments, the composition reduces or prevents neuronal proteinopathy associated with Alzheimer’s disease, such as pathological aggregation or mislocalization of TDP-43. In certain embodiments, the composition is administered orally, intravenously, by another systemic route, and / or chronically. In certain embodiments, the methods further comprise assessing the subject for one or more of: a) a reduction in cerebralvascular basement membrane remodeling; b) a reduction in extracellular matrix accumulation of one or more CAA extracellular matrix protein; c) an increase perivascular clearance of amyloidogenic proteins; d) a reduction in accumulation of collagen IV and fibronectin within cerebral vessel walls; e) attenuated TGF-β and / or WNT signaling-associated vascular fibrosis; f) preservation of vascular smooth muscle cell integrity; g) an increase in intramural periarterial drainage (IPAD) or reduces protein elimination failure angiopathy (PEFA); h) a reduction in aggregation or vascular retention of one or more amyloidogenic proteins selected from cystatin C, amyloid-p, and medin; or i) reduces neuronal TDP-43 pathology associated with Alzheimer’s disease; wherein each of these criteria are compared to an untreated control.

[0016] In certain embodiments, the CAA is Alzheimer’s Disease or a hereditary cerebral amyloid angiopathy. In certain embodiments, the hereditary cerebral amyloid angiopathy is selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF). In certain embodiments, the agent is N-acetyl cysteine or a function derivative thereof in a pharmaceutically acceptable carrier. In certain embodiments, the NAC derivative is selected from NAC-amide, NAC-methyl ester, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt.

[0017] In another aspect, the method comprises methods for treating a cerebral amyloid angiopathy (CAA) which comprise administering an effective amount of a composition comprising N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier, to a patient presenting with the CAA, said agent disrupting amyloid deposits in the brain and / or the walls of blood vessels caused by said CAA, thereby alleviating disease symptoms. In certain embodiments, the CAA is a hereditary cerebral amyloid angiopathy.

[0018] In certain embodiments, said hereditary cerebral amyloid angiopathy is selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis. Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF). In certain embodiments, the CAA is a subtype of HCHWA selected from the Dutch subtype (HCHWA-D), the Italian subtype (HCHWA-It), the Iowa subtype (HCHWA-Io), the Flemish subtype (HCHWA-F), the Piedmont subtype (HCHWA-P), or the Arctic subtype (HCHWA- A).In certain embodiments the subtype is HCHWA-D and the mutation is a E639Q mutation residing in the Ap domain of the amyloid precursor protein (APP). In certain embodiments the subtype is HCHW-It and the mutation is a E639K or A713T mutation residing in the Ap domain of APP. In certain embodiments the subtype is HCHWA-Io and the mutation is a D694N mutation residing in the Ap domain of APP. In certain embodiments the subtype is HCHWA-F and the mutation is a A692G mutation residing in the Ap domain of APP. In certain embodiments the subtype is HCHWA-P and the mutation is a L705V mutation residing in the Ap domain of APP. In certain embodiments the subtype is HCHWA-A and the mutation is a E693G mutation residing in the Ap domain of APP.

[0019] In certain embodiments the CAA is FBD caused by a mutation in the BRI2 / ITM2B gene. In certain embodiments the mutation is a mutation in the stop codon. In certain embodiments, the CAA is TRR caused by a mutation in transthyretin selected from L12P, D18G, A25T, V30G, V30M, T49P, L58R, F64S, Y69H. Y114C, G53R, G53E, G53A, V122I, or E89Q. In certain embodiments, the CAA is FDD caused by a mutation in the BRI2 / ITM2B gene. In certain embodiments, the mutation is a duplication mutation that disrupts the stop codon.

[0020] In certain embodiments, the NAC derivative is selected from NAC-amide, NAC-methyl ester, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt.

[0021] In certain embodiments, the methods further comprise performing a skin biopsy on said subject following treatment to assess reduction in amyloid-cystatin protein aggregates in skin and / or performing an MRI or CAT scan on said subject following treatment to assess reduction in protein aggregates in the cerebral blood vessels. In certain embodiments, the methods further comprise administering an ionophore and / or an anti-inflammatory agent. In certain embodiments, the anti-inflammatory agent is selected from the group consisting of one or more of corticosteroids, aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac. tolmetin, donepezil, fasoracetam, interleukin (IL)-l receptor antagonist, IL-4, IL-6, IL-10, IL-11, IL-13, cytokine receptors for IL-1, tumor necrosis factor-alpha, IL-18 and derivatives and biosimilars thereof. In certain embodiments, the methods further comprise administering one or more of glutathione, monensin, papain, cathepsin B, falcipain, and an siRNA.

[0022] In certain embodiments, said administration reduces protein aggregates in the wall of cerebral blood vessels. In certain embodiments, aid administration increases the dementia ratingscale-2 (DRS-2) overall index total score of the subject by at least 10% when compared to the DRS-2 overall index total score of the subject prior to administration. In certain embodiments, the dementia rating scale-2 (DRS-2) overall index total score of the subject does not change after said administration.

[0023] In another aspect of the invention, methods for identifying therapeutic agents which disrupt amyloid deposits in the brain and / or walls of blood vessels are provided. An exemplary method comprises providing a population of cells which express a nucleic acid encoding a mutant Ap protein, said mutant causing formation of amyloid protein aggregates; and providing a population of cells which express an A0 protein which lacks the mutation. Both populations of cells are contacted with a test agent and assessed to determine whether the agent alters amyloid protein aggregate formation of cells expressing the mutant relative to those expressing the wild type protein, thereby identifying agents which alter amyloid protein aggregate formation. Agents so identified should have efficacy for the treatment of CAAs.

[0024] Another exemplary method comprises providing two populations of cells which express a nucleic acid encoding a mutant Ap protein, said mutant causing formation of amyloid protein aggregates. The first population is contacted with N-acetyl cysteine or a function derivative thereof and the second population is contacted with a test agent. Both populations of cells are then assessed to determine whether the test agent alters amyloid protein aggregate formation at a greater than or equal level as the NAC or a functional derivative. The test agents that decrease amyloid protein aggregate formation at a level equal to, or greater than the decrease of amyloid protein aggregate formation caused by NAC or a functional derivative are identified as therapeutic agents.

[0025] In certain embodiments, the N-acetyl cysteine derivative is selected from NAC-amide, NAC-methyl ester, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt. In certain embodiments, the mutation is selected from any one of the mutations in Table I. In certain embodiments, the mutation is in the APP gene. In certain embodiments, the test agent decreases amyloid protein aggregation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0026] In another aspect of the invention, methods for identifying a therapeutic agent for the treatment of a first cerebral amyloid angiopathy (CAA) are provided. In certain embodiments, the method comprise providing a population of cells from a patient with a second CAA;contacting the cells with a test agent; and detecting whether the test agent alters levels of at least one CAA biomarker selected from fibronectin, collagen IV, vimentin, a-SMA, SMAD2 / 3, or WNT-1. In certain embodiments, the test agents that alter the at least one CAA biomarker level in the cells are identified as therapeutic agents. In certain embodiments, the first CAA is the same as the second CAA. In certain embodiments, the cell is a vascular smooth muscle cell, a pericyte, an endothelial cell, a brain microvascular endothelial cell, an adventitial fibroblast, a myofibroblast, a fibroblast, a dermal fibroblast, an astrocyte, a microglial cell / macrophage, or a neuron.

[0027] In certain embodiments the first CAA is Alzheimer’s disease or a hereditary cerebral amyloid angiopathy selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF). In certain embodiments, the first CAA is selected from anyone of the CAAs discussed above.

[0028] In certain embodiments the second CAA is Alzheimer’s disease or a hereditary cerebral amyloid angiopathy selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF). In certain embodiments, the second CAA is selected from anyone of the CAAs discussed above.

[0029] In certain embodiments, the test agents that decrease levels of at least one of fibronectin, collagen IV, vimentin, SMAD2 / 3, and WNT-1 in the cells are identified as therapeutic agents. In certain embodiments, the test agents that increase levels a-SMA, in the cells of are identified as therapeutic agents.

[0030] Still other aspects and advantages of these compositions and methods for making the compositions and using the compositions are described further in the following detailed description of the preferred embodiments thereof.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1A-1F: Hematoxylin-eosin (HE) staining in cerebral samples in patients with hereditary cystatin C amyloid angiopathy (HCCAA) and Ap-CAA from AD patients (Fig. 1A-1E), and control samples (Fig. IF).Figure 2A-2C: Antibody staining for cystatin C in HCCAA (Fig. 2A) and Ap in Ap-CAA sample from AD (Fig. 2B) and control sample (2C).

[0033] Figure 3A-3F: Antibodies staining for collagen IV in HCCAA (Fig. 3 A and 3D) and A -CAA in AD (Fig. 3B and 3D) in arteries and parenchyma and control (Fig. 3C and 3F)

[0034] Figure 4A-4D: Collagen IV antibody staining in HCCAA and Ap-CAA in AD in severely affected CAA vessels (Fig. 4A-4C) and in plaques (Fig. 4D), showing extensive collagen IV accumulation in all vessel types and plaques.

[0035] Figure 5A-5F: Fibronectin (FN) antibody staining, in hereditary cystatin C amyloid angiopathy (HCCAA) (Fig. 5A and 5D) and Ap-CAA in AD (Fig. 5B and 5E), and control samples (Fig. 5C and 5F).

[0036] Figure 6A-6F: FN antibody staining in HCCAA and Ap-CAA in plaques in AD (Fig. 6A and 6B) and in veins / venules and capillaries (Fig.6C-6E), showing FN accumulation in all vessel types and plaques compared to control (Fig. 6F).

[0037] Figure 7A-7F: Vimentin antibody staining in HCCAA and Ap-CAA in AD in CAA arteries / arterioles, showing fibroblasts in all layers of the vessel wall of larger arteries (Fig. 7A-7B) and thickening layers in intima and adventitia in smaller arteries / arterioles (Fig. 7D-7E) and control (Fig. 7C and 7F).

[0038] Figure 8A-8F: SMA antibody staining in HCCAA and Ap-CAA in AD in CAA arteries / arterioles with SMA positive cells in all layers and few in more affected vessels (Fig. 8A-8B and 8D-8E) and normal SMA cells in control (Fig. 8C and 8F).

[0039] Figure 9A-9I: Comparison of SMA (Fig. 9B, 9E, and 9H) and Vimentin (Fig. 9A, 9D, and 9G co-staining in the vessel walls of sample groups. Co-staining of vimentin (green) and SMA (red) is shown in Fig. 9C, 9F, and 91. All images are at 60x magnification.

[0040] Figure 10A-10F: CD34 and collagen IV antibody staining in brain parenchyma, Comparison of CD34 and collagen IV staining in brain tissue of HCCAA sample (Fig. 10A-10B). CD34 stains faintly in blood vessels within the tissue, and some blood vessels show no staining but, on theother hand, show strong collagen TV staining (yellow arrows). Comparison of the CD34 and collagen IV staining of Ap-CAA in AD brain tissue. (Fig. 10C) and (Fig. 10D). Comparison of the CD34 and collagen IV staining of control brain tissue (Fig. 10E-10F). All images are at 5x magnification.

[0041] Figure 11A-11H: Wnt and smad2 / 3 antibody staining. Wnt antibody staining of the medium and small cerebral arteries of a HCCAA specimen. (Fig. 11 A) Smad2 / 3 antibody staining of the middle and minor cerebral arteries of an HCCAA specimen (Fig. 1 IB). Wnt antibody staining of the middle cerebral artery of an Ap-CAA sample in AD (Fig. 11C), Smad2 / 3 antibody staining of the middle cerebral artery of an A0-CAA specimen in AD (Fig. 11D). Wnt antibody staining of the middle cerebral artery of an Ap-CAA sample in AD (Fig. HE). Smad2 / 3 antibody staining of the middle cerebral artery of an Ap-CAA sample in AD (Fig. 1 IF). Wnt antibody staining of the middle cerebral artery of a control sample (Fig. 11G). Smad2 / 3 antibody staining of the middle cerebral artery of a control specimen (Fig. 11H). All images are at 20x magnification.

[0042] Figure 12A-12F: Antibody staining for Wnt and smad2 / 3. Wnt-1 antibody staining of a large cerebral artery of a HCCAA specimen (Fig. 12A). Smad2 / 3 antibody staining of a large cerebral artery of a HCCAA specimen (Fig. 12B). Wnt-I antibody staining of a large cerebral artery of an Ap-CAA sample in AD (Fig. 12C). Smad2 / 3 antibody staining of a large cerebral artery of an Ap-CAA sample in AD (Fig. 12D). Wnt antibody staining of a large cerebral artery of a control (Fig. 12E). Smad2 / 3 antibody staining of a large cerebral artery of a control (Fig. 12F). All images are at 40x magnification.

[0043] Figure 13A-13F: Double staining of HCCAA sample for cy statin C and SMA (Fig. 13A and 13C) and double staining of Ap-CAA sample in AD for Amyloid-P and SMA (Figs. 13D and 13F). All images are at 60x magnification.

[0044] Figure 14: N-acetylcysteine blocks oligomerization of Cyst-C L68Q

[0045] Figure 15: Testing of new and improved compounds. NAC, NAC amide (NAC-A), or NAC methyl ester (NAC-M) are provided. A representative image from the Western blot and image intensities of regions containing high molecular weight aggregates or monomeric forms of hCC.Figure 16A-16B: NACA crosses the BBB determined by High Performance Liquid Chromatography (HPLC) after administration of (Fig. 16A) NACA (designated as AD4) and (Fig. 16B) NAC. NACA was identified as a new fluorescent peak (absent in control brain samples). The endogenous thiols, cysteine (Cys) and GSH, were identified according to the retention time of standards tagged with monobromobimane, and as with NACA, these peaks were absent in NEM-treated samples. NAC was absent from chromatogram showing no crossing into the brain.

[0046] Figure 17: Amyloid beta precursor protein - Netherlands (Dutch) mutation.

[0047] Figure 18: NAC and NACA treatment of 293T cells transiently transfected with the WT vs E618Q APP mutated plasmids. APP shows multimerization under non-reducing conditions. Importantly, NACA is capable of reducing APP multimers to monomer.

[0048] Figure 19: Homo sapiens integral membrane protein 2B (ITM2B) - Familial British Dementia (FBD).

[0049] Figure 20: NA and NACA treatment of 293T cells transiently transfected with the WT vs Bri2 mutated plasmids. Bri2 shows multimerization under non-reducing conditions. A small difference can be observed between WT and the mutant Bri2 at this stage, the treatment with NAC has some impact on the presence of Bri2 multimers. NAC at lOmM was sufficient to completely render CST3 multimers to monomers. Importantly, NACA is capable of reducing Bri2 multimers to monomer.

[0050] Figure 21A-21B: Structural comparison of NAC and NACA (Fig. 21 A). Due to the substitution of an amide for the carboxylate in NAC, NACA has increased lipophilicity and, therefore, greater cell permeability, bioavailability and BBB permeability than NAC (Fig. 2 IB).

[0051] Figure 22A-22B: Formation of amyloid deposits (Fig. 22 A). Mechanism of action for NAC, NACA and its other derivatives (Fig. 22B).

[0052] Figure 23A-23B. Study flow diagram from a Phase Ila clinical trial in HCCAA patients treated with NAC for 9 months. (Fig. 23A) Study flow diagram showing enrollment of HCCAA patients and analyses. (Fig. 23B) Characteristics of HCCAA patients at baseline.Figure 24A-24E. Results from skin biopsies obtained at baseline (VI) and following 9 months of treatment with NAC (V4). Statistical analysis was done with Wilcoxon matched-pairs signed rank test (p < 0.05) for comparing % immunoreactivity / ROI in biopsy VI to biopsy V4 in 15 patients. Plots were conducted in Graphpad and represent median (and interquartile range (IQR)). (Fig. 24A) Skin biopsy from control / healthy family member showing no L68Q-hCC amyloid complex deposition. In carrier, moderate hCC amyloid complex deposition is seen in the BM, between epidermis and dermis and in upper dermis in VI and less intensive deposition in V4. The plot shows the change in % hCC staining per ROI in the carrier biopsies between V 1 and V4 (p = 0.27), VI median (IQR) = 0.89 (0.51-4.75), V4 median (IQR) = 0.65, (0.19-4.10) and control / C median (IQR) = 0.003 (0.0015-0.01). (Fig. 24B) Skin biopsy from control with collagen IV staining in BM between epidermis and dermis and BMs in dermis. In earner, more intense staining is seen in V 1, especially in the BM between epidermis / dermis and in fibroblasts in upper dermis (arrows) and less extensive staining in seen in V4, mainly in fibroblasts. The plot shows the change in % collagen IV staining per ROI in carrier biopsies (p = 0.0002), VI median (IQR) = 3.69 (2.48-5.16), V4 median (IQR) = 2.60 (1.99-2.97) and Control / C median (IQR) = 1.97 (1.29-2.44). (Fig. 24C) Skin biopsy from control with fibronectin staining in BMs, more intense staining in VI from carrier, especially in fibroblasts in upper dermis (arrows) and less intense staining in fibroblasts in V4. The plot shows the change in % fibronectin staining per ROI in earner biopsies before (VI) and after treatment (V4) (p = 0.01), VI median (IQR) = 3.17 (2.09-5.05), V4 median (IQR) = 2.37 (1.87-3.42) and control / C median (IQR) = 2.95 (2.06-4.29). One control biopsy had intense fibronectin immunoreactivity. (Fig. 24D) Skin biopsy from control with vimentin staining, showing normal fibroblasts in upper dermis, biopsy V 1 from carriers showing an elevated number of fibroblasts in the upper dermis with activated appearance (arrows) and less staining and more normal appearance in fibroblasts in V4. The plot shows the change in % vimentin staining per ROI in carrier biopsies between VI and V4 (p < 0.0001), VI median (IQR) = 1.60 (1.24-2.37), V4 median (IQR) = 1.31 (0.97-1.68) and control / C median (IQR) = 0.88 (0.68-1.08). (Fig. 24E) Skin biopsy from control showing normal fibroblasts with SMAD 2 / 3 immunoreactivity in upper dermis, biopsy VI from earner showing an increased number of fibroblasts with SMAD 2 / 3 immunoreactivity in the upper dermis between VI and V4. The plot shows the change in % SMAD staining per ROI in the carrier biopsies between V 1 and V4 (p = 0.0004), VI median (IQR) = 2.25 (0.55-4.36), V4 median (IQR) = 1.56 (0.20-2.54)and control / C median (IQR) = 1.35 (1.16-1.56). All figures were taken with 20x objective. Scale bar: 100 pm on all figures.

[0053] Figure 25A-25D. Free GSH and GSH: GSSG ratio analysis in HCCAA patients following 9 months of NAC. The plots show the results from analysis for free GSH and GSH: GSSG ratio at baseline (VI) and following 9 months of treatment (V4) for all 15 HCCAA patients and for 6 NAC- (naive) patients. The statistical analysis was done with Wilcoxon test (p < 0.05) and plots were condutcted in Graphpad and represents median (and interquartile range (IQR)). (Fig. 25A) There was a not a significant change in free GSH between V 1 and V4 in HCCAA patients, (p = 0.93), VI median (IQR) = 1.68 (0.90-10.85), V4 median (IQR) = 1.56 (0.76-18.72). (Fig. 25B) There was a not a significant change in GSH: GSSG ratio in HCCAA patients (p = 0.12), VI median (IQR) = 1.64 (0.96-13.52), V4 median (IQR) = 2.01 (0.06-80.53). (Fig. 25C) There was a significant change in free GSH between VI and V4 in NAC- patients (p = 0.03), VI median (IQR) = 6.64 (1.18-18.02), V4 median (IQR) = 18.64 (16.57-18.78) (Fig. 25D) There was a measurable change, albeit not significant, in GSH: GSSG ratio between V 1 and V4 in NAC-patients (p = 0.12), VI median (IQR) =18.26 (1.67-22.15), V4 median (IQR) = 25.31 (12.62-35.40).

[0054] Figure 26A-26C: NAC treatment reduces HMW hCC levels in plasma samples of HCCAA patients. HMW hCC was detected by WB in plasma samples of L68Q-hCC carriers. (Fig. 26A) Representative figure of HMW hCC levels in WB analysis shows reduction in HMW hCC levels following NAC therapy. (Fig. 26B) HMW hCC levels were determined by WB and quantitated by optical density. Bars are means; error bars represent standard deviation; individual data are depicted as dots. Data was normalized to the V 1 time point of each patient for accurate comparison (n = 15 patients for time points VI, V2 and V3, and n = 11 for time point V4). Normal distribution was tested and comparison between the V 1 and V4 sample population were made using two-tailed t-test. There was significant reduction in HMW hCC levels between baseline sample (VI), (mean ± SD = 100 ± 0) and treatment at 9 months (V4), (mean ± SD = 73.6 ± 27.9) with two-tailed student t test, p = 0.001. (Fig. 26C) hCC ratio in L68Q-hCC carriers analyzed by mass spec who were not previously treated with NAC prior to clinical trial enrollment (left group), patients who were on low-medium dose NAC (middle group) vs high dose NAC (right group) upon study enrollment. Bars represent mean and error bars representstandard deviation of hCC ratio between V4 and V 1 and show reduction in HMW levels at V4. Individual data are depicted as dots.

[0055] Fig. 27A-27F: H& E of leptomeningeal vessels. Fig. 27A: Representative small- to medium-sized leptomeningeal arteries / arterioles from HCCAA patient showing classic CAA features, including thickened, homogeneous, largely acellular vessel walls. Residual mural cells are present in the largest vessel (arrow), whereas smaller arterioles are predominantly acellular, with some displaying “double-barrel” splitting. Fig. 27B: Higher-magnification view of Fig. 27A highlighting leptomeningeal arteries / arterioles with acellular walls and “double-barrel” morphology (arrow). Fig. 27C: Leptomeningeal arterioles from HCCAA patient showing a homogenized vessel wall appearance with sparse residual nuclei. Fig. 27D: Leptomeningeal vessel from HCCAA patient exhibiting reactive-appearing mural cells with elongated nuclei distributed throughout the vessel wall. Fig. 27E: Less affected artery showing intimal thickening with increased numbers of cells within the intima (arrows indicate the intimal layer) Fig. 27F: Normal leptomeningeal vessel walls with preserved architecture, intact cellular layers, and normal wall thickness from control tissue. Scale bar: 250 pm.

[0056] Fig. 28A-28F: Cystatin C immunohistochemistry and Congo red. Fig. 28 A: Cystatin C immunostaining of affected leptomeningeal vessels from HCCAA patient, highlighting arteries / arterioles (most prominently involved; black arrow indicates one) and focal parenchymal deposits (red arrow). Fig. 28B: Cystatin C immunoreactivity in focal parenchymal deposits from the same patient as in Fig. 28 A (arrow). Fig. 28C: Cystatin C immunoreactivity in a venule from HCCAA patient (arrow). Fig. 28D: Less affected vessel with residual mural cells, showing circumferential / pericellular cystatin C immunoreactivity around VSMCs (red arrow) and relatively weaker intimal staining (black arrow). Fig. 28E: Higher magnification of affected arteries from HCCAA patient showing cystatin C aggregation involving all vessel wall layers. Fig. 28F: Congo red staining of an affected artery from HCCAA patient demonstrating circumferential amyloid deposition (arrow) with segmental accentuation. Scale bars: 250 pm. Fig. 29A-29C: Immunofluorescence for cystatin C and a-SMA. Fig. 29A: Artery showing circumferential cystatin C aggregation in HCCAA artery within the media of moderately affected artery. Fig. 29B: a-SMA highlights VSMCs in the media and proliferating cells in the thickened intima of the same artery as in Fig. 29A. Fig. 29C: Merge: cystatin C (red) encircles a-SMA-positive cells (green) in the media, with minimal signal in the intima and no spatial overlap (no-colocalization) between a-SMA and cystatin. Scale bar: 100 pm.

[0057] Fig. 30A-30I: COL IV immunohistochemistry. Fig. 30A: COL IV immunostaining in HCCAA patient showing immunoreactivity in all layers of the affected vessel walls. Fig. 30B: Extensive COL IV accumulation in HCCAA patient, with intense staining in a venule (black arrow) and in all layers of a larger artery, including the intima (red arrow). Fig. 30C: COL IV immunoreactivity in a parenchymal plaque (black arrow) in HCCAA patient. Fig. 30D: Normal distribution of COL IV in control vessels, confined to medial basement membranes (red arrow, artery; black arrow, venule). Fig. 30E: Box-and- whisker plots showing the results from quantitative analyses of COL IV immuno staining in leptomeningeal vessels. The whiskers show the 10th and 90th percentiles. Values that fall above the 90th percentile and below the 10th percentile are presented as circles or squares. Quantitative analysis shows higher COL IV immunoreactivity in leptomeningeal arteries / arterioles in HCCAA versus controls (median [IQR], 59.4% [49.5-71.4] vs 33.3% [28.1-39.2]; Mann-Whitney U test, P < 0.0001). Fig. 30F: COL IV staining in the parenchyma showing widespread immunoreactivity across different vessel types. Fig. 30G: Higher magnification showing seemingly elevated COL IV immunoreactivity in an affected arteriole (red arrow) and capillaries (black arrow). Fig. 30H: Control tissue showing normal COL IV staining in parenchymal arterioles and capillaries. Fig.

[0058] 301: Box-and-whisker plots showing the results from quantitative analyses of COL IV immunostaining in parenchymal vessels. The whiskers show the 10th and 90th percentiles. Values that fall above the 90th percentile and below the 10th percentile are presented as circles or squares. Quantitative analysis shows higher COL IV staining in parenchymal vessels in HCCAA versus controls (median [IQR], 2.81 [1.65-4.59] vs 1.94 [1.68-2.19]; Mann-Whitney U test, P = 0.0055). Scale bars: 250 pm.

[0059] Fig. 31A-31D: Immunofluorescence for COL IV and cystatin C. Fig. 31 A: COL IV outlines BMs around VSMCs in HCCAA artery, with denser labeling in the BM at the media-intima interface and additional signal in the thickened intima (arrows). Fig. 3 IB: Cystatin C deposition encircles VSMCs within the media, with little or no signal in the intima. Fig. 31C: Merged image demonstrating spatial overlap of COL IV and cystatin C (yellow) within the media, supporting a close association between these proteins; nuclei are counterstained with DAPI (white). Fig. 3 ID:Representative fluorescence intensity profile across the vessel wall illustrating the relative distribution of COL IV and cystatin C. Scale bar: 100 pm.

[0060] Fig. 32A-321: FN 1 immunohistochemistry in HCCAA. Fig. 32A: FN 1 (E5H6X) immunostaining detected in the vessel wall of affected arteries / arterioles and a venule (black arrow), as well as in parenchymal plaques (red arrow), in HCCAA patient. Fig. 32B: In smaller affected arteries / arterioles, FN 1 staining was often extensive (black arrow), with concomitant staining observed in an adjacent parenchymal plaque (red arrow) in HCCAA patient. Some parenchymal capillaries showed FN 1 immunoreactivity (orange arrow), whereas most were negative. Fig. 32C: Sparse FN1 (E5H6X) immunostaining in all vessel layers observed in affected vessels in HCCAA patient. Fig. 32D: In control tissue, FN1 (E5H6X) immunoreactivity was largely confined to the media and the intimal basement membrane of arteries / arterioles. Fig. 32E:

[0061] Immuno staining with the FN1 (IST-9) antibody showing a similar spatial distribution as seen with FN1 (E5H6X) immunoreactivity in an affected artery from HCCAA patient. Fig. 32F: The same artery as shown in Fig. 32E, stained with the corresponding negative control (FLEX, IR750). Fig. 32G: FN1 (IST-9) immunoreactivity detected within and surrounding parenchymal plaques. Fig. 32H: In control tissue, FN1 (IST-9) immunoreactivity was largely confined to the media and the intimal basement membrane of arteries / arterioles. Fig. 321: Box-and-whisker plots showing the results from quantitative analyses of FN1(E5H6X) immuno staining in leptomeningeal arteries / arterioles. The whiskers show the 10th and 90th percentiles. Values that fall above the 90th percentile and below the 10th percentile are presented as circles or squares. Quantitative analysis showed that FN 1 immunoreactivity (% immunoreactive area per RO I) was higher in HCCAA patients (median [IQR], 31.9% [19.4-43.5]) than in controls (18.6% [14.02-49.5]; Mann-Whitney U test, P < 0.0001). Scale bars: 250 pm.

[0062] Fig. 33A-33B: Amyloid-P (AP) immunohistochemistry in Alzheimer’s disease-associated CAA. Fig. 33A: Ap immunostaining demonstrating deposition within affected leptomeningeal vessels, consistent with vascular amyloid angiopathy. Fig. 33B: Ap immunoreactivity in parenchymal plaques showing dense extracellular plaque deposition. Scale bar: 250 pm.

[0063] Fig. 34A-34G: COL IV and FN1 immunohistochemistry in Alzheimer’s disease-associated CAA. Fig. 34A: COL IV immunostaining in AD-CAA showing marked BM thickening in affected leptomeningeal vessels. Fig. 34B: COL IV immunoreactivity within parenchymalamyloid plaques (arrow) and in capillaries. Fig. 34C: Box-and-whisker plots showing quantitative analysis of COL IV immunostaining in leptomeningeal vessels. The whiskers show the 10th and 90th percentiles. Values that fall above the 90th percentile and below the 10th percentile are presented as circles or squares. Quantitative analysis demonstrated higher COL IV immunoreactivity in leptomeningeal vessels from AD-CAA cases compared with controls (mean ± SD: 56.42 ± 11.43 vs 34.20 ± 8.45, respectively; range 23.54-79.81 vs 16.84-55.16; Mann-Whitney U test, P < 0.0001). Fig. 34D: Box-and-whisker plots showing the results from quantitative analyses of COL IV immuno staining in parenchymal vessels. The whiskers show the 10th and 90th percentiles. Values that fall above the 90th percentile and below the 10th percentile are presented as circles or squares. Quantitative analysis showed no significant difference in COL IV immunoreactivity (% immunoreactive area per ROI) between AD (A -CAA) patients and controls (median [IQR], 1.95 [1.56-2.43] vs 1.94 [1.68-2.19], respectively; Mann-Whitney U test, P = 0.8745). Fig. 34E: FN1 immuno staining in an affected leptomeningeal artery demonstrating extensive vessel wall labeling. Fig. 34F; FN1 immunoreactivity within parenchymal plaques (arrow), appearing stronger than COL IV staining and similar to the pattern observed in HCCAA. Fig. 34G: Box-and-whisker plots showing the results from quantitative analyses of FN1 (E5H6X) immunostaining in leptomeningeal arteries / arterioles. The whiskers show the 10th and 90th percentiles. Values that fall above the 90th percentile and below the 10th percentile are presented as circles or squares. Quantitative analysis showed that FN 1 immunoreactivity (% immunoreactive area per ROI) was higher in AD-CAA cases (median [IQR], 25.77% [19.99-35.07]) than in controls (18.62% [14.02-27.26]; Mann-Whitney U test, P < 0.0001). Scale bar: 250 pm.

[0064] Fig. 35A-35J: Vimentin and a-SMA immunohistochemistry. Fig. 35A: Vimentin immunoreactivity detected across vessel wall layers in HCCAA patient, with enrichment in the thickened intima and irregular medial staining characterized by large, elongated cells. Fig. 35B: Affected artery from HCCAA patient showing vimentin immunoreactivity in all vessel wall layers, most prominently within the intima (arrow). Fig. 35C; Smaller arteries / arterioles from HCCAA patient often showed little or no medial vimentin staining, with only scattered positive cells; in some vessels, more pronounced labeling was observed in the intima and adventitia. In the upper left comer, a parenchymal plaque is shown with surrounding vimentin-positive fibroblast-like cells and astrocytes. Fig. 35D: In control tissue, vimentin immunoreactivity waslargely restricted to the intima and media, with stronger staining in the media and thinner labeling in the intima. Fig. 35E: Box-and-whisker plots showing the results from quantitative analyses of vimentin immunostaining in leptomeningeal vessels. The whiskers show the 10th and 90th percentiles. Values that fall above the 90th percentile and below the 10th percentile are presented as circles or squares. Quantitative analysis showing lower vimentin immunoreactivity (% immunoreactive area per RO I) in HCCAA patients compared with controls (median [IQR], 6.25% [1.10-12.05] vs 16.30% [8.70-21.80]; Mann-Whitney U test, P < 0.0001). Fig. 35F: a-SMA immunoreactivity present across all vessel wall layers in some larger arteries from HCCAA patient, with prominent intimal labeling (black arrow); in smaller arteries / arterioles, a-SMA-positive medial cells were often reduced or absent (red arrow). Fig. 35G: Higher-magnification view of a-SMA staining in Fig. 35F showing thick labeling in the intima (white arrow) and discontinuous venous staining (black arrow). Fig. 35H: a-SMA immunoreactivity in a vessel from a HCCAA patient, with absence of staining in an affected arteriole (arrow). Fig.

[0065] 351: In control tissues. a-SMA staining of vessel walls was uniform and continuous. Fig. 35 J: Box-and-whisker plots showing the results from quantitative analyses of a-SMA immunostaining in leptomeningeal vessels. The whiskers show the 10th and 90th percentiles. Values that fall above the 90th percentile and below the 10th percentile are presented as circles or squares. Quantitative analysis showing lower a-SMA immunoreactivity (% immunoreactive area per ROI) in HCCAA patients compared with controls (median [IQR], 6.35% [1.98-14.55] vs 16.64% [12.40-21.17]; Mann-Whitney U test, P < 0.0001). Scale bars: 250 pm.

[0066] Fig. 36A-36C: Immunofluorescence for a-SMA and COL IV. Fig. 36A: a-SMA labels VSMC in an HCCAA artery. Fig. 36B: COL IV positivity along the media in the same artery. Fig. 36C: Merge shows spatial overlap of a-SMA and COL IV (yellow). Scale bar: 100 pm.

[0067] Fig. 37A-37C: Immunofluorescence staining of vimentin and a-SMA. Fig. 37A: Vimentin staining in an artery from an HCCAA patient; arrow indicates a positive cell. Fig. 37B: a-SMA staining in the same artery as in Fig. 37A, showing a similar distribution; arrow marks the same cell. Fig. 37C: Dual immunofluorescence shows spatial overlap of vimentin and a-SMA (yellow); arrow indicates the same double-positive cell. Scale bar: 100pm.

[0068] Fig. 38A-38L: WNT-1 and SMAD2 / 3 immunohistochemistry. Fig. 38A: SMAD2 / 3 immunoreactivity detected in cells across all vessel wall layers in affected vessels from HCCAApatient. Fig. 38B: SMAD2 / 3 staining in control tissue showing labeling confined to the media and intima, with dense, regular medial staining and orderly cellular morphology. Fig. 38C: pSMAD2 / 3 immunoreactivity detected in cells across all vessel wall layers in affected vessels from HCCAA patient. Fig. 38D: pSMAD2 / 3 staining in control tissue showing labeling in the media and intima with dense, regular medial staining and orderly cellular morphology. Fig. 38E: Higher-magnification view of SMAD2 / 3 staining in a larger artery from HCCAA patient, showing immunoreactivity across all vessel wall layers. Fig. 38F: The same vessel as shown in Fig. 38E, stained for pSMAD2 / 3. demonstrating more prominent labeling across the vessel wall. Fig. 38G: Negative control staining of the same vessel shown in Figs. 38E-38F, showing no immunoreactivity. Fig. 38H: WNT-1 immunostaining showing a spatial distribution similar to that of SMAD2 / 3 and pSMAD2 / 3, with labeling across all vessel wall layers in affected vessels. Fig. 381: WNT-1 staining in control tissue showing labeling confined to the media and intima, with dense, regular medial staining and orderly cellular morphology. Fig. 38 J: Quantitative analysis demonstrated that SMAD2 / 3 immunoreactivity (% immunoreactive area per RO I) did not differ significantly between HCCAA patients and controls (median [IQR], 4.03% [2.20-6.64] vs 4.71% [3.10-7.19]: Mann-Whitney U test, P = 0.1193). Box-and- whisker plots showing the results from quantitative analyses of vimentin immunostaining in leptomeningeal vessels. The whiskers show the 10th and 90th percentiles. Values that fall above the 90th percentile and below the 10th percentile are presented as circles or squares. Fig. 38K: Quantitative analysis demonstrated that pSMAD2 / 3 immunoreactivity (% immunoreactive area per ROI) did not differ significantly between HCCAA patients and controls (median [IQR], 3.99% [2.24-6.26] vs 4.71% [3.14-7.27]; Mann-Whitney U test, P = 0.0855). Fig. 38L: Quantitative analysis demonstrated that WNT-1 immunoreactivity (% immunoreactive area per ROI) did not differ significantly between HCCAA patients and controls (median [IQR], 4.01% [2.24-8.10] vs 4.76% [3.11-7.35]; Mann-Whitney U test, P = 0.1265). Scale bars: 250 pm.

[0069] Fig. 39A-39E: Cellular alterations and signaling-associated markers in AD-associated CAA. Fig.

[0070] 39A-39B: Vimentin immunoreactivity in AD-CAA showing expanded staining within the vessel wall, extending beyond the adventitia into additional layers of affected vessels. Fig. 39C:

[0071] SMAD2 / 3 immunostaining demonstrating positive vascular cells within the vessel wall of an AD-CAA-affected vessel. Fig. 39D: WNT-1 immunoreactivity in AD-CAA showing labeling of vascular cells across vessel wall layers. Fig. 39E: pSMAD2 / 3 immuno staining in AD-CAAdemonstrating a distribution similar to SMAD2 / 3 and WNT-1, consistent with activated signaling in affected vessels. Scale bar: 250 jam.

[0072] Fig. 40A-40C: Ap deposition in relation to vascular smooth muscle cells in AD with associated CAA. Fig. 40A: An artery showing circumferential Ap deposition within the media of a moderately affected AD-CAA vessel. Fig. 40B: a-Smooth muscle actin (a-SMA) immunostaining highlighting VSMCs in the media of the same artery shown in Fig. 40A. Fig. 40C: Merged image demonstrating Ap (green) encircling a-SMA-positive cells (red) in the media and no spatial overlap (no co-localization) between Ap and a-SMA.

[0073] Fig. 41A-41F: Collagen la immunohistochemistry. Fig. 41A: Collagen la immunoreactivity in HCCAA brain tissue showing prominent adventitial staining and additional labeling within other layers of affected vessel walls. Fig. 4 IB: Collagen la staining in AD-CAA demonstrating a similar distribution and intensity to HCCAA, with adventitial and vessel wall immunoreactivity. Fig. 41C: Control brain tissue showing collagen la staining confined predominantly to the adventitial layer of leptomeningeal vessels (arrow). Fig. 41D: Collagen la staining is also observed in parenchymal vessels, most prominent in affected vessels. Fig. 41E: Collagen la staining in parenchymal vessels in AD-CAA demonstrating a similar distribution and intensity to HCCAA. Fig. 41F: Minimal staining observed in parenchymal vessels from control tissue. Scale bar: 250 pm.

[0074] Fig. 42A-42C: Collagen Illa immunohistochemistry. Fig. 42A: Collagen Illa immunoreactivity in HCCAA brain tissue showing increased adventitial staining with extension into additional layers of affected vessel walls. Fig. 42B: Collagen Illa staining in AD-CAA with demonstrating a distribution similar to that observed in HCCAA. Fig. 42C: Control brain tissue showing thin, discontinuous collagen Illa staining restricted to the adventitial connective tissue (arrow), with no staining of parenchymal vessels. Scale bar: 250 pm.

[0075] Fig. 43A-43B: Medin amyloid immunohistochemistry. Fig. 43A: Medin immunoreactivity in 28-year-old HCCAA patient showing amyloid deposition within the vessel wall, predominantly localized to the medial layer of affected arteries / arterioles. Fig. 43B: Medin immuno staining in AD-CAA demonstrating a similar vascular distribution to that observed in HCCAA. Scale bar: 250 pm.Fig. 44A-44C: Phosphorylated TDP-43 (pTDP-43) immunohistochemistry. Fig. 44A:

[0076] Representative pTDP-43 immuno staining in HCCAA brain tissue showing neurons with cytoplasmic pTDP-43-positive aggregates. Fig. 44B: pTDP-43 immuno staining in AD demonstrating prominent cytoplasmic aggregates. Fig. 44C: Control brain tissue showing absence of cytoplasmic pTDP-43 aggregates and preservation of normal nuclear TDP-43 localization (arrow). Scale bar: 250 pm.

[0077] DETAILED DESCRIPTION OF THE INVENTION

[0078] The immunostaining of brain tissue samples from autopsy specimens reveals the similarities in the pathology of cerebral vessels in HCCAA and Ap-CAA affected Alzheimer’s patients. We demonstrate similar pattern of accumulation of basement membrane proteins as well as the presence of myofibroblasts that are involved with making these proteins and are present in both HCCAA and Ap-CAA brain samples. We also show that activation of the TGF-P and Wnt signaling pathways is present in both diseases, resulting in fibrosis associated with brain scarring and atrophy. The data reveal increased deposits of basement membrane proteins collagen IV and fibronectin compared to controls. The deposition of amyloid followed the same pattern around smooth muscle cells within vessel walls in both diseases. Myofibroblasts, positive for SMA and vimentin were seen in Ap-CAA as well as HCCAA and an activation of both TGF-P and Wnt signaling pathways was observed as well. We also show that NACA demonstrates comparable effects of breaking up high molecular weight amyloid protein complexes in cellbased assays that have been transfected with mutations in the APP gene encoding Ap-proteins, responsible for the Dutch type of CAA, which is highly similar in presentation to the Icelandic type of CAA (HCCAA) and strongly simulating Alzheimer's disease, caused by toxic amyloid Ap-protein oligomers. The drug also has the same effects on the BRI2 mutation that causes Familial British Dementia, another hereditary form of CAA due to comparable protein aggregation.

[0079] Collectively, these results show that there are strong similarities in the pathologies of Ap-CAA including HCCAA, D-CAA, and FBD. Therefore, NACA, and other NAC derivatives, are a potent therapeutic for Ap-CAA and Alzheimer's disease patients, most of whom have extensive CAA brain vessel wall changes that are identical to the more rare hereditary forms of the disease. This is the first time NACA, a prodrug of NAC, and related NAC-derivatives, areshown to dissolve protein aggregates in biological samples from rare familial forms of CAAs as well as in samples from patients with Alzheimer's disease.

[0080] Additionally, herein we demonstrate that the disease pathology is identical between the hereditary forms of CAAs (such as HCCAA and HCHWA) and AD. We also show in a Phase II clinical trial that N-acetylcysteine (NAC) reverses the amyloid aggregation and dissolves amyloid deposits from the skin in HCCAA patients, strongly indicating that NAC or derivatives of NAC will also benefit other CAAs and AD. Given the superiority of NAC amide (NACA) in crossing the blood brain barriers and getting into cells over NAC, where the disease-related action of the drug takes place, in certain embodiments, NACA is the preferred molecule for breakthrough treatment of HCCAA and other CAAs, including AD.

[0081] The following definitions are provided to aid in understanding the subject matter regarded as the invention.

[0082] In this invention, “a” or “an” means “at least one” or “one or more,” etc., unless clearly indicated otherwise by context. The term “or” means “and / or” unless stated otherwise. In the case of a multiple-dependent claim, however, use of the term “or” refers to more than one preceding claim in the alternative only.

[0083] “Treatment,” as used herein, covers any administration or application of a therapeutic for disease in a mammal, including a human, and includes inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, partially or fully relieving the disease, preventing the onset of the disease, or preventing a recurrence of symptoms of the disease. Example treatments include administration at least one NAC derivative at efficacious doses.

[0084] The terms “inhibition” or “inhibit” refer to a decrease or cessation of any event (such as fibril formation) or to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and / or amount as compared to a reference. It is not necessary that the inhibition or reduction be complete. For example, in certain embodiments, “reduce” or “inhibit” refers to the ability to cause an overall decrease of 20% or greater. In another embodiment, “reduce” or “inhibit” refers to the ability to cause anoverall decrease of 50% or greater. In yet another embodiment, “reduce” or “inhibit” refers to the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.

[0085] The term “inhibitor” refers to an agent that slows down or prevents a particular chemical reaction, signaling pathway or other process, or that reduces the activity of a particular reactant, catalyst, or enzyme.

[0086] The terms “patient” and “subject” are used interchangeably to mean a mammal, including human.

[0087] " Nucleic acid" or a "nucleic acid molecule" as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3’ direction.

[0088] With reference to nucleic acids of the invention, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.

[0089] When applied to RNA, the term "isolated nucleic acid" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

[0090] A "replicon" is any genetic element, for example, a plasmid, cosmid, bacmid, phage or virus, that is capable of replication largely under its own control. A replicon may be either RNA or DNA and may be single or double stranded.

[0091] A "vector" is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element. Exemplary vectors of the invention includewithout limitation, adenoviral-based vectors, adeno-associated viral vectors and retroviral vectors.

[0092] An "expression operon" refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.

[0093] The term "isolated protein" or "isolated and purified protein" is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure" form. " Isolated" is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into, for example, immunogenic preparations or pharmaceutically acceptable preparations.

[0094] The term "substantially pure" refers to a preparation comprising at least 50-60% by weight of a given material (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-95% by weight of the given compound. Purity is measured by methods appropriate for the given compound (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0095] The term "tag," "tag sequence" or "protein tag" refers to a chemical moiety, either a nucleotide, oligonucleotide, polynucleotide or an amino acid, peptide or protein or other chemical, that when added to another sequence, provides additional utility or confers useful properties, particularly in the detection or isolation, to that sequence. Thus, for example, a homopolymer nucleic acid sequence or a nucleic acid sequence complementary to a capture oligonucleotide may be added to a primer or probe sequence to facilitate the subsequent isolation of an extension product or hybridized product. In the case of protein tags, histidine residues (e.g., 4 to 8 consecutive histidine residues) may be added to either the amino- or

[0096] carboxy-terminus of a protein to facilitate protein isolation by chelating metal chromatography. Alternatively, amino acid sequences, peptides, proteins or fusion partners representing epitopesor binding determinants reactive with specific antibody molecules or other molecules (e.g., flag epitope, c-myc epitope, transmembrane epitope of the influenza A virus hemaglutinin protein, protein A, cellulose binding domain, calmodulin binding protein, maltose binding protein, chitin binding domain, glutathione S-transferase, and the like) may be added to proteins to facilitate protein isolation by procedures such as affinity or immunoaffinity chromatography. Chemical tag moieties include such molecules as biotin, which may be added to either nucleic acids or proteins and facilitates isolation or detection by interaction with avidin reagents, and the like. Numerous other tag moieties are known to, and can be envisioned by, the trained artisan, and are contemplated to be within the scope of this definition.

[0097] As used herein, the terms "reporter," "reporter system", "reporter gene," or "reporter gene product" shall mean an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is a readily measurable, e.g., by biological assay, immunoassay, radioimmunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods. The nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product. The required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like.

[0098] The terms "transform", "transfect", "transduce", shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion and the like.

[0099] The introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism. In bacterial, yeast, plant and mammalian cells, for example, the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid. Alternatively, the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell ororganism. In other manners, the introduced nucleic acid may exist in the recipient cell or host organism only transiently.

[0100] A "clone" or "clonal cell population" is a population of cells derived from a single cell or common ancestor by mitosis.

[0101] A "cell line" is a clone of a primary cell or cell population that is capable of stable growth in vitro for many generations.

[0102] N-acetyl cysteine and its derivatives

[0103] “N-acetyl cysteine (NAC)” is a derivative of cysteine that acts to reduce disulfide bonds associated with fibril formation present in neurodegenerative disorders such as HCCAA and Alzheimer’s disease. While NAC and ester derivatives are exemplified herein, other NAC derivatives are known in the art and described in the following patent documents; US3242052, US3591686, US3647834, US3749770, US4016287, US4132803, US4276284, US4331648, US4708965, US4711780. US4721705, US4724239, US4827016, US4859653. US4868114, US4876283, DE150694C, EP0219455A2, EP0269017A2, EP0280606A1, EP0304017A2, and EP0339508A1 which are incorporated herein by reference. However, here we show for the first time that both NAC and its derivatives, including NACA, decrease the levels of ECM proteins as well as decreasing the deposits of amyloids and the levels of cystatin C amyloid complex dimers in circulation. The chemical structure of NAC is shown in Fig. 15.

[0104] A NAC derivative refers to a compound that is produced from NAC through a chemical reaction, thereby creating a substance with modified or partially substituted atoms within the NAC parent molecule, resulting in a new compound with a slightly different structure and properties. In certain embodiments, the modification from NAC comprises replacing a single functional group. Exemplary NAC derivatives include, without limitation NAC-A, NAC-M, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt.

[0105] In certain embodiments, the derivative of NAC is N-acetyl cysteine amine. N-acetyl cysteine amine, (abbreviated NACA or NAC-A), is the amide form of NAC with potent antioxidant and anti-inflammatory activities we show here dissolves toxic amyloid aggregates into harmless monomers. NACA has superior pharmacokinetic properties and bioavailability when compared to NAC. In cellular modelling studies NACA had a more than 10-fold higherpotency in preven ting / dispersing hCC aggregates than NAC. NACA had a 4.6-fold higher bioavailability compared to NAC (mice). The chemical structure of NACA is shown in Fig. 15.

[0106] In certain embodiments, the derivative of NAC is N-acetyl cysteine methyl ester. N-acetyl cysteine methyl ester, (abbreviated NAC-M or NACM), is an esterfied form of NAC with enhanced cellular permeability, thereby facilitating more efficient intracellular delivery. Once inside the cell, NACM is hydrolized into NAC. The chemical structure of NACM is shown in Fig. 15.

[0107] In certain embodiments, the derivative of NAC is N-acetyl cysteine ethyl ester. N-acetyl cysteine ethyl ester, (abbreviated NAC-E, NACE, NAC-ET, or NACET), is another esterfied form of NAC with drastically increased lipophilicity and vastly improved pharmacokinetics. NACET is rapidly absorbed into cells after oral administration but reaches low concentrations in the plasma.

[0108] Hereditary cerebral amyloid angiopathies and Ap-CAAs

[0109] Cerebral amyloid angiopathy (CAA) is a group of diseases in which amyloid deposits accumulate in cerebral blood vessel walls, which ultimately leads to degeneration and rupture of vessel wall structure, causing cerebral hemorrhages ranging from microbleeds to massive hemorrhages. Amyloid deposit aggregates are dependent on hydrogen disulfide bridges to maintain stability. Disrupting these hydrogen and disulfide bridges can prevent amyloid aggregate formulation. In certain embodiments, the CAA is a hereditary cerebral amyloid angiopathy. In certain embodiments, the patient has Alzheimer’s Disease (AD). Various dementia rating scales (DRS) are used to capture the memory loss of these patients, which is identical across CAAs and AD, with over 80% of AD cases having identical CAA pathology. The DRS-2 test is widely used and includes 24 subtests that are used to generate five subscales including Attention, Initiation / Perseveration, Construction, Conceptualization, and Memory, which when summed together provide an overall index total score of mental status / global cognition. The DRS-2 has been revised to include updated age- and education- corrected normative data, therefore can be used for both young and elderly affected CAA patients.

[0110] Hereditary cerebral amyloid angiopathies (hereditary CAAs) are a group of rare inherited disorders affecting the central nervous system (CNS). The defining, shared pathology is the formation of amyloid deposits in cerebral blood vessels (most often in arterial vessels) due to thepropensity of a mutated protein to form pathogenic aggregates / amyloid fibrils. Symptoms can vary depending on the disease type / subtype, although generally they present with cerebral angiopathies leading to cognitive decline (progressive dementia). Most, types / subtypes and cases of hereditary CAAs cause repeated episodes of clinically manifested hemorrhagic or ischemic strokes or infarcts that directly contribute to the neurological, neurocognitive, and / or psychiatric symptoms. Sporadic forms of CAAs exist, where the genetic architecture appears to be polygenic arising from contributions of many variants of low effect sizes without any obvious inheritance patterns. In contrast, hereditary CAAs are monogenic (autosomal dominant) and tend to be more severe with an earlier age of onset, resulting in lethality in young adults in some types / subtypes. While over 25 human proteins have been shown to form amyloid deposits, 6 of these contribute to the various hereditary CAAs, including both the Icelandic and the Dutch types of hereditary cerebral hemorrhage with amyloidosis (HCHWA).

[0111] Among hereditary CAAs, is the hereditary cerebral hemorrhage with amyloidosis (HCHWA) that currently includes 8 distinct sub types, each named based on the geographical location where the disease was first identified. The Icelandic sub type (HCHWA Icelandic) also referenced herein as HCCAA is an often-fatal disease in young adults where amyloids in cerebral vessels arise from aggregation of misfolded, mutated forms of human cystatin C (hCC). The other 7 HCHWA sub types, each arising from a different mutation in the amyloid precursor protein (APP) within the Ap domain, include the Dutch, Italian (E22K), Italian (A42T), Iowa, Flemish, Piedmont, and Arctic sub types. Collectively, there are at least 26 known pathogenic mis sense mutations in APP that have been reported to date, however the other ones fall outside or on the edge of the Ap domain and instead are considered hereditary forms of Alzheimer’s disease (AD) and are more likely to demonstrate microbleeds than hemorrhagic strokes. A list of known hereditary CAAs and their corresponding mutations is provided in Table 1.

[0112] Table 1: List of currently known autosomal dominant hereditary cerebral amyloid angiopathies (CAAs) with the corresponding gene, expressed protein, mutation, and amyloidogenic entity.disease Sub-type Gene Protein Amytoktcrgente Matattonfs)

[0113] Protein / Peptide

[0114] Hereditary Icwwtew CTSG Hernan Mutated hCC CSSQ

[0115] cyteatin C

[0116] henwihage (hCC)

[0117] with

[0118] amyteidasis

[0119] (MCHWA)

[0120] HO* Dutch AW Arsyiote Mutated A.® (£22Q}

[0121] precursor

[0122] prteein (A??')

[0123] HCHhvA Mian AWfW Mutated Ag (£32k}

[0124] wm Mias (A42Tii AW APP Mutated Ag -< A42T) A?ur

[0125] HCWA iowa AW APP Mutated AS OisSMtJ

[0126] HCHWA fetish AW. APP Mutated Ag (A21G) AAS2G

[0127] MCHWA APP ASP. Mutated Ag {tSAvj L7SSV

[0128] HCHWA Artek? AW ArP Mutated Ag(£23G) g£<$3«

[0129] MeSnpnvassuiar TTP TAn-Ahyrstsa Mutated 7TR Wisww amyididosis (W V30M, C5SA, F8$S< WH, >< HC?G5GA; G535, G53A, V1221. gSSQ

[0130]

[0131] aiSCASA Sub-type ®ene Protein Amyipidognnic Mutatkmfs)

[0132] Protein / Poptirfe

[0133] Genefc Gten CAA vadants. Prior pattern.

[0134] t>wease (ArPSa) Yt &5X- i 2W, t326X ■ 12SV hsnsM WA) SA12 Mutated AS< > Mujabofi:rs step >::sdC-8 Sntdh feadshs ti aouno derrsenba add mAensrsn

[0135] Farfsiird (k / A) sstc Mutated ADen th-nudeadde duototw uWsh disrupts step <sxfc»s dementia ( 7S7„79«dupnTAAnTGn restettep in li amino add extension

[0136] hamfei WA} GS?t Gehsaiin AGei D ISM, GisPYs mot anryinidtsia- it; GJ*:. P432R, ASS IP, and hmmsh type A.ia7fe

[0137] (W)

[0138]

[0139] In certain embodiments, the hereditary CAA is Hereditary cy statin C amyloid angiopathy (HCCAA). HCCAA is an Icelandic CAA disease in which a mutation L68Q in the gene coding for cystatin C (CST3) causes the protein to deposit in vessel walls, leading to cerebral hemorrhages and death of affected carriers around the age of 30. The pathology of HCCAA includes an increase in basement membrane proteins such as collagen IV, fibronectin (FN), laminin, and proteoglycan aggrecan within cerebral vessel walls. This causes a substantial loss ofcells within the walls, while other cells differentiate into undesirable extracellular matrix (ECM) protein secreting myofibroblasts.

[0140] As used herein, "human cystatin C (hCC)" refers to a protein which functions as cysteine protease inhibitor that belongs to the cystatin superfamily. hCC is a secretory type 2 cystatin and is expressed in all nucleated human cells. L68Q-hcc refers to a mutated hCC wherein a leucine at position 68 is substituted for a glutamine variant.

[0141] In certain embodiments, the hereditary CAA is HCHWA. All of the HCHWA sub-types arise from mutations in the Ap domain of APP. These subtypes share common CAA pathology resulting from deposition of A amyloids in cerebral vessels, which consist predominantly of the Ap40 isoform, with a small amount of Ap42, although this ratio varies depending on the subtype. The propensity for the CAA to result in clinically evident cerebral hemorrhages also varies with sub-type. Brain parenchymal tissues also show deposits of amyloid plaques, but the clinical consequence of this varies considerably among sub-types.

[0142] In certain embodiments, the HCHWA is the Dutch subtype of HCHWA (HCHWA-Dutch, HCHWA-D). HCHWA-D is the most common and most-studied of HCHWA sub-type. It arises from a pathogenic mutation in the APP gene encoding the amyloid precursor protein (APP). The E693Q mutation resides within the Ap domain in APP. This mutated form of Ap (Ap E22Q, or “Dutch” AP) is more susceptible to forming toxic abnormal amyloid fibrils forming deposits in the CNS vasculature. HCHWA-D was the first sub-type of HCHWA to be characterized. The Dutch sub-type (E22Q) exhibits many similarities to HCCAA clinically, although disease presentation usually occurs a bit later around 40-65 years of age. Both HCCAA and HCHWA-D are characterized by severe CAA causing recurring, symptomatic intracerebral hemorrhages (ICH) and strokes often leading to early death. Microinfarcts that can be seen radiologically are also common. Cognitive decline (dementia) is usually observed, although specific behavioral or psychiatric problems have not been reported. Occasionally, cognitive decline arises as the first symptom later in life in the absence of other clinical symptoms.

[0143] Parenchymal deposition of amyloid (Ap42) plaques is present in HCHWA-D patients, but this is relatively limited and shows minimal neurofibrillary pathology, where deposition in cerebrovascular walls is much more prominent. The damage arising from CAA is considered to be the major and disproportionate contributor to disease pathology in comparison to the parenchymal deposition. Damage to white matter (leukoencephalopathy) is also observedradiologically and suspected to arise in part from CAA along the long, perforating arterioles; however, cognitive decline apparently does NOT correlate with the number of focal lesions nor the amount of white matter hyperintensities (WMH) detected via magnetic resonance imaging (MRI). Notably, other proteins are found to be co-localized with the Ap plaques in parenchymal tissues and in amyloid-laden vascular walls, including unmutated (human) cystatin C (hCC).

[0144] In certain embodiments, the HCHWA is the Italian subtype of HCHWA (HCHWA-Italian, HCHWA-It). This form of HCHWA is characterized by an onset of 50 years of age, symptoms include both dementia and lobar intracerebral hemorrhage. HCHWA-It can be caused by a E693K or A713T mutation in the Ap domain of APP. HCHWA-It is associated with increased accumulation of amyloid-beta protein in the walls of the arteries and capillaries of the meninges, cerebellar cortex and cerebral cortex, leading to the weakening and eventual rupture of these vessels.

[0145] In certain embodiments, the HCHWA is the Iowa subtype of HCHWA (HCHWA-Iowa, HCHWA-Io). This form of HCHWA is characterized by an onset between 50-66 years of age, memory impairment, myoclonic jerks, expressive dysphagia, short-stepped gait, personality changes and lobar intracerebral hemorrhages. HCHWA-Io is often caused by a D694N mutation in the Ap domain of APP. HCHWA-Io also results in increased accumulation of amyloid-P protein in the walls of the arteries and capillaries of the meninges, cerebellar cortex and cerebral cortex, leading to the weakening and eventual rupture of these vessels.

[0146] In certain embodiments, the HCHWA is the Flemish subtype of HCHWA (HCHWA-Flemish, HCHWA-F). This form of HCHWA is characterized by age of onset between 35-55 years, progressive dementia, and intracerebral hemorrhage. HCHWA-F can be caused by a A692G mutation in the Ap domain of APP. The Flemish mutation is associated with variable pathology, but generally involves amyloid deposition in the blood vessels of the brain containing large amyloid cores.

[0147] In certain embodiments, the HCHWA is the Piedmont subtype of HCHWA (HCHWA-Piedmont, HCHWA-P). This form of HCHWA is characterized by onset between 50-70 years of age, recurrent lobar intracerebral hemorrhages and cognitive decline. HCHWA-P can be caused by a L705V mutation in the Ap domain of APP. This mutation also causes an increased accumulation of amyloid-beta protein in the walls of the arteries and capillaries of the meninges,cerebellar cortex and cerebral cortex, leading to the weakening and eventual rupture of these vessels.

[0148] In certain embodiments, the HCHWA is the Arctic subtype of HCHWA (HCHWA-Arctic, HCHWA- A). This form of HCHWA is characterized by onset between 54-61 years of age, and progressive Alzheimer’s disease-like dementia. HCHWA-A can be caused by a E693G mutation in the A0 domain of APP. This mutation also causes an increased accumulation of amyloid-beta protein in the walls of the arteries and capillaries of the meninges, cerebellar cortex and cerebral cortex, leading to the weakening and eventual rupture of these vessels.

[0149] Besides HCHWA, a number of other hereditary CAAs involving other genes have also been reported to date. These include, without limitation, Familiar British Dementia (FBD), Familial Danish dementia (FDD), transthyretin (TTR) amyloidosis, Genetic Prion disease, and Familial amyloidosis - Finnish type (FAF).

[0150] In certain embodiments, the hereditary CAA is FBD. FBD has been identified in several British families and the disease results from comparable aggregation and precipitation of the BRI2 protein, with comparable presentation to that of HCCAA. This form of hereditary CAA arises from a mutation in the stop codon of the BRI2 / ITM2B gene that results in the translation of an extended form of the BRI2 precursor protein, which is cleaved into the pathogenic form of the ABri2 peptide that forms neurotoxic amyloid fibrils.

[0151] Aside from angiopathy, accumulation of ABri amyloids in cerebral vessels result in ischemic white matter damage evident in autopsy and imaging, but hemorrhagic strokes have not been observed. ABri amyloid plaques are large, diffuse, and stain weakly with Congo Red. They are found in the parenchyma of the cerebellum, cerebellar cortex, dentate gyrus, and hippocampus. Small plaques that are strongly congophilic are also found in the hippocampus. Neurofibrillary tangles with hyper-phosphorylated tau show signs of degeneration, and clinical manifestations are considered to be a primary effect of neuronal loss due to toxicity of the ABri peptide rather than angiopathy. A significant amount of the ABri peptide found in amyloid deposits are found to be modified post-translationally at the N-terminal glutamate into pyroglutamate, which likely contributes to ABri pathogenicity. ABri amyloid fibrils are also found systemically and accumulate in blood vessels of the pancreas, myocardium, adrenal glands, lung, liver, spleen, and skeletal muscle and in parenchymal as well for most of theseorgans; however, no pathogenic or clinically noticeable impacts have been observed except in the CNS.

[0152] In certain embodiments, the hereditary CAA is FDD. FDD is characterized by progressive cataracts, loss of hearing, cerebellar ataxia, paranoid psychosis, dementia, atrophy of brain tissue, chronic diffuse encephalopathy, and the presence of exceedingly thin and nearly totally demyelinated cranial nerves. As with FBD, FDD is an autosomal dominant disorder caused by a mutation in the BRI12 gene.

[0153] In certain embodiments, the hereditary CAA is FAF. FAF is characterized by extracellular accumulation of amyloid and by clinical symptoms and signs of polyneuropathy. FAF, an autosomal dominant trait, belongs to those rare monogenic disorders which occur with increased frequency in the Finnish population. In FAP syndromes the accumulating protein is a transthyretin variant or gelsolin. FAF can be caused by any one or more of the mutations identified in Table 1.

[0154] In certain embodiments, the hereditary CAA is TTR amyloidosis. TTR amyloidosis refers to a progressive condition characterized by the buildup of amyloids (amyloidosis) in the body's organs and tissues. These protein deposits most frequently occur in the peripheral nervous system, which is made up of nerves that connect the brain and spinal cord to muscles and sensory cells for detection of sensations including touch, pain, heat, and sound. The two major forms of TTR amyloidosis are the neuropathic form and the cardiac form. The neuropathic form of transthyretin amyloidosis primarily affects the peripheral and autonomic nervous systems, resulting in peripheral neuropathy and difficulty controlling bodily functions. Impairments to bodily functions can include sexual impotence, diarrhea, constipation, problems with urination, a sharp drop in blood pressure upon standing, heart and kidney problems, various eye problems, and carpal tunnel syndrome. The cardiac form of transthyretin amyloidosis affects the heart. People with cardiac amyloidosis may have an abnormal heartbeat (arrhythmia), an enlarged heart (cardiomegaly), or orthostatic hypertension. These abnormalities can lead to progressive heart failure and death. Occasionally, people with the cardiac form of transthyretin amyloidosis have mild peripheral neuropathy. TTR amyloidosis can be caused by one or more of the mutations identified in Table 1.Methods and Uses for Treating Ap-CAAs

[0155] Encompassed herein are methods of treating an Ap-CAA, comprising administering an effective amount of NAC or a functional derivative thereof to a patient in need thereof. Also encompassed herein are methods of treating a CAA, comprising preventing or reducing amyloid / tau protein aggregation in the patient, particularly in the brain. In certain embodiments, the methods of treating a CAA comprise administering a composition that disrupts hydrogen and disulfide bonds between amyloid dimers and oligomers, thereby disrupting amyloid deposition in the brain and blood vessels and alleviating CAA symptoms. In certain embodiments, the composition disrupts amyloid deposition in cerebral blood vessels. In certain embodiments, the methods comprise reducing cerebral vascular basement remodeling in the subject. In certain embodiments, the methods comprise reducing extracellular matrix accumulation of one or more CAA extracellular matrix proteins.

[0156] The term “treatment,” as used herein, includes any administration or application of a therapeutic for a disease or disorder in a subject, and includes inhibiting the disease, arresting its development, relieving the symptoms of the disease, or preventing occurrence or reoccurrence of the disease or symptoms of the disease.

[0157] In some embodiments, the treatment methods comprise identifying or diagnosing a subject as having a genetic alteration causative of the Ap-CAA, and administering a NAC or a functional derivative thereof to the identified or diagnosed subject. In other embodiments, the subject has a different disease associated with pathological fibril formation, including but not limited to Alzheimer’s disease. In certain embodiments, the CAA is a hereditary cerebral amyloid angiopathy, optionally selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF). In certain embodiments, the CAA is HCHWA selected from the Dutch subtype (HCHWA-D), the Italian subtype (HCHWA-It), the Iowa subtype (HCHWA-Io), the Flemish subtype (HCHWA-F), the Piedmont subtype (HCHWA-P), or the Arctic subtype (HCHWA-A). In certain embodiments, the patient has one of the CAAs and mutations listed in table 1 above.

[0158] In certain embodiments, the treatment methods comprise administering a composition comprising administering a composition that disrupts hydrogen and disulfide bonds between amyloid protein dimers and oligomers. In certain embodiments, disrupting the hydrogen anddisulfide bonds between these proteins treats and / or prevents amyloid deposition in the brain. Tn certain embodiments, the treatment ameliorates symptoms of the CAA. In certain embodiments, the patient has one of the CAAs discussed above. In certain embodiments, the composition that disrupts hydrogen and disulfide bonds comprises N-acetyl cysteine and / or one of its derivatives discussed above.

[0159] In certain embodiments, the treatment methods comprise increasing in vivo glutathione levels. In certain embodiments, increasing glutathione levels reduces disulfide bonds in the amyloid deposits in the brain of the patient. In certain embodiments, the treatment ameliorates symptoms of the CAA. In certain embodiments, the patient has one of the CAAs discussed above. In certain embodiments, the composition that increases glutathione levels comprises N-acetyl cysteine and / or one of its derivatives discussed above. In certain embodiments, the treatment increases a dementia rating scale score of the subject. In certain embodiments, the treatment increases the dementia rating scale-2 (DRS-2) score of the subject. In certain embodiments, the treatment increases one of the cognitive domain scores of the DRS-2 assessment. In certain embodiments, the cognitive domain is selected from Attention, Initiation / Perseveration, Construction, Conceptualization, and Memory. In certain embodiments, the DRS-2 overal index total score is increased. In certain embodiments, any one of these scores is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In certain embodiments, any one of these scores is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or 144 points. In certain embodiments any one of these scores remains the same. In certain embodiments, any one of these scores remains the same for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or longer.

[0160] In certain embodiments, the methods for reducing cerebral vascular basement membrane remodeling in a subject in need thereof, comprise administering to said subject, an effective amount of a composition comprising N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier. In certain embodiments, administration of the composition reduces cerebral vascular basement membrane remodeling in the subject. In certain embodiments, the patient has one of the CAAs discussed above.The phrase “cerebral vascular basement membrane” or “cerebral vascular BM” refers to the specialized, thin layer of extracellular matrix, that is crucial for supporting blood-brain barrier (BBB) integrity and brain fluid drainage. Cerebral vascular basement membrane-associated proteins include, without limitation, collagen IV and fibronectin, as well as signaling molecules involved in vascular remodeling and fibrosis, such as vimentin, a-smooth muscle actin (a-SMA), SMAD2 / 3, phosphorylated SMAD2 / 3, and WNT-1. In certain embodiments, the methods disclosed herein increase levels of collagen IV, fibronectin, vimentin and a-smooth muscle actin (a-SMA). In certain embodiments, the methods disclosed herein reduce activation and / or expression of SMAD2 / 3, phosphorylated SMAD2 / 3, WNT-1 and vimentin.

[0161] The phrase “cerebral vascular basement membrane remodeling” refers to any change in the structure, composition, or organization of the cerebral vascular basement membrane that compromises the integrity of the BBB. In certain embodiments, remodeling can include degradation or synthesis of basement membrane proteins, thickening or thinning of the membrane, and / or altered interactions with cellular components.

[0162] In certain embodiments, the compositions described herein attenuates vascular fibrosis associated signaling. The phrase “vascular fibrosis associated signaling” refers to one or more biochemical, molecular, or cellular signal transduction pathways that are activated, modulated, or dysregulated in the context of vascular fibrosis. In certain embodiments, such signaling influences the accumulation, deposition, or organization of extracellular matrix components (e.g., collagen IV, fibronectin, collagen la, collagen Illa) promotes myofibroblast and vascular smooth muscle activation (e.g., a-smooth muscle actin (a-SMA) and vimentin expression) and contributes to vascular wall thickening, stiffness, or remodeling. In certain embodiments, vascular fibrosis associated signaling includes, without limitation, TGF-P signaling and WNT signaling.

[0163] In certain embodiments, the methods for reducing extracellular matrix accumulation of one or more CAA extracellular matrix proteins in a subject in need thereof, comprise administering to said subject, an effective amount of a composition comprising N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier. In certain embodiments, administration of the composition reduces reducing extracellular matrix accumulation of one or more CAA extracellular matrix proteins in the subject. In certain embodiments, the patient has one of the CAAs discussed above.The phrase “extracellular accumulation of one or more extracellular matrix proteins” as used herein, refers to the presence, increase, deposition, retention, or persistence of one or more extracellular matrix (ECM) proteins within the extracellular space of a cell, tissue, organ, or biological system, whether under physiological or pathological conditions. In certain embodiments, ECM proteins include, without limitation, collagens (e.g., collagen I, III, IV), fibronectin, laminins, elastin, proteoglycans, and other structural or regulatory matrix components. In preferred embodiments, the ECM proteins are selected from collagen IV or fibronectin.

[0164] The total treatment dose or doses (when two or more targets are to be modulated) can be administered to a subject as a single dose or can be administered using a fractionated treatment protocol, in which multiple / separate doses are administered over a more prolonged period of time, for example, over the period of a day to allow administration of a daily dosage or over a longer period of time to administer a dose over a desired period of time. One skilled in the art would know that the amount of therapeutic agent required to obtain an effective dose in a subject depends on many factors, including the age, weight and general health of the subject, as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan would adjust the particular dose so as to obtain an effective dose for treating an individual having the A^-CAA.

[0165] The effective dose of therapeutic agent(s) will depend on the mode of administration, and the weight of the individual being treated. The dosages described herein are generally those for an average adult but can be adjusted for the treatment of children. The dose will generally range from about 0.001 mg to about 10.000 mg.

[0166] In an individual suffering from a more severe form of the disease, administration of therapeutic agents can be particularly useful when administered in combination, for example, with a conventional agent for treating such a disease. The skilled artisan would administer therapeutic agent(s), alone or in combination and would monitor the effectiveness of such treatment using routine methods such as neurological or pulmonary function determination, radiologic or immunologic assays, or, where indicated, histopathologic methods.

[0167] Administration of the pharmaceutical preparation is preferably in an “effective amount” this being sufficient to show benefit to the individual. This amount prevents, alleviates, abates, or otherwise reduces the severity of CAA symptoms in a patient. Treatment of patients havingCAA with an efficacious amount of NAC or a functional derivative thereof may produce improvements in neurological function, respiratory function, tapering of concomitant medication usage, or increased survival.

[0168] The pharmaceutical preparation is formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art.

[0169] Dosage units may be proportionately increased or decreased based on the weight of the patient. Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.

[0170] Pharmaceutical compositions that are useful in the methods of the invention may be administered systemically in parenteral, oral solid and liquid formulations, subcutaneously, intradermally, intramuscularly, sublingually, topically, intraperitoneal, nasally, percutaneous, respiratory, ophthalmic, suppository, aerosol, topical or other known routes of administration. In addition to the agent(s) useful for treating a CAA, the pharmaceutical compositions may contain pharmaceutically-acceptable carriers and other ingredients known to enhance and facilitate drug administration. Thus, such compositions may optionally contain other components, such as adjuvants, e.g., aqueous suspensions of aluminum and magnesium hydroxides, and / or other pharmaceutically acceptable carriers, such as saline. Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to deliver / administer the appropriate agent to a patient according to the methods of the invention. The use of nanoparticles to deliver such agents, as well as cell membrane permeable peptide earners that can be used are described in Crombez et al.. Biochemical Society Transactions v35:p44 (2007).

[0171] The pharmaceutical compositions can also comprise anti-inflammatory agents for coadministration to further alleviate symptoms of amyloid disease. These include, without limitation, corticosteroids, aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac, tolmetin, interleukin (IL)-l receptor antagonist, IL-4, IL-6, IL-10, IL-11, IL-13,cytokine receptors for IL- 1, tumor necrosis factor-alpha, IL-18 and derivatives and biosimilars thereof.

[0172] In certain embodiments, the methods disclosed herein preserve vascular smooth muscle cell integrity and / or prevent phenotypic switching of vascular smooth muscle cells to fibroblastlike states. In certain embodiments, the methods further improve intramural periarterial drainage (IPAD) and / or reduces protein elimination failure angiopathy (PEFA). In certain embodiments, the methods reduce vascular retention of amyloidogenic proteins, such as amyloid-P, cy statin C, and / or medin. In certain embodiments, the methods reduce or prevents at least one neuronal proteinopathy associated with Alzheimer’s disease. In certain embodiments, the neuronal proteinopathy is the pathological aggregation and / or mislocalization of TDP-43. In certain embodiments, the subject is assessed for one or more of a reduction in cerebral vascular basement membrane remodeling; a reduction in extracellular matrix accumulation of one or more CAA extracellular matrix protein; an increase perivascular clearance of amyloidogenic proteins; a reduction in accumulation of collagen IV and fibronectin within cerebral vessel walls; attenuated TGF-β and / or WNT signaling-associated vascular fibrosis; preservation of vascular smooth muscle cell integrity; an increase in intramural periarterial drainage (IPAD) or reduces protein elimination failure angiopathy (PEFA); a reduction in aggregation or vascular retention of one or more amyloidogenic proteins selected from cystatin C, amyloid-β, and medin; or reduces neuronal TDP-43 pathology associated with Alzheimer’s disease. In certain embodiments the change in each of these criteria is compared to an untreated control.

[0173] Screening Methods

[0174] In another aspect of the invention, methods for identifying therapeutic agents which disrupt amyloid deposits in the brain and / or walls of blood vessels are provided. An exemplary method comprises providing a population of cells which express a nucleic acid encoding a mutant Ap protein, said mutant causing formation of amyloid protein aggregates; and providing a population of cells which express an Ap protein which lacks the mutation. Both populations of cells are contacted with a test agent and assessed to determine whether the agent alters amyloid protein aggregate formation of cells expressing the mutant relative to those expressing the wild type protein, thereby identifying agents which alter amyloid protein aggregate formation. Agents so identified should have efficacy for the treatment of CAAs.Another exemplary method comprises providing two populations of cells which express a nucleic acid encoding a mutant Ap protein, said mutant causing formation of amyloid protein aggregates. The first population is contacted with N-acetyl cysteine or a function derivative thereof and the second population is contacted with a test agent. Both populations of cells are then assessed to determine whether the test agent alters amyloid protein aggregate formation at a greater than or equal level as the NAC or a functional derivative. The test agents that decrease amyloid protein aggregate formation at a level equal to, or greater than the decrease of amyloid protein aggregate formation caused by NAC or a functional derivative are identified as therapeutic agents.

[0175] In certain embodiments, the N-acetyl cysteine derivative is selected from NAC-amide, NAC-methyl ester, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt. In certain embodiments, the mutation is selected from any one of the mutations in Table 1. In certain embodiments, the mutation is in the APP gene. In certain embodiments, the test agent decreases amyloid protein aggregation by at least 10%. 20%, 30%, 40%, 50%, 60%. 70%, 80%, 90%, or 100%.

[0176] Another exemplary method comprises a method for identifying a therapeutic agent for the treatment of a first cerebral amyloid angiopathy (CAA) using a population of cells from a patient with a second CAA. This population of cells is contacted with a test agent and assessed to determine whether the test agent alters levels of at least one CAA biomarker selected from fibronectin, collagen IV, vimentin, a-SMA, SMAD2 / 3, or WNT-1. The test agents that alter at least one CAA biomarker level in the cells are identified as therapeutic agents. In certain embodiments, the first CAA is the same as the second CAA. In certain embodiments, the cell is a vascular smooth muscle cell, a pericyte, an endothelial cell (optionally a brain microvascular endothelial cell), an adventitial fibroblast, a myofibroblast, a fibroblast (including a dermal fibroblast), an astrocyte, a microglial cell / macrophage, or a neuron, optionally wherein one or more of the foregoing are derived from induced pluripotent stem cells.

[0177] In certain embodiments the first CAA is Alzheimer’s disease or a hereditary cerebral amyloid angiopathy selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF). In certain embodiments, the first CAA is selected from anyone of the CAAs discussed above.In certain embodiments the second CAA is Alzheimer’s disease or a hereditary cerebral amyloid angiopathy selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF). In certain embodiments, the second CAA is selected from anyone of the CAAs discussed above.

[0178] In certain embodiments, the test agents that decrease levels of at least one of fibronectin, collagen IV, vimentin, SMAD2 / 3, and WNT-1 in the cells are identified as therapeutic agents. In certain embodiments, the test agents that increase levels a-SMA, in the cells of are identified as therapeutic agents.

[0179] As used herein, the phrase “CAA biomarker” refers to any refers to any biological, biochemical, molecular, cellular, physiological, or functional characteristic that is measurable, detectable, or quantifiable in a biological system and that is indicative of, correlated with, or predictive of the CAA. A biomarker may comprise, without limitation, a nucleic acid, a polypeptide or protein, a metabolite, lipid, carbohydrate, small molecule, ion, cellular phenotype, signaling pathway activity, or physiological parameter, or any combination thereof.

[0180] In certain embodiments, the biomarker is directly or indirectly modulated by exposure to a test agent and is useful for screening, identifying, characterizing, ranking, or selecting pharmaceutical agents based on their biological activityl. The biomarker may exhibit a change in level, activity, localization, structure, modification, interaction, or functional state in response to such exposure.

[0181] The term “test agents” or “test compounds” refers to any agent or compound that is evaluated for potential biological activity by inclusion in the screening assays described herein.

[0182] The term “modulate” as used herein refers to increasing / promoting or decreasing / inhibiting a particular cellular, biological or signaling function associated with the normal activities of the mutation containing molecules described herein or the proteins encoded thereby. For example, the term modulate refers to the ability of a test compound or test agent to interfere with amyloid protein aggregate formation.

[0183] The following materials and methods are provided to facilitate the practice of the present invention and the following Examples are provided to illustrate certain embodiments of the invention. It is not intended to limit the invention in any way.EXAMPLE I: COMPARISON OF HCCAA AND OTHER Ap-CAA TISSUES Samples from 10 healthy controls, 10 HCCAA patients and 10 Alzheimer’s patients with extensive Ap-CAA were stained with Hematoxylin & Eosin (HE) to examine tissue structure and to compare alterations in vessel wall and parenchyma structures between groups. In HE stains, a large thickening of the vascular walls of HCCAA patients was observed and cells were reduced in numbers and distributed irregularly around vessel walls, some vessel walls were homogeneous (e. hyalinization) (Fig. 1) and some veins were ruptured with leaflets into the brain tissue. Also in some cases, a double-barrel blood vessel is formed that is newly formed within the vessel (Fig. 1). In Ap-CAA samples there was also thickening of the vascular walls and cells were reduced in numbers and distributed irregularly around vessel walls but to a lesser extent compared to HCCAA. This was mostly seen in small and medium-sized arteries and arterioles (Fig. 1), and larger arteries were not as significantly abnormal as seen in HCCAA (Fig. 1).

[0184] Cerebral arteries in normal children have a normal, cell-rich appearance as distinct septa appear between layers of vessel walls (Fig. 1).

[0185] To evaluate amyloid deposits, we counterstained all HCCAA samples with cystatin C antibody that capture the cystatin C amyloid complexes in HCCAA patients, and all Ap-CAA samples were counterstained with amyloid-P antibody to confirm amyloid deposition. Cystatin C amyloid deposits were seen in almost all arteries and arterioles of HCCAA patients (Fig. 2A), also in veins and capillaries to some extent (Fig. 2B). There were also amyloid plaques seen in the brain (Fig. 2A).

[0186] In Ap-CAA samples, we observed amyloid-P deposits in arteries / arterioles, veins and in some samples also in capillaries. Deposits were to a greater extent in small and medium-sized arteries and arterioles with some deposits were also seen in larger arteries and veins. In vessels with deposits to a lesser degree, the deposits were confined to a middle layer (Fig. 2C). Amyloid plaques were also seen in the brain tissue of the Ap-CAA patients.

[0187] To compare collagen IV accumulation between HCCAA and other Ap-CAA, collagen IV staining was performed. Collagen IV was observed in all layers of the vessel wall in all HCCAA samples, where the staining was particularly evident in the arteries and arterioles of the rib and scleral area. These arteries tend to be completely closed or have formed a double barrel, whereas larger arteries showed less staining in the extravasation of the vessel walls. Severe thickening ofthe veins was also observed and when examined at maximum magnification, circular staining was seen around cells, especially in the middle layer of the vessel walls, in basement membrane around smooth muscle cells. Considerably stronger staining was also observed in the basement membrane up to insertion (Fig. 3A). Staining was also markedly intense in the capillaries and arterioles / arterioles within of the brain tissue (Fig. 3D). In Ap-CAA samples, similar staining was observed for collagen IV relative to HCCAA. Coloring was evident in all layers of the vessel wall in the arteries and arterioles of the scrotal and scleral area, also seen in large arteries strong staining in the basement membrane of the middle layer around the smooth muscle cells and a strong thickening in the basement membrane. Little or no occluded arterioles and vessels with double circulation were seen (double-barrel) compared to HCCAA (Fig. 3B). Staining within brain tissue was not as strong within the capillaries / arterioles / arterioles (Fig. 3E) though collagen IV was observed in parenchymal plaques in all stained samples, staining was greater in plaques in HCCAA samples (Fig. 4). In controls, staining was observed mainly in the basement membrane in the middle layer and at the insertion. The staining was considerably less and paler than in patient samples while the basement membrane around the smooth muscle cells in the middle layer of the arterial cord- and of the skin area was much more distinct (Fig. 3C). There were also noticeably fewer staining capillaries / arterioles / arterioles within brain tissue (Fig. 3F).

[0188] Next, all samples were stained with fibronectin antibody. The distribution was similar in both HCCAA and other Ap-CAA, wherein fibronectin was observed in all vessel types. In HCCAA samples staining was seen in all layers of vessel walls, but it was, however, not as continuous as collagen IV staining and not as intense in vessel walls (Fig. 5). In memory arteries and arterioles, however, continuous staining similar to collagen IV staining was observed, especially in vessels that showed a strong effect of cytoreduction (Fig. 5). Staining was seen in capillaries within brain tissue but was very faint and absent in some capillaries (Fig. 6). Staining was also seen in the plaques and was considerably greater and darker than the collagen IV staining (Fig. 6A). In the Ap-CAA sample, staining was observed in all layers of the vessel wall though the coloring was spotty and paler than in HCCAA (Fig. 6B). Prominent thickening of the veins was also observed (Fig 6). Staining was seen in some of the capillaries within the brain tissue, but not all, and, in samples with staining, the staining was not intense (Fig. 6). In contrast, intense staining was observed in plaque which was considerably greater and darker than the staining with collagen IV (Fig. 6). In control samples fibronectin staining was only observed inthe medial layer and basement membrane of the arterial inlay. In the medial layer, staining was regular and around smooth muscle cells. On the other hand, it was considerably more irregular in both HCCAA and Ap-CAA and cells were easier to distinguish in controls (Fig. 1). In controls, faint and low staining was observed in a few capillaries within the brain tissue (Fig. 6C and 6F).

[0189] Samples were stained with both vimentin and SMA antibodies to examine the distribution and appearance of fibroblast cells in the vessel wall. In HCCAA samples, staining for vimentin was observed in all layers of vessel walls. Thickening was seen in the inlay, irregular staining was seen in the middle layer and irregular shaped cells, large and elongated with an activated appearance. In larger arteries, many positive cells with an activated appearance were observed throughout the vessel wall (Fig. 7A). Arteries / arterioles often showed little or no staining in the middle layer, only a few positive cells but sometimes thick staining in both intima and adventitia (Fig. 7D), the same vessels that had little or no endothelial staining (CD34). In Ap-CAA, staining was similar to both HCCAA and controls. Some thickening was also observed in staining in the vessel wall inlay in Ap-CAA but not as high as in HCCAA (Fig. 7B and 6E). In control samples, staining was only observed in the lining and middle layer of the vessel walls. The staining was considerably greater in the middle layer than in the inlay while the cells were coiled and tightly and regularly packed together (Fig. 7 ).

[0190] SMA staining was seen in all layers of the vessel walls of some larger arteries in HCCAA samples, with thick staining was in the inlay and in smaller arteries / arterioles. SMA positive cells completely disappeared from the vessel wall (Fig. 8A). In larger vessels, there was staining in the thickened inlay, and in the middle and outer layers the cells in the vessel wall were irregularly stained and had a stretched, activated appearance (Fig. 8B). In the veins, staining was persistent and in some arteries with less abnormality, cell proliferation was observed despite cytoreduction in other vessels. In Ap-CAA samples, positive SMA staining was usually only seen in the inlay and middle layer (Fig. 8B), and the staining was either lighter or darker compared to controls, i.e. more extensive in less affected CAA vessels and loss of SMA cells in more affected CAA vessels. Additionally, the shape of cells often more irregular and the staining more uniform when compared to controls. In smaller arteries / arterioles in the CAA sample, there was staining in all layers of the vessel walls but thick staining was only seen in the inlay (Fig. 8B). Veins often had an eroded layer of staining (Fig. 8). In control samples uniform and continuous staining was observed in the vessel walls. In the arteries / arterioles there was staining in the inlayand medial layer (Fig. 8C and 4F). In the veins control samples, the staining formed a thin, continuous layer of cells (Fig. 8C and 4F).

[0191] Double staining using both vimentin and SMA was performed and the samples were examined under a confocal microscope. In HCCAA samples, a high cytoreduction in the middle layer was observed, positive cells sat on a rod around the vessel wall, and some co-staining was observed in the remaining cells and in cells with a fibroblast activated (stretched) appearance (Fig. 9A-9C). In Ap-CAA samples, positive cells were not as regular as in control samples but significant cell reduction was not observed when compared to the reduction observed in HCCAA cells. Co-staining was observed in cells within the vessel wall which had an activated (stretched) appearance (Fig. 9D-9F). The control showed dense staining for both vimentin and SMA in the medial layer of the vessel wall. Some co-staining was seen, but positive cells for both vimentin and SMAs did not show an activated (stretched) appearance (Fig. 9G-9I).

[0192] There is a decrease in the number of vascular endothelial cells in HCCAA. Visual inspection of brain tissue revealed collagen IV staining and an increase in the number of capillaries and small arteries / arterioles in both HCCAA and other Ap-CAA samples relative to controls. Therefore, we stained all samples with CD34 marker that targets endothelial cells and compared the results with collagen staining of brain tissue. In HCCAA samples, CD34 antibody staining was light, and no staining was observed in all vessels, including vessels that show thick and continuous collagen IV staining (Fig. 10A). The collagen IV staining, on the other hand, was very dark and a lot of small arteries / arterioles with a lot of thickening within the brain tissue were observed. Capillaries also showed thickening (Fig. 10B). In Ap-CAA samples, the CD34 staining was darker, with very few small blood vessels having little or no staining (Fig. 10C). The collagen IV staining was not as dark as in HCCAA samples but, in Ap-CAA samples, the small arterioles / arterioles and capillaries showed thickening (Fig. 10D). Controls included CD34 staining that is darker than in HCCAA and all blood vessels showed staining. However, the staining appeared to be much less in these tissues when compared to the other sample groups (Fig. 10E). No thickening of capillaries was seen in collagen IV staining and considerably less of small arteries / arterioles were observed within brain tissue (Fig. 10F).

[0193] To evaluate if activated cells in the vessel wall of patients were positive for the TGF-b / smad pathways and Wnt / b-catenin, we stained all samples with antibodies for both smad2 / 3 and Wnt-1. In HCCAA samples, the Wnt-1 staining was distributed throughout all layers of thevessel walls and both the middle and outer layers were irregular when compared to control samples. Positive cells were scattered throughout both the middle and outer layers with some vessels showing dark, continuous staining in a thicker inlay than the control samples (Fig. 11A and 12A). In A0-CAA samples the Wnt-1 staining was similar to other sample groups. The staining was dense and regular in the larger arteries / arterioles though it was limited to the middle layer and sometimes the inlay. In smaller arteries / arterioles irregular and fainter staining was seen in all layers of the vessel walls (Fig. 11C, HE and 12C). In control samples, Wnt-1 staining was present in the lining and medial layer of vessel walls and the coloring was dense and regular in the middle layer. In some vessels there was continuous staining in the inlay (Fig. 11G and 12E).

[0194] In HCCAA samples, smad2 / 3 staining was distributed throughout all layers of the vascular wall, like Wnt-1. When the samples were compared, they appeared to be in the same cell types. In the middle layer and outer layer, the coloring was spotted and irregular and the coloring in the inlay was dark, thick and continuous. The staining was in most cells of the middle layer and outer layer, and cells often had an activated, elongated appearance (Fig. 11B and 12B).

[0195] In Ap-CAA samples, the appearance of smad2 / 3 staining differed from vessel to vessel. In larger vessels and some smaller arteries / arterioles, staining was dense and regular in the middle layer similar to control samples. In other arteries / arterioles with CAA appearance, staining was distributed irregularly in the middle and outer layer, similar to HCCAA samples. Core staining was also either in the middle layer and outer layer or only limited to the middle layer with the nuclei appearing either normal in appearance or stretched. There was thick and dark continuous staining of the intravascular Ap-CAA in some veins, while narrower with lighter staining in others (Fig. HD, 1 IF and 12D). Smad2 / 3 staining of control samples showed very similar staining to Wnt-1. The coloring was in the middle layer and inlay, dense and regular medial layer and cells had a normal, regular appearance (Fig. 11H and 12F).

[0196] To examine if the deposition pattern of Ap in Ap-CAA is similar to HCCAA, double staining for cystatin C and SMA was performed in HCCAA samples and double staining for Ap and SMA was performed on Ap-CAA samples. Endoscopy of cystatin C / SMA and Ap / SMA showed a very similar pattern in both cases. Both cystatin C and Ap showed rich staining in the medial layer of larger arteries, in basement membrane around smooth muscle cells (Fig. 13B and 13E). Additionally, the deposition pattern was very clearly seen around the cells (Fig. 13A and13D). No co-staining was observed with SMA and cystatin C / A0 indicating that the proteins are not present in the cytoplasm SMA positive cells (Fig. 13C and 13F)

[0197] EXAMPLE II: ANALYSIS OF NAC DERIVATIVES

[0198] We found that glutathione (GSH) completely blocked amyloid aggregation (Fig. 14). GSH is well tolerated and a precursor version of it (NAC) can be given as a tablet. The oligomers essentially disappeared, suggesting there is no new precipitation of amyloid in the brain and brain vessels and a skin biomarker confirmed response to therapy in patients. The main effects of NAC treatment in our Phase II study were that no major strokes occurred for the treatment duration, no decline in cognitive function and marked reduction in amyloid and related biomarkers in the skin. Higher local concentrations of the active drag NAC in the brain are likely to improve the effect of treatment.

[0199] Superior pharmacokinetic properties of NACA, compared to NAC, result in higher local concentration of the active substance, NAC. in the brain where drug activity is most relevant. The data suggest that increased local NAC concentrations should reduce the occurrence of, and / or prevent further intracranial hemorrhages. This enables effective once daily dosing, rather than 3X daily dose required for NAC, resulting in improved compliance and adherence to a drag regimen that is likely to entail long term treatment, possibly, over an entire lifetime. NACA has also been shown to accumulate more readily in the CNS compared to NAC (Fig. 15) when introduced orally or via intraperitoneal injection in an animal model. After 15 minutes, NACA and NAC peaks could be detected in the brain, whereas after oral administration of NAC, no substantial elevation of NAC in the brain of mice could be detected.

[0200] Based on the in vitro studies presented, NACA exhibits greater ability to prevent or resolve pathogenic aggregation of mutant L68Q hCC into toxic oligomers / amyloids compared to NAC. NACA is a potent reducing agent which converts disulfide bonds into free thiols. Notably. hCC has 4 cysteine residues that can form two intramolecular disulfide bonds or intermolecular bridges between monomeric units. The high molecular weight (HMW) L68Q hCC aggregates are likely dependent on disulfide bridges to maintain stability, which is disrupted by NACA. In vivo, the ability of NACA to break disulfide bonds and generate free thiols can be mediated directly by NACA or through its metabolic products. Evidence of the ability of NACA to interrupt disulfides include (a) the formation of mixed disulfides in tissues during the development of an LCMSbioanalytical assay for NACA and (b) the cystine-depletion activity of NACA in human cystinosis cell culture. NACA is metabolized to NAC, which is then metabolized into the amino acid, L-cysteine, a precursor to the cellular redox regulator, glutathione. Administration of NACA or NAC is known to increase / replenish glutathione levels, and one of the biological functions of glutathione is to reduce disulfide bonds. Consequently, NACA exerts similar effects in preventing / resolving other amyloid structures where the amyloidogenic peptides contain cysteine residues that can form disulfide bonds. Table 2 lists the number of cysteine residues for each of the hereditary CAA amyloidogenic peptides.

[0201] Table 2: Enumeration of cysteine residues (with potential to form disulfide bonds) in amyloidogenic entities of various hereditary CAAs.

[0202] Amyloidogenic Disease Cysteine Residues in peptide / protein sequence

[0203] L68Q Cystatin C HCCAA 4

[0204] Ap40and Ap42 HCHWA (A|3 sub-types) 0

[0205] TTR ATTRv 1

[0206] PrPSc Genetic prion diseases 2

[0207] ABri FBD 2

[0208] ADan FDD 2

[0209] AGel FAF 2

[0210]

[0211] Each of the primary structures of the amyloidogenic peptides / proteins listed in Table 2 comprise cysteine residues, and thus possess the capacity to form disulfide bonds, except for 0-amyloid (A0). Even though A0 lacks cysteine residues, it includes hydrogen bonds and pathogenic effects of amyloid / oligomer formation in A0 forms of HCHWA is still disrupted by NACA through disruption of the hydrogen bonds and by other mechanisms. In this regard, addition of the amino acid cysteine (which is also a metabolite of NACA) into an amyloid aggregation assay with A 040 or A042 in vitro inhibited fibril formation and instead produced a “dead-end” aggregate that were non-toxic. These results indicate that structurally similar derivatives / analogs of cysteine (e.g., NACA) may exhibit similar inhibitory activities. Sites of A0 aggregation in the CNS also have unusually high concentrations of metals, including Cu(II) and Fe(III), which have been implicated in the pathogenesis of A0 diseases. Removal of metals by soluble chelators can prevent A0 aggregation. NAC and NACA have been shown to exhibitnoticeable metal chelating properties, which could be utilized for the purposes of inhibiting Ap aggregation and resulting toxicity by removing / lowering the amount of available metals. The damaging effects of A have been attributed at least in part to the actions of soluble di-tyrosine crosslinked dimers, which are formed under oxidative conditions where reactive oxygen species (ROS) generate free radicals. These ROS are formed at least in part from the interaction between Ap and metal ions, such as iron (Fe). copper (Cu), and zinc (Zn), thus the chelating properties of NACA reduce the formation of di-tyrosine dimers.

[0212] Human cystatin C (hCC) is found to co-localize with Ap amyloids in cerebral vasculature in the HCHWA-Dutch. Additionally, in sporadic cases of CAA, hCC deposition in cerebral vessels is found only in those subjects experiencing clinically evident ICH, and the hCC in these sporadic cases are WT that do not contain the L68Q mutation. Sporadic CAA rarely results in ICH. The apparent colocalization of hCC only at sites of ICH in subjects with hereditary CAAs suggests a role in the pathogenesis (trigger). In vitro studies show that WT hCC can form aggregates that increase Ap fibrillation and is cytotoxic, whereas monomeric hCC inhibits Ap fibril formation. We have demonstrated that NACA prevents / disrupts hCC and Ap protein aggregation, thereby indicating that NACA has therapeutic effects in Ap sub-types of HCHWA, other CAAs and Alzheimer's disease through its mechanism of action of pre venting / ameliorating protein aggregation.

[0213] Additionally, in vitro data demonstrates that the amide analog. NACA, has > 10-fold higher potency in preventing / dispersing hCC aggregates than NAC. Moreover, its higher bioavailability and ability to penetrate the BBB and cross over cell membranes compared to NAC shows that NACA will be a more effective drug. Additionally, the data indicates that NACA is effective in treating other hereditary CAAs beyond HCCAA as well as Alzheimer's disease. The ability of NACA to interrupt disulfides bridges and interfere with hydrogen bonds, supports the efficacy of NACA in the treatment of hereditary CAAs and Alzheimer's disease, in which the respective amyloidogenic peptides contain cysteine residues. Thus, in reference to HCHWAs and Alzheimer's disease arising from Ap mutations, several additional characteristics of NACA are beneficial (thiophilic interactions, metal chelating properties, prevention of hCC aggregation) and contribute to the inhibition of the pathogenic aggregation of Ap. Overall, our studies reported implicate NACA therapy as an effective in the treatment of patients with hereditary CAAs as well as Alzheimer's Disease.EXAMPLE II: FURTHER ANALYSIS OF BASEMENT MEMBRANE AND EXTRACELLULAR MATRIX REMODELING IN HCCAA AND ALZHEIMER’S DISEASE PATIENTS

[0214] Hereditary cystatin C amyloid angiopathy (HCCAA) is a familial form of cerebral amyloid angiopathy (CAA), a progressive cerebral amyloid vasculopathy that causes recurrent, often severe intracerebral hemorrhages and premature death in young adults. It is caused by a mutation in the CST3 gene which leads to cystatin C aggregation. Cystatin C aggregation is also found in peripheral tissues, such as the skin. Basement membrane (BM) remodeling is implicated in the disease in both cerebral vessels and the skin. We examined temporal relationships between BM expansion, extracellular matrix remodeling (collagen la. collagen Illa, fibronectin (FN1), collagen IV (COL IV), vascular and neuronal cellular alterations, signaling-associated markers (SMAD2 / 3, pSMAD2 / 3, WNT-1), and amyloid aggregation (cystatin C, Ap and Medin) in HCCAA (as representative rare, inherited CAA) and in its common form of Alzheimer's Disease (AD) as the common sporadic form of CAA.

[0215] The data presented herein indicate that BM remodeling was prominent. FN1 and COL IV were expanded beyond the BM into all vessel-wall layers, with FN1 immunoreactivity -71% higher in leptomeningeal arteries / arterioles in HCCAA versus controls. COL IV was elevated across arteries, arterioles, veins, venules, and capillaries, even in vessels without detectable cystatin C. In leptomeningeal arteries / arterioles, COL IV was -79% higher, and in parenchymal vessels (including capillaries and veins / venules) COL IV was -45% higher. We also observed proliferation of cells with (myo)fibroblast-like phenotype (a-SMA7vimentin+), together with intimal COL IV accumulation in cystatin C-negative vessels, these data indicate that BM expansion precedes cystatin C aggregation, consistent with prior observations in skin.

[0216] Comparable results were obtained in brain tissue samples from AD patients, indicating that similar vascular and extracellular matrix alterations accompany the more common form of sporadic CAA.

[0217] Together, these findings indicate that early BM expansion with COL IV and FN 1 accumulation, along with broader extracellular matrix remodeling, including increased deposition of interstitial collagens such as collagen la and collagen Illa and altered vascular cellular organization, may impair perivascular drainage and create a permissive environment forcystatin C and other amyloid aggregation in both rare (HCCAA) and common (AD) forms of CAAs.

[0218] The data indicate that targeting oxidative stress and pro-fibrotic signaling with therapies such as N-acetylcysteine amide (NACA) to restore matrix homeostasis limits aggregation of cystatin C / amyloid and related protein aggregation, thereby preventing, reducing symptoms of, and treating both rare and common forms of CAAs, most prominently Alzheimer's Disease.

[0219] Materials and Methods

[0220] The following materials and methods are provided to facilitate the practice of the present invention. It is not intended to limit the invention in any way.

[0221] Samples

[0222] Post-mortem brain tissue samples, formalin-fixed, paraffin-embedded (FFPE), were obtained from 20 individuals diagnosed with HCCAA (11 females and 9 males), aged 15-79 years (mean age 33.9 years) and 20 controls (7 females and 13 males), aged 17-40 years (mean age 33.2 years) without any diagnosed neurological disorders. Because HCCAA is an ultra-rare disease with orphan status, the availability of autopsy material is limited, and most specimens are from archival collections. A comparative analysis was performed using postmortem brain tissue from AD patients with concomitant A0-CAA (8 females and 2 males), aged 63-88 years (mean age 78 years). To ensure optimal comparability across cases, the cerebral cortex was selected for analysis, with a primary focus on leptomeningeal vessels.

[0223] All tissue samples underwent standard histological processing and were embedded in paraffin blocks. From each paraffin-embedded tissue block, serial sections were cut: one section at 1.5 pm thickness for Hematoxylin and Eosin (H& E) staining and others at 3 pm thickness for immunohistochemical analyses. Sections were mounted on Starfrost microscope slides and dried overnight at 37°C. Subsequently, slides were incubated at 60°C for 60 minutes, followed by deparaffinization in xylene and alcohol, rendering them ready for staining procedures.

[0224] Hematoxylin and Eosin (H& E) and Conge) red staining

[0225] H& E staining was performed to assess the general histological architecture, highlighting cellular and extracellular components. Congo red staining was performed and images captured on a Leica DM6000 equipped with a Crest Optics Cicero spinning disc and TeledynePhotometries Prime 95B using a 20x, NA 0.8, PL APO, Air objective lens and Visiview acquisition software version 6.0. Images where then adjusted and prepared for manuscript using FIJI (Fiji Is just ImageJ) (Schindelin et al. 2012).

[0226] Immunohistochemistry

[0227] Immunohistochemical staining was conducted to detect specific antigens, including COL IV, FN1, collagen la, collagen Illa, vimentin, WNT-1, SMAD2 / 3, phosphorylated SMAD2 / 3 (pSMAD2 / 3), alpha-smooth muscle actin (a-SMA), amyloid p, Medin, cystatin C and phosphorylated TDP43. Staining for COL IV, WNT-1, and SMAD2 / 3 were performed manually with primary antibodies incubated overnight at 4°C. Other antibodies were processed using the Dako Autostainer Link 48 system. Antibody dilutions were prepared using EnVision FLEX Antibody Diluent (see Table 3), except for vimentin, which was pre-diluted by the manufacturer. A corresponding negative control (EnVision FLEX IR750, Agilent), in which the primary antibody was omitted, was included in all staining runs. All procedures were carried out at the Department of Pathology, Landspitali University Hospital.

[0228] Antigen retrieval was achieved using EnVision FLEX High pH (pH 9) target retrieval solution for FN1, vimentin, a-SMA, and cystatin C, pSMAD2 / 3 and Low pH (pH 6) solution for Wnt-1, and SMAD2 / 3. Slides were immersed in the appropriate buffer and heated in a water bath at 98-99°C. For COLIV, proteinase K treatment (1:19 dilution in target retrieval buffer) at 37°C was employed. To block endogenous peroxidase activity and prevent non-specific binding, sections were treated with 3% hydrogen peroxide. Primary antibodies were then applied (see table 2). Detection was facilitated using secondary antibodies conjugated to horseradish peroxidase (HRP) within a dextran polymer, visualized with diaminobenzidine (DAB) chromogen, producing a brown precipitate at antigen sites. Slides were counterstained with hematoxylin.

[0229]

[0230] Table 3: Primary antibodies and IHC conditions (FFPE)

[0231]

[0232] Antibody 5 Manufacturer Host / Type Antigen retrieval Dilution Primary incubation i / Cat. no.

[0233] COL IV. Sign a. Cl 926. Mouse, Proteinase K 1:500. Overnight at 4 C monoclonal

[0234]

[0235] FN1 § Cell Signaling,: Rabbit, i pH 9 i 1:40 1 60 min 1 j E5H6X 1 monoclonal:: 1

[0236]

[0237] s FN1 IST-9 j Santa Cruz, sc-; Mouse i pH 9 i 1:30 i 30 min::: 59826: monoclonal i:

[0238]

[0239] Vimentin § Agilent, IR630 Mouse,: pH 9 Ready-to-use 1 30 min

[0240] : monoclonal;;

[0241]

[0242]

[0243] : WNT-1 § Santa Cruz, sc-; Mouse, i pH 6 i 1:30 Overnight at 4 °C;: § 514531; monoclonal i:;

[0244]

[0245] : SMAD2 / 3 s Santa Cruz, sc-: Mouse,: pH 6 i 1:: Overnight at 4 °C:

[0246] § 133098 monoclonal:::

[0247]

[0248] pSMAD2 / 3: Invitrogen,: Mouse; pH 9 i 1:100: 30 min:: § PA5-110155: monoclonal; i:

[0249]

[0250] i a-SMA Dako, M0851 j Mouse, 1 pH 9 j 1:300 I 30 min:::: monoclonal:::: Cy statin C: Sigma,: Rabbit,: pH 9 j 1:100 i 30 min::: HPA013143: polyclonal:::: Ap; Agilent, M0872: Mouse, i pH 9 1:100: 60 min:: j: monoclonal i;;:: Medin > 6B3: Mouse, i pH 9 i 1:500: 30 min:::: monoclonal i;;::

[0251]

[0252] Phospho-: Invitrogen,: Rabbit,: pH 9 j 1:400: 30 min: TDP-43 j # 80007 polyclonal i

[0253]

[0254]

[0255] : Collagen I; Cell Signaling,: Rabbit,:::: i i pH 6 1: 100 i 30 min:: (COL1A1); #72026: monoclonal::::; Cell Signaling,::::: Collagen III: Rabbit,::: i: #63034 / i i pH 6 1: 100: 30 min i: (COL3A1):: monoclonal;;:

[0256] 5 63034S

[0257]

[0258] Dual Immunofluorescence Staining

[0259] To visualize co-localization of antigens, dual immunofluorescence staining was performed. Antigen retrieval was conducted using EnVision FLEX High pH (pH 9) target retrieval solution at 98-99 °C. Primary antibodies were incubated overnight at 4 °C.Subsequently, slides were incubated with DAPI nuclear stain and two secondary antibodies for 60 minutes (room temperature). Secondary antibodies included Alexa Fluor 488 Goat anti-rabbit IgG (Thermo Fisher: 1:1000), Alexa Fluor 488 Goat anti-mouse IgGl (Thermo Fisher; 1:1000), and Alexa Fluor 647 Goat anti-mouse IgG2a (Thermo Fisher; 1:1000). Stained sections were examined, and images captured using an Olympus FV1200 confocal microscope. Image processing was performed with FIJI (Fiji Is just ImageJ) (Schindelin et al., 2012).

[0260] Image Acquisition and Analysis

[0261] All stained slides beside Congo red and immunofluorescence-stained slides were digitized using a NanoZoomer XR scanner (HAMAMATSU) Digital images were reviewed with NDP.view2 software (HAMAMATSU), and regions of interest were saved as TIFF files. Image analysis was conducted using FIJI (ImageJ) (Schindelin et al., 2012).

[0262] For quantification of COL IV, FN1, vimentin, a-SMA, Wnt-1, SMAD2 / 3, and p-SMAD2 / 3 staining intensity and distribution within vessel walls. Two FN1 antibodies were evaluated. As the CST E5H6X antibody provided stronger and more consistent FN1 immunoreactivity across vessel walls and parenchymal deposits compared with IST-9, only E5H6X was used for quantitative analyses. Images acquired at a pixel resolution of 0.226 pm / pixel were analyzed. A total of 100 small- to medium-sized leptomeningeal arteries / arterioles were randomly selected under blinded conditions. The outcome for each vessel was DAB-positive area fraction (%) of the vessel-wall ROI. Per case, vessel-level values were averaged to yield one measurement. The percentage of the vessel wall area exhibiting DAB staining was calculated / Region of interest (ROI), the method was previously described in (Snorradottir et al.. 2013).

[0263] For COL IV parenchymal staining, each case, three equal-sized cortical regions of interest (RO Is) were analyzed to quantify vascular staining as COL IV-positive area fraction. Color images were converted to CIELAB color space and the b* channel was extracted and duplicated. Brightness / contrast was adjusted to the minimum and maximum histogram limits and applied, images were converted to 16-bit, and brightness / contrast was adjusted once more and applied. Segmentation used to adjust threshold with dark background enabled to isolate COL IV positive signal, the threshold was kept constant across all images. The resulting mask was binarized and refined with Process, binary, close. Particles were quantified with analyze particles using a size threshold >10 (calibrated units; no upper limit) and circularity 0.00-1.00, with show:Overlay for quality control. Measurement settings were area, area fraction, mean gray value, limit to threshold, and display label. For each ROI, the primary outcome was COL IV positive area fraction (%),all threshold and particle analysis parameters were standardized across images, and overlays were inspected to confirm accurate vessel segmentation.

[0264] Statistical analyses were performed in GraphPad Prism (version 10.6.1). Because the data were not normally distributed (confirmed by Shapiro-Wilk and Kolmogorov-Smirnov tests), group comparisons between patients and controls were made using the Mann-Whitney U test (two-tailed), with significance set at P < 0.05. Immunoreactivity values (% immunoreactive area per ROI) were analyzed for COL IV, FN1, vimentin, a-SMA, SMAD2 / 3, pSMAD2 / 3 and Wnt-1. Results were visualized as box-and-whisker plots (GraphPad 10.6.1) displaying the median and interquartile range (IQR).

[0265] Results

[0266] H& E

[0267] H& E staining confirmed the typical acellular, homogenized appearance of CAA-laden vessel walls (Fig. 27A-27D). Smaller affected arteries / arterioles often contained few or no residual cells, whereas larger, less affected arteries retained cells across the intima, media, and adventitia (Fig. 27C-27E). Among these, very few vessels showed near-normal architecture; instead, many vessels across all sizes exhibited altered cellular organization, with cells appearing more scattered throughout the vessel wall and, in some cases, extending into the intimal layer (Fig. 27E, arrows), rather than maintaining the normal layered arrangement observed in control vessels (Fig. 27F).

[0268] Cystatin C

[0269] Across HCCAA cases, we observed severe cystatin C deposition, parenchymal cystatin C deposits, and focal parenchymal cystatin C plaques (Fig.28A-28E). Cystatin C aggregation was detected in all vessel types, with smaller arteries and arterioles the most affected (Fig. 28A and 28D). With improved staining and imaging, we also observed greater venous involvement (Fig.

[0270] 28C, arrow), while capillary involvement remained minimal. In larger arteries with less advanced pathology, cystatin C immunoreactivity was often observed at or adjacent to the BM surrounding VSMCs and / or along the internal elastic lamina, consistent with early pathogenic involvement of these structures, forming circular pericellular labeling around the smooth-muscle-associated BM. This pattern was evident with cystatin C immunostaining and Congo red staining (visualized with a Texas Red filter; Fig. 28D and 28F and Fig. 29). In larger and less affected arteries, typically 1-6 vessels per brain section across patients, cystatin C immunoreactivity was initially confined to BM-associated structures, whereas with progression it extended into the adventitia and, at later stages, into the intima and toward the endothelium (Fig. 28D). No cystatin C co-localization with a-SMA was detected by multiplex immunofluorescence (Fig. 29A-29C). The same analysis demonstrated intimal thickening, as seen in HE stained sections, with an increased number of a-SMA-immunoreactive cells in intima in affected vessels (Fig. 29B-29C), consistent with the alterations observed by H& E staining and then cystatin C staining surrounding a-SMA-immunoreactive cells in the media (Fig. 29C). These findings are consistent with our skin biopsy data, where cystatin C closely associates with BMs and lies near fibroblasts (Snorradottir et al., 2017).

[0271] Collagen IV (COL IV)

[0272] COLIV immunostaining was evident in all layers of affected vessels (Fig. 30A-30B). Leptomeningeal arterial / arteriolar COL IV immunoreactivity was elevated in HCCAA (median [1QRJ, 59.4% [49.5-71.4]) compared with controls (33.3% [28.1-39.2], Mann-Whitney U test, P < 0.0001) (Fig. 30E). Beyond leptomeningeal vessels, a novel and consistent finding was that COL IV deposition was detected in parenchymal plaques (Fig. 30C) and appeared increased along the walls of most parenchymal vessels (Fig. 30F-30G). In the current cohort, increased COL IV immunoreactivity extended beyond arteries / arterioles to involve veins and capillaries (Fig. 30B, 30F, 30G). The thickened BM rendered vessel profiles more prominent in HCCAA sections compared with controls (Fig. 30D and 30H). Quantitative analysis confirmed that COL IV immunoreactivity in parenchymal vessels (% immunoreactive area per ROI) was higher in HCCAA patients than in controls (median [IQR], 2.81 [1.65-4.59] vs 1.94 [1.68-2.19], Mann-Whitney U test, P - 0.0055) (Fig. 301).

[0273] Notably, COL IV immunoreactivity was observed within the intimal layer of affected leptomeningeal arteries in some vessels lacking detectable cystatin C in this compartment, suggesting that BM expansion and COL IV deposition may occur prior to more extensive cystatin C accumulation (Fig. 30B and 31 A). Overall, these findings indicate that COL IV accumulation represents an early pathological event. Our prior skin studies demonstrated that cystatin C aggregation is consistently closely associated with COL IV-positive BM, with clearco-localization

[0047] ; a similar pattern was observed in cerebral vessels, where early cystatin C aggregation appeared to localize to the BM and co-localize with COL IV (Fig. 31A-31D).

[0274] Fibronectin (FN1)

[0275] In HCCAA, FN1 immunostaining was detected across all major vessel types, a novel finding in this disease (Fig. 32A-32C). Using the CST E5H6X antibody, FN1 staining involved all vessel wall layers but compared with COL IV, was less continuous and generally lower in intensity (Fig. 32A-32C). In smaller affected arteries / arterioles, FN1 labeling often formed a continuous circumferential ring resembling COL IV, particularly in vessels with marked cellular depletion (Fig. 32B). Some parenchymal vessels showed dense FN1 staining, whereas many were negative, especially capillaries (Fig. 32B), in contrast to the more uniform COL IV labeling of parenchymal vessels. In parenchymal cystatin C plaques, FN1 immunoreactivity was readily detected with E5H6X and appeared stronger than COL IV when assessed with E5H6X (Fig. 32A-32B).

[0276] In parallel experiments using the IST-9 antibody, FN1 staining in leptomeningeal vessels showed a similar spatial distribution but was generally weaker and less distinct (Fig. 32E), particularly within parenchymal plaque-like deposits (Fig. 32G). Overall, E5H6X provided stronger and cleaner FN1 immunoreactivity across tissue compartments. Because FN1 immunoreactivity in parenchymal vessels was heterogeneous and both FN 1 antibodies exhibited higher background staining in the parenchyma compared with COL IV, quantitative analyses were restricted to leptomeningeal vessels and were not performed for parenchymal vessels.

[0277] In control brains, FN1 was largely confined to the media and intimal basement membrane of leptomeningeal arteries and accompanying veins, with regular circumferential labeling around vascular smooth muscle cells and only scant capillary staining (Fig. 32D and 32H).

[0278] Given the superior signal-to-noise ratio and consistency of FN I detection obtained with the CST E5H6X antibody, quantitative analyses in leptomeningeal arteries / arterioles were perf ormed exclusively using E5H6X. Quantitative analysis showed that FN 1 immunoreactivity (% immunoreactive area per ROI) was higher in HCCAA patients (median [IQR], 31.9% [19.4-43.5]) than in controls (18.6% [14.02-49.5], Mann-Whitney U test, P < 0.0001), Fig. 321).

[0279] Ap, COL IV and FN1 in AD with associated CAAImmunohistochemical staining for amyloid-P (AP) confirmed the presence of cerebral amyloid angiopathy in Alzheimer’s disease (AD) cases, with prominent Ap deposition in leptomeningeal and cortical vessels as well as within parenchymal plaques (Fig. 33). Vascular Ap immunoreactivity was observed along vessel walls, consistent with AD-associated CAA, while parenchymal plaques showed dense extracellular Ap accumulation.

[0280] Following confirmation of Ap pathology, extracellular matrix remodeling was assessed by COL IV and FN 1 (E5H6X) immunostaining. Immunohistochemical analysis revealed prominent BM remodeling similar to that observed in HCCAA. COL IV immunoreactivity was markedly increased in affected leptomeningeal vessels and in parenchymal vessels, especially capillaries, outlining thickened vessel walls with expanded BM staining (Fig. 34A). In addition, COL IV was detected within parenchymal amyloid plaques (Fig. 34B). Quantitative analysis demonstrated higher COL IV immunoreactivity in leptomeningeal vessels from AD (Ap-CAA) cases compared with controls (mean ± SD: 56.42 ± 11.43 vs 34.20 ± 8.45, respectively; range 23.54-79.81 vs 16.84-55.16; Mann-Whitney U test, P = 0.0055) (Fig. 34C) but was not significantly different in parenchymal vessels (Fig. 8d), though capillaries in some cases showed thicker labeling (Fig. 34B).

[0281] FN 1 immunostaining in AD (Ap-CAA) demonstrated robust labeling of affected leptomeningeal arteries, with staining extending across vessel wall layers (Fig. 34E). Notably, FN 1 immunoreactivity was also prominent within parenchymal plaques and appeared stronger than COL IV staining in these deposits, mirroring the pattern observed in HCCAA (Fig. 34F).

[0282] Together, these findings indicate that both BM expansion and FN 1 accumulation are shared features of vascular and parenchymal pathology in AD (Ap-CAA). Quantitative analysis showed that FN 1 immunoreactivity (% immunoreactive area per ROI) was higher in AD (Ap-CAA) cases (median [IQR]. 25.77% [19.99-35.07]) than in controls (18.62% [14.02-27.26]; Mann-Whitney U test, P < 0.0001). (Fig. 34G).

[0283] Vimentin

[0284] Vimentin immunoreactivity was detected across all vessel wall layers, with prominent staining in the thickened intima and in cells displaying elongated nuclei. In the media, staining was heterogeneous, and vimentin-positive cells often appeared enlarged and elongated, consistent with altered cellular morphology (Fig. 35A-35B). Larger arteries contained numerous vimentin-positive cells distributed throughout the vessel wall, whereas smaller arteries / arteriolesfrequently showed little or no medial staining, with only scattered positive cells, although in some vessels more pronounced labeling was observed in the intima and adventitia (Fig. 35B-35C). In controls, vimentin was restricted to the intima and media, stronger in the media and thinner in the intima, with spindle-shaped cells arranged closely and regularly (Fig. 35D).

[0285] Quantitative analysis showed that vimentin immunoreactivity (% immunoreactive area per ROI) was lower in HCCAA patients (median [IQR], 6.25% [1.10-12.05]) than in controls (16.30% [8.70-21.80]), P < 0.0001 (Mann-Whitney U test), Fig. 35E)).

[0286] a-SMA

[0287] a-SMA immunostaining was present across all vessel wall layers in some larger arteries, with prominent labeling in the thickened intima; in contrast, smaller arteries / arterioles often showed markedly reduced or absent a-SMA-positive cells within the vessel wall (Fig. 35F-35H). In larger vessels, a-SMA-positive cells were irregularly distributed and frequently displayed elongated morphology across the intima, media, and adventitia (Fig. 35F-35G). Venous a-SMA staining was sometimes discontinuous (Fig. 35G, arrow).

[0288] In control tissue, vessel wall staining was uniform and continuous: arteries / arterioles exhibited regular a-SMA labeling in the intima and media with densely and orderly arranged spindle-shaped cells, while veins displayed a thin, continuous cellular layer (Fig. 351).

[0289] Quantitative analysis demonstrated that a-SMA immunoreactivity (% immunoreactive area per ROI) was significantly lower in HCCAA patients than in controls (median [IQR], 6.35% [1.98— 14.55] vs 16.64% [12.40-21.17]; Mann-Whitney U test, P < 0.0001; Fig. 35 J).

[0290] In some patient arteries, increased cellularity was observed across multiple vessel wall layers; however, overall a-SMA immunoreactivity remained reduced compared with controls, indicating persistent loss of normal smooth muscle cell content even in larger, less affected vessels. No co-localization was detected between a-SMA-positive cells and cystatin C (Fig. 30). In contrast. a-SMA-positive cells showed a close spatial association with COL IV, supporting the interpretation that a-SMA-expressing cells are associated with areas of COL IV accumulation (Fig. 36).

[0291] Additionally, confocal double labeling revealed scattered residual cells with substantial co-labeling of vimentin and a-SMA (Fig. 37).

[0292] SMA 1)2 / 3, pSmad2 / 3 and WNT-1To assess signaling pathways in residual vascular cells, sections were stained for WNT-1, SMAD2 / 3, and phosphorylated SMAD2 / 3 (pSMAD2 / 3), indicative of activation of canonical TGF-P signaling. SMAD2 / 3 immunoreactivity was detected in cells distributed across all vessel wall layers in HCCAA, including the media, adventitia, and intima (Fig. 38A, 38E). In control vessels, SMAD2 / 3 labeling was primarily confined to the media and intima, with dense, regular medial staining and orderly cellular morphology (Fig. 38B). Quantitative analysis demonstrated that SMAD2 / 3 immunoreactivity (% immunoreactive area per RO I) did not differ significantly between HCCAA patients and controls (median [IQR], 4.03% [2.20-6.64] vs 4.71% [3.10-7.19]; Mann-Whitney U test, P = 0.1193) (Fig. 38J).

[0293] pSMAD2 / 3 showed a similar spatial distribution to total SMAD2 / 3 but appeared more prominent in affected vessels, with labeling of cells across the vessel wall layers (Fig. 38C, 38F). In control tissue, pSMAD2 / 3 staining resembled that of SMAD2 / 3, being mainly restricted to the media and intima (Fig. 38D). Staining with the corresponding negative control in place of the primary antibodies showed no immunoreactivity (Fig. 38G). Quantitative analysis demonstrated that pSMAD2 / 3 immunoreactivity (% immunoreactive area per ROI) did not differ significantly between HCCAA patients and controls (median [IQR], 3.99% [2.24-6.26] vs 4.71% [3.14-7.27]; Mann-Whitney U test, P = 0.0855; Fig. 38K).

[0294] WNT-1 immunoreactivity displayed a distribution broadly similar to that of SMAD2 / 3 and pSMAD2 / 3 in HCCAA vessels, with labeling of cells across the vessel wall (Fig. 38H). In control vessels, WNT-1 staining was largely confined to medial and intimal cells (Fig. 36J). Quantitative analysis demonstrated that WNT-1 immunoreactivity (% immunoreactive area per ROI) did not differ significantly between HCCAA patients and controls (median [IQR], 4.01% [2.24-8.10] vs 4.76% [3.11-7.35]; Mann-Whitney U test, P = 0.1265; Fig. 38L).

[0295] Notably, SMAD2 / 3, pSMAD2 / 3 and Wnt-1 immunoreactivity was not reduced in HCCAA vessels despite overall reduced cellularity.

[0296] Cellular alterations, signaling-associated markers in AD with associated CAA

[0297] Loss of a-smooth muscle actin (a-SMA) positive vascular smooth muscle cells is a well-established feature of AfFCAA. Consistent with this, AD-CAA samples showed reduced and discontinuous a-SMA immunoreactivity in affected vessels (data not shown), reflecting vascular smooth muscle cell depletion. Vimentin immunostaining expanded within the vessel wall in AD-CAA. Vimentin-positive cells were observed not only in the adventitial compartment but also extending into additional vessel wall layers, indicating altered vascular cellular organization in affected vessels (Fig. 39A-39B). This pattern closely resembled the vimentin distribution observed in HCCAA.

[0298] Analysis of signaling-associated markers demonstrated the presence of SMAD2 / 3-positive cells within affected vessel walls in AD-CAA (Fig. 39C). Similarly, WNT-1 immunoreactivity was detected in vascular cells across vessel wall layers (Fig. 39D).

[0299] Phosphorylated SMAD2 / 3 (pSMAD2 / 3) staining showed a comparable distribution, indicating activation of SMAD-associated signaling pathways within remodeled vessels (Fig. 39E). The localization and overall staining patterns of SMAD2 / 3, pSMAD2 / 3, and WNT-1 in AD-CAA were similar to those observed in HCCAA.

[0300] In AD-associated CAA, Ap deposition was observed circumferentially within the media of affected arteries, surrounding VSMCs (Fig. 40). Dual immunofluorescence demonstrated that A localized around a-SMA-positive VSMCs, with no evidence of co-localization between Ap and a-SMA (Fig. 40), consistent with HCCAA (Fig. 29).

[0301] Together, these findings indicate that AD-associated CAA shares key features of vascular cellular alteration and signaling-associated marker expression with HCCAA, supporting the presence of common vascular responses across sporadic and hereditary forms of CAA.

[0302] Interstitial collagen remodeling in HCCAA and AD with associated CAA Immunohistochemical analysis of interstitial collagens revealed comparable patterns of extracellular matrix remodeling in HCCAA and AD brain tissue. In HCCAA samples, collagen la immunoreactivity was increased in affected vessels, with prominent staining in the adventitial layer and additional immunoreactivity extending into other layers of the vessel wall in affected arteries (Fig. 41A). In addition, collagen la staining was detected in parenchymal vessels, a feature not observed for collagen Illa (Fig. 4 ID).

[0303] A similar staining pattern for collagen la was observed in AD samples with associated CAA and in parenchymal vessels (Fig. 41B and 41E). In control brain tissue, collagen la immunoreactivity was largely confined to the adventitial layer of leptomeningeal vessels, with minimal or absent staining in other vessel wall layers and minimal staining in parenchymal vessels (Fig. 41C and 41F).Collagen Illa immunoreactivity also showed increased deposition in affected vessels in both HCCAA and AD-CAA. In these cases, collagen Illa staining was most prominent in the adventitia but, similar to collagen la. extended into additional vessel wall layers in affected arteries (Fig. 42A-42B). In contrast to collagen la, collagen Illa staining was not observed in parenchymal vessels (data not shown). Control tissue exhibited thin, discontinuous collagen Illa staining restricted to the adventitial connective tissue, with no extension into the vessel wall (Fig.

[0304] 42C).

[0305] Together, these findings indicate that both HCCAA and AD-associated CAA are characterized by increased deposition of interstitial collagens la and Illa within affected vessel walls, reflecting broader extracellular matrix remodeling beyond BM expansion, while collagen la additionally involves parenchymal vessels.

[0306] Medin amyloid deposition in HCCAA and Alzheimer’ s disease-associated CAA

[0307] Medin amyloid immunohistochemistry demonstrated vascular amyloid deposition in both HCCAA and AD samples with associated Ap-CAA. In HCCAA brain tissue, medin immunoreactivity was detected within affected cerebral vessels, predominantly localizing to the medial layer of arteries and arterioles (Fig. 43A). A similar pattern of medin deposition was observed in AD-CAA samples, with medin-positive material distributed within the vessel wall (Fig. 43B). In contrast, control brain tissue showed little to no medin immunoreactivity within cerebral vessels (data not shown).

[0308] Neuronal TDP-43 pathology in HCCAA and Alzheimer’s disease

[0309] Phosphorylated TDP-43 (pTDP-43) immunostaining was examined to assess pathological TDP-43 proteinopathy in HCCAA, AD, and control brain tissue. Brain tissue from HCCAA patients showed focal pTDP-43 positive neurons characterized by cytoplasmic accumulation and aggregate-like inclusions, often accompanied by reduced or absent nuclear staining (Fig. 44A). This pattern is consistent with pathological redistribution of TDP-43 from the nucleus to the cytoplasm. Notably, such pTDP-43 pathology was observed in very young HCCAA patients; the representative image shown (Fig. 44) is from a 29-year-old individual.

[0310] In AD samples, pTDP-43 pathology was more prominent, with numerous neurons exhibiting dense cytoplasmic pTDP-43-positive inclusions (Fig. 44B). Aggregates were observed within neuronal cell bodies and occasionally within neuritic processes, consistent withestablished AD-associated TDP-43 proteinopathy. The representative image is from a 78-year-old patient with advanced dementia and concomitant AD-associated CAA.

[0311] In contrast, control samples, pTDP-43 immunoreactivity was absent or minimal, with preservation of normal nuclear localization of total TDP-43 and no cytoplasmic inclusions (Fig.

[0312] 44C, arrow).

[0313] Results

[0314] Alzheimer’s disease is the most common cause of dementia accounting for approximately 70% of cases, while CAA is a leading cause of spontaneous lobar intracerebral hemorrhage in older adults. These two pathologies frequently co-occur, with CAA identified in over 80% of autopsy-confirmed AD cases (Attems & Jellinger, 2014; Greenberg et al., 2020; Jakel et al., 2022). CAA itself is common in aging populations, with moderate to severe pathology identified in approximately 23% of autopsied general cohorts and in about 6% of cognitively normal older individuals (Jakel et al., 2022). Beyond its role in hemorrhagic stroke, CAA is increasingly recognized as a contributor to cognitive impairment and dementia, independent of overt bleeding and even after accounting for coexisting AD pathology (Attems et al., 2011; Greenberg et al., 2020; Koemans et al., 2023).

[0315] Among survivors of CAA-related lobar ICH, the annual recurrence rate is approximately 7.4% (Charidimou et al., 2017). In contrast, HCCAA, a familial form of CAA, causes recurrent, life-altering hemorrhages in young adults and is associated with early-onset dementia and markedly reduced survival, with a mean age at death of approximately 30 years (Snorradottir, Hakonarson, et al.. 2025). To investigate the pathogenic mechanisms underlying this rapid disease progression, we analyzed brain autopsy samples from HCCAA, AD patients with CAA and from control individuals without diagnosed neurological disorders.

[0316] Our analysis focused on leptomeningeal vessels, which are both highly affected and readily comparable to control tissue, as well as on adjacent cortical parenchyma near the sulci. In sporadic Ap-related CAA, leptomeningeal and cortical arteries are commonly involved early; however, a capillary-predominant form (CAA type 1) has been described, in which capillaries represent the earliest and primary site of amyloid deposition (Thai et al., 2002). Given the extreme rarity of HCCAA and the archival nature of many specimens, this targeted approach was selected. To place our findings in a broader disease context, we also analyzed brain tissue frompatients with Alzheimer’s disease-associated CAA. Using this framework, we report multiple novel and shared pathological features across both hereditary and sporadic CAAs, as revealed most prominently by the novel results from the AD samples.

[0317] Skin biopsies from HCCAA patients provides a window into earlier or more slowly evolving stages of pathogenesis, whereas the brain reflects end-stage disease pathology (Benedikz et al., 1990; Snorradottir et al., 2017). Our findings indicate that the pathological processes observed in the skin are recapitulated in the brain and are driven in both tissues by BM remodeling and cystatin C aggregation. Taken together, these observations establish HCCAA as a unique human model of CAA and AD pathogenesis, characterized by early onset, severe cerebrovascular involvement and the rare ability to track early pathogenic events longitudinally through serial skin biopsies.

[0318] Amyloid Aggregation in HCCAA and other CAAs / AD

[0319] Our results consistently demonstrate that the earliest detectable accumulation of mutant cystatin C occurs within the BM (Snorradottir et al., 2017; Snorradottir et al., 2013). In both skin and brain, the BM represents the initial niche for deposition, with a close spatial association between cystatin C aggregates and COL IV -rich BM structures. Importantly, early BM remodeling and BM-associated amyloid deposition are also well established features of sporadic Ap-related CAAs, such as seen in AD, where vascular BMs have been shown to thicken and accumulate extracellular matrix proteins prior to or in parallel with amyloid deposition (Wyss-Coray et al., 2000). Extending earlier mapping of widespread cystatin C deposition across multiple brain regions and the broad distribution of hemorrhages in HCCAA (Osk Snorradottir et al., 2015), the current findings fulfill established Vonsattel / Thal criteria for severe CAA, including extensive amyloid deposition, focal vessel-wall fragmentation, microaneurysm formation, hemorrhagic change, and fibrinoid necrosis (Thai et al., 2002; Thai et al.. 2008;

[0320] Vonsattel et al., 1991).

[0321] Our data further refine the vascular sequence of involvement. In both HCCAA and A -associated CAA (AD), we observed early amyloid deposition in close association with vascular BMs. Morphologically, early cystatin C and Ap deposition localizes circumferentially around Vascular Smooth Muscle Cells (VSMCs) within the associated BM / internal elastic lamina, forming a pericellular pattern. With disease progression, deposits extend outward toward the adventitia and inward toward the endothelium, accompanied by loss of intimal cellularity. Thisstepwise pattern, supported by Congo red, amyloid deposits, and COL IV positive BMs surrounding a-SMA positive VSMCs immuno staining with confocal confirmation, underscores progressive vascular remodeling and identifies the BM as a plausible early therapeutic target for interventions with drugs that reduce oxidative stress such, as NAC and NACA.

[0322] Medin amyloid deposition in HCCAA and Alzheimer’s disease-associated CAA

[0323] Medin amyloid, a peptide derived from the C-terminal region of milk fat globule-EGF factor, 8 (MFGE8, also known as lactadherin), accumulates in the vasculature of the majority of individuals over 50 years of age, making it the most prevalent form of amyloid currently identified [32, 65]. We show that medin represents a common vascular amyloid species across rare hereditary CAA in very young individuals (even under 30 years) and in sporadic AD-associated CAA, indicating that it is involved in diverse forms of cerebral amyloidosis. Taken together, these results indicate that therapies that reduce oxidative stress (e.g., NAC or NACA) would attenuate fibrotic vascular responses and extracellular matrix accumulation, thereby limiting the extracellular milieu that favors vascular amyloid retention.

[0324] Col IV and 'Nl remodeling

[0325] Concordant with amyloid accumulation, we observed marked extracellular matrix (ECM) remodeling, characterized by expansion of COL IV and FN 1 beyond their normal confinement to the BM and into all layers of the vessel wall in HCCAA and AD-associated CAA. Importantly, increased COL IV was evident even in vessels lacking overt cystatin C deposits in HCCAA, i.e. in parenchymal capillaries and in young individuals, indicating that BM remodeling is an early and independent pathogenic even. Our findings demonstrate increased COL IV and FN 1 immunoreactivity in AD-associated CAA. COL IV accumulation is known to increase arterial stiffness, impair microvascular perfusion, and heighten susceptibility to microbleeds, hemorrhage, and dementia (Rabkin, 2023). In addition to vascular compartments, COL IV and FN1 were detected within parenchymal plaque-like deposits. Notably, FN1 plaque- associated staining was robust when assessed with the CST E5H6X antibody but appeared weaker using the IST-9 antibody in HCCAA parenchymal plaques, despite comparable FN1 localization in leptomeningeal vessels. Overall, the presence of BM-associated extracellular matrix proteins within plaques supports a close relationship between extracellular matrix remodeling and amyloid pathology, processes that may be mutually reinforcing in CAA.ECM constituents, including heparan sulfate proteoglycans, COL IV, and FN1, can nucleate and stabilize amyloid fibrils within parenchymal plaques, capillary and arteriolar BMs, perivascular spaces, and vessel walls (Howe et al., 2020; van Horssen et al., 2003). In parallel, BM thickening and ECM expansion impair perivascular protein clearance, while profibrotic signaling pathways such as TGF-P and WNT drive vascular cells toward an ECM-producing phenotype, together accelerating plaque growth, CAA progression, and cognitive decline even in the absence of overt hemorrhage (Greenberg et al., 2020; Tarasoff-Conway et al., 2015; Weller et al., 2008).

[0326] Perivascular clearance and protein elimination failure

[0327] We observed no co-localization between cystatin C and a-SMA in HCCAA or Ap and a-SMA, indicating that impaired perivascular (intramural) clearance along BM pathways rather than intracellular accumulation within VSMCs. In healthy individuals, cystatin C is abundant in cerebrospinal fluid (CSF), whereas carriers of the CST3-L68Q mutation exhibit markedly reduced CSF levels (Lofberg & Grubb, 1979; Lofberg et al., 1987). Similarly, in Alzheimer’s disease and Ap-associated cerebral amyloid angiopathy, CSF levels of amyloid-p, particularly AP42, are reduced, reflecting sequestration and deposition of Ap within brain parenchyma and cerebral vessel walls (Blennow et al., 2010). Therefore, our data indicates that BM remodeling promotes retention of cystatin C within vascular BMs, where it can act as a scaffold for aggregation. Such sequestration provides a plausible explanation for reduced CSF cystatin C and the relative scarcity of cystatin C dimers in CSF compared with plasma in HCCAA (Bjamadottir et al., 2001).

[0328] VSMC loss, phenotypic switching, and impaired drainage

[0329] In HCCAA, we observed pronounced loss of contractile VSMCs, with cystatin C failing to co-localize with a-SMA. COL IV accumulation in HCCAA and AD-CAA vessels co-localized with cells exhibiting a myofibroblast-like phenotype (a-SMA+ / vimentin+) that were also positive for WNT-1, SMAD2 / 3 and pSMAD2 / 3. Additionally, the magnitude of VSMC depletion highlights the severity of vessel degeneration and our data indicates that it is exacerbated by BM remodeling and local cystatin C aggregation. Under cellular stress, including oxidative stress. VSMCs can undergo phenotypic switching toward fibroblast-like states driven by TGF-P, WNT, and ROS signaling (Badran et al., 2020; Sorokin et al., 2020). As old age is not a factor inHCCAA patients whereas BM remodeling is linked to age in sporadic CAA and AD (Howe et al., 2020), our data indicates that activated TGF-p / WNT signaling is involved in the early part of the pathogenesis.

[0330] Interstitial collagen remodeling in CAA

[0331] In addition to BM expansion marked by COL IV and FN1, we observed increased deposition of interstitial collagens la and Illa within affected vessel walls in both HCCAA and Alzheimer’s disease-associated CAA. In control tissue, collagen la and Illa were largely confined to the adventitial compartment, whereas in affected vessels both collagens extended beyond the adventitia into additional layers of the vessel wall, consistent with fibrotic vascular remodeling. Notably, collagen la, but not collagen Illa, was also detected in parenchymal vessels. Collagen la often reflects more mature and rigid fibrosis (Ricard-Blum, 2011).

[0332] Our data indicates that CAA is associated not only with BM thickening but also with broader extracellular matrix reorganization involving interstitial collagens, which contributes to increased vessel stiffness and reduced compliance.

[0333] The increased deposition of interstitial collagens la and Illa occurred alongside the presence of pSMAD2 / 3 and wnt-1 positive cells within affected vessel walls, indicating that activation of TGF-P-associated signaling in regions undergoing extracellular matrix remodeling. This spatial association is consistent with the established role of TGF-p / SMAD signaling in promoting fibrotic extracellular matrix responses in vascular disease. Importantly, the similar patterns observed in HCCAA and AD-associated CAA indicate that interstitial collagen remodeling represents a shared vascular response across distinct forms of CAA.

[0334] TGF-p and WNT-1

[0335] Collectively, the expansion of COL IV and FN1 across vessel-wall layers reflects a shift toward a profibrotic ECM milieu, which can stiffen vessels and hinder perivascular clearance. At the cellular level, vascular cells exhibit phenotypic transitions toward activated fibroblast or myofibroblast states marked by WNT-1, SMAD2 / 3 and pSMAD2 / 3 positivity. While these observations are correlative, they are consistent with dysregulated WNT / TGF-P signaling as a key contributor to matrix overproduction and vascular remodeling. Experimental studies demonstrate that WNT-1 promotes fibroblast proliferation, survival, and ECM production, inducing collagens, FN1, periostin, and a-SMA (Konigshoff et al., 2009; Song et al., 2014).WNT-1 synergizes with TGF-P, enhancing SMAD signaling, while TGF-P reciprocally upregulates WNT ligands, creating a self-sustaining profibrotic feedback loop (Akhmetshina et al., 2012).

[0336] Cystatin C itself modulates TGF-P signaling by binding TpRII and dampening downstream SMAD activation, thereby inhibiting fibroblast differentiation and EMT / EndMT processes (Sokol et al., 2005; Sokol & Schiemann, 2004). In CST3-L68Q carriers, mutant cystatin C undergoes aberrant intracellular processing, reducing secretion and likely lowering extracellular levels (Bjarnadottir et al., 1998; Olafsson & Grubb, 2000; Thorsteinsson et al.. 1992).

[0337] TDP-43 pathology in HCCAA and Alzheimer’s

[0338] Pathological pTDP-43 immunoreactivity was defined by cytoplasmic aggregation with concomitant nuclear clearance, distinguishing it from the normal nuclear localization of pTDP-43 observed in control tissue. This pattern reflects a loss of physiological nuclear TDP-43 function and the emergence of a pathological proteinopathy, which is increasingly recognized as a frequent comorbidity in Alzheimer’s disease and related dementias (Jo et al., 2020; Meneses et al., 2021). The presence of pTDP-43 pathology in HCCAA, including in very young patients, suggests that neuronal stress and protein homeostasis disturbances may arise early in the disease course, potentially secondary to chronic vascular dysfunction, impaired perivascular clearance, or extracellular matrix remodeling. Our findings indicate that vascular pathology and extracellular matrix remodeling contribute to a permissive environment for secondary neuronal proteinopathies. Importantly, TDP-43 contains redox-sensitive cysteine residues and can form aberrant disulfide-linked oligomers under oxidative conditions, a process implicated in pathological aggregation (Cohen et al., 2012). Redox-modulating agents such as NAC or NACA, which enhance intracellular glutathione and reduce oxidative stress, could therefore interfere with disulfide-dependent protein aggregation and prevent / treat these devastating conditions.

[0339] Therapeutic implications

[0340] Our data indicates that therapeutic strategies aimed at limiting profibrotic signaling, reducing oxidative stress, and improving ECM turnover would be effective for treating a CAA and / or AD. Oxidative stress amplifies TGF-P-mediated fibrogenesis, in part through glutathione depletion, whereas glutathione restoration attenuates TGF-P signaling and matrix accumulation(Liu & Gaston Pravia, 2010). WNT / 0-catenin signaling further cooperates with TGF-0 to sustain fibroblast activation (Liu et al., 2022).

[0341] Our data indicates that N-acetylcysteine (NAC) and its analogue NACA exert dual effects by reducing oxidative stress and disrupting disulfide-linked cystatin C oligomers (March et al., 2021).

[0342] Taken together, our findings teach a model in which early vascular cell phenotypic transitions toward fibroblast- and myofibroblast-like states, accompanied by activation of WNT and TGF-P signaling, are associated with BM remodeling in HCCAA. This remodeled extracellular matrix, in turn, creates a permissive environment for cystatin C aggregation and subsequent amyloid deposition within the vascular basement membrane. These converging processes highlight rational therapeutic targets, including attenuation of profibrotic signaling, normalization of extracellular matrix turnover, preservation of vascular compliance, and reduction of cystatin C aggregation. Notably, HCCAA uniquely enables paired brain and skin analyses, allowing serial skin biopsies to serve as accessible mechanistic readouts in early-phase clinical trials. HCCAA thus provides a powerful human model for dissecting fundamental pathogenic mechanisms relevant to other forms of cerebral amyloid angiopathy, including Alzheimer’s disease, together with therapeutic interventions using NAC or NACA.

[0343] References

[0344] Akhmetshina, A., Palumbo, K., Dees, C., Bergmann, C., Venalis, P., Zerr, P., Horn, A., Kireva, T., Beyer, C., Zwerina, J., Schneider, H., Sadowski, A., Riener, M. O., MacDougald, O. A., Distler, O., Schett. G.. & Distler, J. H. (2012). Activation of canonical Wnt signalling is required for TGF-beta-mediated fibrosis. Nat Commun, 3, 735. https: / / doi.org / 10.1038 / ncommsl734

[0345] Aldea, R., Weller, R. O., Wilcock, D. M„ Carare. R. O., & Richardson, G. (2019).

[0346] Cerebrovascular Smooth Muscle Cells as the Drivers of Intramural Periarterial Drainage of the Brain. Front Aging Neurosci, 11, 1. https: / / doi.org / 10.3389 / fnagi.2019.00001

[0347] Attems, J., Jellinger, K., Thai, D. R., & Van Nostrand, W. (2011). Review: sporadic cerebral amyloid angiopathy. Neuropathol Appl Neurobiol, 37(1), 75-93. https: / / doi.org / 10.1111 / j.1365-2990.2010.01137.xAttems, J., & Jellinger, K. A. (2014). The overlap between vascular disease and Alzheimer's disease-lessons from pathology. BMC Med, 12, 206. https: / / doi.org / 10.1186 / sl2916-014-0206-2

[0348] Badran, A., Nasser, S. A., Mesmar, J., El-Yazbi, A. F., Bitto, A., Fardoun, M. M., Baydoun, E., & Eid, A. H. (2020). Reactive Oxygen Species: Modulators of Phenotypic Switch of Vascular Smooth Muscle Cells. Int J Mol Sci, 21(22). https: / / doi.org / 10.3390 / ijms21228764 Bavarsad Shahripour, R., Harrigan, M. R., & Alexandrov, A. V. (2014). N-acetylcysteine (NAC) in neurological disorders: mechanisms of action and therapeutic opportunities. Brain Behav, 4(2), 108-122. https: / / doi.org / 10.1002 / brb3.208

[0349] Benedikz, E., Blondal, H., & Gudmundsson, G. (1990). Skin deposits in hereditary cystatin C amyloidosis. Virchows Arch A Pathol Anat Histopathol, 417(4), 325-331.

[0350] https: / / doi.org / l 0.1007 / BF01605784

[0351] Biffi, A. (2022). Main features of hereditary cerebral amyloid angiopathies: A systematic review. Cereb Circ Cogn Behav, 3. 100124. https: / / doi. Org / 10.1016 / j.cccb.2022.100124 Bjamadottir, M., Nilsson, C., Lindstrom, V., Westman, A., Davidsson, P., Thormodsson, F., Blondal, H., Gudmundsson, G., & Grubb, A. (2001). The cerebral hemorrhage-producing cystatin C variant (L68Q) in extracellular fluids. Amyloid-Journal of Protein Folding Disorders, 8(1), 1-10. https: / / doi.org / Doi 10.3109 / 13506120108993809

[0352] Bjamadottir, M„ Wulff. B. S., Sameni, M., Sloane, B. F., Keppler, D., Grubb, A., & Abrahamson, M. (1998). Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH / 3T3 cells. Mol Pathol, 51(6), 317-326. https: / / doi.org / 10.1136 / mp.5L6.317

[0353] Blennow, K., Hampel, H., Weiner, M., & Zetterberg, H. (2010). Cerebrospinal fluid and plasma biomarkers in Alzheimer disease. Nat Rev Neurol, 6(3), 131-144. https: / / doi. Org / 10.1038 / nrneurol.2010.4

[0354] Blondal, H., Gudmundsson, G., Benedikz, E., & Johannesson, G. (1989). Dementia in hereditary cystatin C amyloidosis. Prog Clin Biol Res, 317, 157-164. https: / / www.ncbi.nlm.nih.gov / pubmed / 2602413

[0355] Carare, R. O., Hawkes, C. A., Jeffrey, M., Kalaria, R. N., & Weller, R. O. (2013).

[0356] Review: cerebral amyloid angiopathy, prion angiopathy, CADASIL and the spectrum of proteinelimination failure angiopathies (PEFA) in neurodegenerative disease with a focus on therapy. Neuropathol Appl Neurobiol, 39(6), 593-611. https: / / doi.org / 10.llll / nan.12042

[0357] Carare, R. O., Teeling, J. L., Hawkes, C. A., Puntener, U., Weller, R. O., Nicoll. J. A., & Perry, V. H. (2013). Immune complex formation impairs the elimination of solutes from the brain: implications for immunotherapy in Alzheimer's disease. Acta Neuropathol Commun, 1, 48. https: / / doi.org / 10.1186 / 2051 -5960- 1 -48

[0358] Charidimou, A., Imaizumi, T., Moulin, S., Biffi, A., Samarasekera, N., Yakushiji, Y., Peeters, A., Vandermeeren, Y., Laloux, P., Baron, J. C., Hernandez-Guillamon, M., Montaner, J., Casolla, B., Gregoire, S. M., Kang, D. W„ Kim, J. S., Naka, H., Smith, E. E., Viswanathan, A.,... Werring, D. J. (2017). Brain hemorrhage recurrence, small vessel disease type, and cerebral microbleeds: A meta-analysis. Neurology, 89(8), 820-829. https: / / doi.org / 10.1212AVNL.0000000000004259

[0359] Cohen, T. J., Hwang, A. W., Unger, T., Trojanowski, J. Q., & Lee, V. M. (2012). Redox signalling directly regulates TDP-43 via cysteine oxidation and disulphide cross-linking. EMBO J, 31(5), 1241-1252. https: / / doi.org / 10.1038 / emboj.2011.471

[0360] Goumans, M. J., & Ten Dijke, P. (2018). TGF-beta Signaling in Control of Cardiovascular Function. Cold Spring Harb Perspect Biol, 10(2). https: / / doi.org / 10.1101 / cshperspect.a022210

[0361] Grand Moursel, L., Munting, L. P„ van der Graaf, L. M„ van Duinen, S. G., Goumans, M. T. H., Ueberham, U., Natte, R., van Buchem, M. A., van Roon-Mom, W. M. C., & van der Weerd, L. (2018). TGFbeta pathway deregulation and abnormal phospho-SMAD2 / 3 staining in hereditary cerebral hemorrhage with amyloidosis-Dutch type. Brain Pathol, 28(4), 495-506. https: / / doi.org / 10. Ill 1 / bpa.12533

[0362] Grand Moursel, L., van der Graaf, L. M., Bulk, M., van Roon-Mom, W. M. C., & van der Weerd, L. (2019). Osteopontin and phospho-SMAD2 / 3 are associated with calcification of vessels in D-CAA, an hereditary cerebral amyloid angiopathy. Brain Pathol, 29(6), 793-802. https: / / doi.org / 10. Ill 1 / bpa.12721

[0363] Greenberg, S. M., Bacskai, B. J., Hernandez-Guillamon, M., Pruzin, J., Sperling, R., & van Veluw, S. J. (2020). Cerebral amyloid angiopathy and Alzheimer disease - one peptide, two pathways. Nat Rev Neurol, 16(1), 30-42. https: / / doi.org / 10.1038 / s41582-019-0281-2Greenberg, S. M., & van Veluw, S. J. (2024). Cerebral Amyloid Angiopathy. Stroke, 55(5), 1409-1411. https: / / doi.org / 10.1161 / STROKEAHA.124.044293

[0364] Grubb, A., Jensson, O., Gudmundsson, G., Arnason, A., Lofberg, H., & Malm, J. (1984). Abnormal-Metabolism of Gamma-Trace Alkaline Microprotein - the Basic Defect in Hereditary Cerebral-Hemorrhage with Amyloidosis. New England Journal of Medicine, 311(24), 1547-1549. https: / / doi.org / Doi 10.1056 / Nejml98412133112406

[0365] Hawkes, C. A., Jayakody, N., Johnston, D. A., Bechmann, I., & Carare, R. O. (2014). Failure of perivascular drainage of beta-amyloid in cerebral amyloid angiopathy. Brain Pathol, 24(4), 396-403. https: / / doi.org / 10.1111 / bpa.12159

[0366] Held, F., Morris, A. W. J., Pirici, D., Niklass, S., Sharp, M. M. G., Garz, C., Assmann, A., Heinze. H. J., Schreiber, F„ Carare, R. O., & Schreiber, S. (2017). Vascular basement membrane alterations and beta-amyloid accumulations in an animal model of cerebral small vessel disease. Clin Sci (Lond), 131(10), 1001-1013. https: / / doi.org / 10.1042 / CS20170004 Howe, M. D., McCullough, L. D., & Urayama, A. (2020). The Role of Basement Membranes in Cerebral Amyloid Angiopathy. Front Physiol, 11, 601320. https: / / doi.org / 10.3389 / fphys.2020.601320

[0367] Jakel, L„ De Kort, A. M„ Klijn, C. J. M„ Schreuder, F„ & Verbeek, M. M. (2022).

[0368] Prevalence of cerebral amyloid angiopathy: A systematic review and meta-analysis. Alzheimers & Dementia, 18(1), 10-28. https: / / doi.org / 10.1002 / alz.12366

[0369] Jo, M., Lee, S„ Jeon, Y. M„ Kim, S„ Kwon, Y„ & Kim, H. J. (2020). The role of TDP-43 propagation in neurodegenerative diseases: integrating insights from clinical and experimental studies. Exp Mol Med, 52(10), 1652-1662. https: / / doi.org / 10.1038 / sl2276-020-00513-7 Koemans, E. A., Chhatwal, J. P., van Veluw, S. J., van Etten, E. S., van Osch, M. J. P., van Walderveen, M. A. A., Sohrabi, H. R., Kozberg, M. G., Shirzadi, Z., Terwindt, G. M., van Buchem, M. A., Smith, E. E., Werring, D. J., Martins, R. N., Wermer, M. J. H., & Greenberg, S. M. (2023). Progression of cerebral amyloid angiopathy: a pathophysiological framework. Lancet Neurol, 22(7), 632-642. https: / / doi.org / 10.1016 / S1474-4422(23)00114-X

[0370] Konigshoff, M., Kramer, M., Balsara, N., Wilhelm, J., Amarie, O. V., Jahn, A., Rose, F., Fink, L., Seeger, W., Schaefer, L., Gunther, A., & Eickelberg, O. (2009). WNTl-inducible signaling protein- 1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis. J Clin Invest, 119(4), 772-787. https: / / doi.org / 10.1172 / JCI33950Kozberg, M. G., Munting, L. P., Hanlin, L. H., Auger, C. A., van den Berg, M. L., Denis de Senneville, B., Hirschler, L., Wamking, J. M., Barbier, E. L., Farrar, C. T., Greenberg, S. M., Bacskai, B. J., & van Veluw, S. J. (2025). Vasomotion loss precedes impaired cerebrovascular reactivity and microbleeds in cerebral amyloid angiopathy. Brain Commun, 7(3), fcaf186. https: / / doi.org / 10.1093 / braincomms / fcaf186

[0371] Liu, J„ Xiao. Q.. Xiao, L. Niu, C„ Li, Y„ Zhang. X.. Zhou, Z„ Shu. G.. & Yin, G. (2022). Wnt / beta-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct Target Ther, 7(1), 3. https: / / doi.org / 10.1038 / s41392-021-00762-6

[0372] Liu, R. M„ & Gaston Pravia, K. A. (2010). Oxidative stress and glutathione in TGF-beta-mediated fibrogenesis. Free Radic Biol Med, 48(1), 1-15. https: / / doi. Org / 10.1016 / j.freeradbiomed.2009.09.026

[0373] Lofberg, H., & Grubb, A. O. (1979). Quantitation of gamma-trace in human biological fluids: indications for production in the central nervous system. Scand J Clin Lab Invest, 39(7), 619-626. https: / / doi.org / 10.3109 / 00365517909108866

[0374] Lofberg, H., Grubb, A. O., Nilsson, E. K., Jensson, O., Gudmundsson, G., Blondal, H., Amason, A., & Thorsteinsson, L. (1987). Immunohistochemical characterization of the amyloid deposits and quantitation of pertinent cerebrospinal fluid proteins in hereditary cerebral hemorrhage with amyloidosis. Stroke, 18(2), 431-440. https: / / doi. Org / 10.1161 / 01.str.18.2.431 March, M. E., Gutierrez-Uzquiza, A., Snorradottir, A. O., Matsuoka, L. S., Balvis, N. F., Gestsson, T., Nguyen, K., Sleiman, P. M. A., Kao, C., Isaksson, H. J., Bragason, B. T., Olafsson, E., Palsdottir, A., & Hakonarson, H. (2021). NAC blocks Cystatin C amyloid complex aggregation in a cell system and in skin of HCCAA patients. Nat Commun, 12(1), 1827. https: / / doi.org / l 0.1038 / s41467-021 -22120-4

[0375] Massague, J. (2012). TGFbeta signalling in context. Nat Rev Mol Cell Biol, 13(10), 616-630. https: / / doi.org / 10.1038 / nrm3434

[0376] Meneses, A., Koga, S., O'Leary, J., Dickson, D. W., Bu, G., & Zhao, N. (2021). TDP-43 Pathology in Alzheimer's Disease. Mol Neurodegener, 16(1), 84. https: / / doi.org / 10.1186 / sl3024-021-00503-x

[0377] Nusse, R., & Clevers, H. (2017). Wnt / beta-Catenin Signaling, Disease, and Emerging Therapeutic Modalities. Cell, 169(6), 985-999. https: / / doi. Org / 10.1016 / j.cell.2017.05.016Olafsson, T., & Grubb, A. (2000). Hereditary cystatin C amyloid angiopathy. Amyloid-Journal of Protein Folding Disorders, 7(1), 70-79. https: / / doi.org / 10.3109 / 13506120009146827 Olichney, J. M., Hansen, L. A., Hofstetter, C. R., Lee, J. H., Katzman, R., & Thai. L. J. (2000). Association between severe cerebral amyloid angiopathy and cerebrovascular lesions in Alzheimer disease is not a spurious one attributable to apolipoprotein E4. Arch Neurol, 57(6), 869-874. https: / / doi. Org / 10.1001 / archneur.57.6.869

[0378] Ontawong, A., Nehra, G., Maloney, B. J., Vaddhanaphuti, C. S., Bauer, B., & Hartz, A. M. S. (2025). N-Acetylcysteine Attenuates Abeta-Mediated Oxidative Stress, Blood-Brain Barrier Leakage, and Renal Dysfunction in 5xFAD Mice. Int J Mol Sci, 26(9). https: / / doi.org / 10.3390 / ijms26094352

[0379] Osk Snorradottir, A., Isaksson, H. J., Kaeser, S. A., Skodras, A. A., Olafsson, E„ Palsdottir, A., & Thor Bragason, B. (2015). Parenchymal cystatin C focal deposits and glial scar formation around brain arteries in Hereditary Cystatin C Amyloid Angiopathy. Brain Research, 1622, 149-162. https: / / doi. Org / 10.1016 / j.brainres.2015.06.019

[0380] Palsdottir, A., Abrahamson, M., Thorsteinsson, L., Arnason, A., Olafsson, I., Grubb, A., & Jensson, O. (1988). Mutation in cystatin C gene causes hereditary brain haemorrhage. Lancet, 2(8611), 603-604. https: / / doi.org / ! 0.1016 / s0140-6736(88)90641- 1

[0381] Palsdottir, A., Helgason, A., Palsson, S., Bjomsson, H. T., Bragason, B. T., Gretarsdottir, S., Thorsteinsdottir, U., Olafsson, E„ & Stefansson, K. (2008). A drastic reduction in the life span of cystatin C L68Q carriers due to life-style changes during the last two centuries. PLoS Genet, 4(6), el000099. https: / / doi.org / 10.1371 / joumal.pgen.1000099

[0382] Palsdottir, A., Snorradottir, A. O., & Hakonarson, H. (2022). [Did ketogenic diet in past centuries protect against the consequence of the cystatin L68Q mutation in carriers of HCCAA?]. Laeknabladid, 108(12), 553-557. https: / / doi.org / 10.17992 / lbl.2022.12.721

[0383] Palsdottir, A., Snorradottir, A. O., & Thorsteinsson, L. (2006). Hereditary cystatin C amyloid angiopathy: genetic, clinical, and pathological aspects. Brain Pathol, 16(1), 55-59. https: / / doi. Org / 10.llll / j.1750-3639.2006.tb00561.x

[0384] Rabkin, S. W. (2023). Collagen type IV as the link between arterial stiffness and dementia. Am J Transl Res, 15(10), 5961-5971.

[0385] https: / / www.ncbi.nlm.nih.gov / pubmed / 37969177Ricard-Blum, S. (2011). The collagen family. Cold Spring Harb Perspect Biol, 3(1), a004978. https: / / doi. Org / 10.l 101 / cshperspect.a004978

[0386] Schindelin, J., Arganda-Carreras, I., Frise. E„ Kaynig, V., Longair, M., Pietzsch, T„ Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K„ Tomancak, P., & Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods. 9(7). 676-682. https: / / doi.org / 10.1038 / nmeth.2019 Snorradottir, A. O., Gutierrez-Uzquiza, A., Bragado, P., March, M. E., Kao, C., Arkink, E. B., Jonsdottir, S., Sigurdardottir, A., Isaksson, H. J., Mariasdottir, H. L., Bjorgvinsdottir, O. Y., Kowal, N. M., Heimisdottir, H. L., Sverrisdottir, A., Palsdottir, A., Bjornsson, H. T., & Hakonarson, H. (2025). N-Acetylcysteine for Hereditary Cystatin C Amyloid Angiopathy: A Nonrandomized Clinical Trial. JAMA Neurol, 82(5), 486-494. https: / / doi.org / 10.1001 / jamaneurol.2025.0326

[0387] Snorradottir, A. O., Hakonarson, H., & Palsdottir, A. (2024). The historical background of hereditary cystatin C amyloid angiopathy: Genealogical, pathological, and clinical manifestations. Brain Pathol, el3291. https: / / doi.org / 10.1111 / bpa.13291

[0388] Snorradottir, A. O., Hakonarson, H., & Palsdottir, A. (2025). The historical background of hereditary cystatin C amyloid angiopathy: Genealogical, pathological, and clinical manifestations. Brain Pathol, 35(2), e13291. https: / / doi.org / 10.1111 / bpa.13291 Snorradottir, A. O., Isaksson, H. J., Ingthorsson, S., Olafsson, E., Palsdottir, A., & Bragason, B. T. (2017). Pathological changes in basement membranes and dermal connective tissue of skin from patients with hereditary cystatin C amyloid angiopathy. Lab Invest, 97(4), 383-394. https: / / doi.org / 10.1038 / labinvest.2016.133

[0389] Snorradottir, A. O., Isaksson, H. J., Kaeser, S. A., Skodras, A. A., Olafsson, E., Palsdottir, A., & Bragason, B. T. (2013). Deposition of collagen IV and aggrecan in leptomeningeal arteries of hereditary brain haemorrhage with amyloidosis. Brain Research, 1535, 106-114. https: / / doi. Org / 10.1016 / j.brainres.2013.08.029

[0390] Sokol, J. P., Neil, J. R., Schiemann, B. J., & Schiemann, W. P. (2005). The use of cystatin C to inhibit epithelial-mesenchymal transition and morphological transformation stimulated by transforming growth factor-beta. Breast Cancer Res, 7(5), R844-853.

[0391] https: / / doi.org / 10.1186 / bcrl312Sokol, J. P., & Schiemann, W. P. (2004). Cystatin C antagonizes transforming growth factor beta signaling in normal and cancer cells. Mol Cancer Res, 2(3), 183-195. https: / / www.ncbi.nlm.nih.gov / pubmed / 15037657

[0392] Song, P„ Zheng, J. X., Liu, J. Z„ Xu, L, Wu, L. Y„ Liu, C„ Zhu, Q„ & Wang, Y. (2014). Effect of the Wntl / beta-catenin signalling pathway on human embryonic pulmonary fibroblasts. Mol Med Rep. 10(2), 1030-1036. https: / / doi.org / 10.3892 / mmr.2014.2261

[0393] Sorokin, V., Vickneson, K., Kofidis, T., Woo, C. C., Lin, X. Y., Foo, R., & Shanahan, C. M. (2020). Role of Vascular Smooth Muscle Cell Plasticity and Interactions in Vessel Wall Inflammation. Front Immunol, 11, 599415. https: / / doi.org / 10.3389 / fimmu.2020.599415 Tarasoff-Conway, J. M., Carare, R. O., Osorio, R. S., Glodzik, L., Butler, T., Fieremans, E., Axel, L., Rusinek, H., Nicholson, C.. Zlokovic, B. V., Frangione, B., Blennow. K.. Menard. J., Zetterberg, H., Wisniewski, T., & de Leon, M. J. (2015). Clearance systems in the brainimplications for Alzheimer disease. Nat Rev Neurol, 11(8), 457-470.

[0394] https: / / doi.org / 10.1038 / nrneurol.2015.119

[0395] Thai, D. R., Ghebremedhin, E., Rub, U., Yamaguchi, H., Del Tredici, K., & Braak, H. (2002). Two types of sporadic cerebral amyloid angiopathy. J Neuropathol Exp Neurol, 61(3), 282-293. https: / / doi.org / 10.1093 / jnen / 6L3.282

[0396] Thai, D. R., Griffin, W. S., de Vos, R. A., & Ghebremedhin, E. (2008). Cerebral amyloid angiopathy and its relationship to Alzheimer's disease. Acta Neuropathol. 115(6), 599-609. https: / / doi.org / 10.1007 / s00401-008-0366-2

[0397] Thomsen, M. S., Routhe, L. J., & Moos, T. (2017). The vascular basement membrane in the healthy and pathological brain. J Cereb Blood Flow Metab, 37(10), 3300-3317. https: / / doi.org / 10.1177 / 0271678X17722436

[0398] Thorsteinsson, L., Georgsson, G., Asgeirsson. B., Bjarnadottir, M., Olafsson, I., Jensson, O., & Gudmundsson, G. (1992). On the role of monocytes / macrophages in the pathogenesis of central nervous system lesions in hereditary cystatin C amyloid angiopathy. J Neurol Sci, 108(2), 121-128. https: / / doi.org / 10.1016 / 0022-510x(92)90042-j

[0399] Turner, N., Toklu, H. Z., Muller-Delp, J. M., Oktay, S., Ghosh, P., Strang, K., Delp, M. D., & Scarpace, P. J. (2014). The effects of aging on the functional and structural properties of the rat basilar artery. Physiol Rep, 2(6). https: / / doi.org / 10.14814 / phy2.12031van Horssen, J., Wesseling, P., van den Heuvel, L. P., de Waal, R. M., & Verbeek, M. M. (2003). Heparan sulphate proteoglycans in Alzheimer's disease and amyloid-related disorders. Lancet Neurol. 2(8). 482-492. https: / / doi.org / 10.1016 / sl474-4422(03)00484-8

[0400] Ventura- Antunes, L., Nackenoff, A., Romero-Fernandez, W., Wang, Y., Bosworth, A. M., Prusky, A., Wang, E., Carvajal-Tapia, C., Shostak, A., Harmsen, H., Mobley, B., Maldonado, J.. Womble, N., Solopova, E„ Snider, J. C., Merryman, W. D„ Lippmann, E. S., & Schrag, M. (2025). Arteriolar degeneration and stiffness in cerebral amyloid angiopathy are linked to Abeta deposition and lysyl oxidase. Alzheimers & Dementia, 21(6), e70254. https: / / doi.org / 10.1002 / alz.70254

[0401] Vonsattel, J. P., Myers, R. H., Hedley- Whyte, E. T., Ropper, A. H., Bird, E. D., & Richardson, E. P., Jr. (1991). Cerebral amyloid angiopathy without and with cerebral hemorrhages: a comparative histological study. Ann Neurol, 30(5), 637-649. https: / / doi.org / 10.1002 / ana.410300503

[0402] Wei, Z„ Caty, J., Whitson, J., Zhang, A. D„ Srinivasagan, R„ Kavanagh, T. J., Yan, H„ & Fan, X. (2017). Reduced Glutathione Level Promotes Epithelial-Mesenchymal Transition in Lens Epithelial Cells via a Wnt / beta-Catenin-Mediated Pathway: Relevance for Cataract Therapy. Am J Pathol, 187(11), 2399-2412. https: / / doi. Org / 10.1016 / j.ajpath.2017.07.018 Weller, R. O., Subash, M., Preston, S. D., Mazanti, L, & Carare, R. O. (2008).

[0403] Perivascular drainage of amyloid-beta peptides from the brain and its failure in cerebral amyloid angiopathy and Alzheimer's disease. Brain Pathol, 18(2), 253-266.

[0404] https: / / doi.org / 10.1111 / j.1750-3639.2008.00133. X

[0405] Whitehead, M„ Yusoff, S., Ahmad, S., Schmidt, L„ Mayr, M., Madine, J., Middleton, D„ & Shanahan, C. M. (2023). Vascular smooth muscle cell senescence accelerates medin aggregation via small extracellular vesicle secretion and extracellular matrix reorganization. Aging Cell, 22(2), el3746. https: / / doi.org / 10.1111 / acel.13746

[0406] Wyss-Coray, T., Lin, C., Sanan, D. A., Mucke, L., & Masliah, E. (2000). Chronic overproduction of transforming growth factor-betal by astrocytes promotes Alzheimer's diseaselike microvascular degeneration in transgenic mice. Am J Pathol, 156(1), 139-150. https: / / doi.org / 10.1016 / s0002-9440( 10)64713-x

[0407] Yurchenco, P. D. (2011). Basement membranes: cell scaffoldings and signaling platforms. Cold Spring Harb Perspect Biol, 3(2). https: / / doi.org / 10.1101 / cshperspect.a004911Embodiments:

[0408] Embodiment 1. A method for treating a cerebral amyloid angiopathy (CAA) in a subject in need thereof, comprising administering to said subject, an effective amount of a composition comprising an agent that disrupts both hydrogen and disulfide bonds between amyloid dimers and / or oligomers, thereby disrupting amyloid deposition in the brain and / or the walls of blood vessels.

[0409] Embodiment 2. The method of embodiment 1, wherein the blood vessels are cerebral blood vessels.

[0410] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the treatment ameliorates symptoms of the CAA in said patient.

[0411] Embodiment 4. The method of any one of embodiments 1-3, wherein the CAA is Alzheimer's Disease or a hereditary cerebral amyloid angiopathy.

[0412] Embodiment 5. The method of embodiment 4, wherein the hereditary cerebral amyloid angiopathy is selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF).

[0413] Embodiment 6. The method of anyone of embodiment 1-5, wherein the agent is N-acetyl cysteine or a function derivative thereof in a pharmaceutically acceptable carrier.

[0414] Embodiment 7. The method of embodiment 6, wherein N-acetyl cysteine derivative is selected from NAC-amide, NAC-methyl ester, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt.

[0415] Embodiment 8. A method for treating a cerebral amyloid angiopathy (CAA) comprising administering an effective amount of a composition comprising N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier, to a patient presenting with the CAA, said agent disrupting amyloid deposits in the brain and / or the walls of blood vessels caused by said CAA, thereby alleviating disease symptoms.Embodiment 9. The method of embodiment 8, wherein said CAA is a hereditary cerebral amyloid angiopathy.

[0416] Embodiment 10. The method of embodiment 9, wherein said hereditary cerebral amyloid angiopathy is selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF).

[0417] Embodiment 11. The method of embodiment 10, wherein the CAA is a subtype of HCHWA.

[0418] Embodiment 12. The method of embodiment 11, wherein the HCHWA is the Dutch subtype (HCHWA-D), the Italian subtype (HCHWA-It), the Iowa subtype (HCHWA-Io), the Flemish subtype (HCHWA-F), the Piedmont subtype (HCHWA-P), or the Arctic subtype (HCHWA-A).

[0419] Embodiment 13. The method of embodiment 12, wherein the subtype is HCHWA-D and the mutation is a E639Q mutation residing in the A domain of the amyloid precursor protein (APP).

[0420] Embodiment 14. The method of embodiment 12, wherein the subtype is HCHW-It and the mutation is a E639K or A713T mutation residing in the Ap domain of APP.

[0421] Embodiment 15. The method of embodiment 12, wherein the subtype is HCHWA-Io and the mutation is a D694N mutation residing in the A domain of APP.

[0422] Embodiment 16. The method of embodiment 12, wherein the subtype is HCHWA-F and the mutation is a A692G mutation residing in the Ap domain of APP.

[0423] Embodiment 17. The method of embodiment 12, wherein the subtype is HCHWA-P and the mutation is a L705V mutation residing in the Ap domain of APP.

[0424] Embodiment 18. The method of embodiment 12, wherein the subtype is HCHWA-A and the mutation is a E693G mutation residing in the Ap domain of APP.

[0425] Embodiment 19. The method of embodiment 10, wherein the FBD is caused by a mutation in the BRI2 / ITM2B gene.

[0426] Embodiment 20. The method of embodiment 19, wherein the mutation is a mutation in the stop codon.Embodiment 21. The method of embodiment 10, wherein the TRR is caused by a mutation in transthyretin selected from L12P, D18G, A25T, V30G, V30M, T49P, L58R, F64S, Y69H. Y114C. G53R. G53E. G53A. V122I, or E89Q.

[0427] Embodiment 22. The method of embodiment 10, wherein the FDD is caused by a mutation in the BRI2 / ITM2B gene.

[0428] Embodiment 23. The method of embodiment 22, wherein the mutation is a duplication mutation that disrupts the stop codon.

[0429] Embodiment 24. The method of any one of the preceding embodiments, wherein N-acetyl cysteine derivative is selected from NAC-amide, NAC-methyl ester, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt.

[0430] Embodiment 25. The method of any of the preceding embodiments further comprising

[0431] i) performing a skin biopsy on said subject following treatment to assess reduction in amyloid protein aggregates in skin; and / or

[0432] ii) performing an MRI, CAT scan, or PET scan on said subject following treatment to assess reduction in protein aggregates in the cerebral blood vessels.

[0433] Embodiment 26. The method of any of the preceding embodiments further comprising administration of an ionophore.

[0434] Embodiment 27. The method of any of the preceding embodiments further comprising administration of an anti-inflammatory agent.

[0435] Embodiment 28. The method of embodiment 27, wherein said anti-inflammatory agent is selected from the group consisting of one or more of corticosteroids, aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac, tolmetin, donepezil, fasoracetam, interleukin (IL)-l receptor antagonist, IL-4, IL-6, IL-10, IL-11, IL-13, cytokine receptors for IL-1, tumor necrosis factor- alpha, IL- 18 and derivatives and biosimilars thereof.Embodiment 29. The method of any one of the preceding embodiments, further comprising administration of one or more of glutathione, monensin, papain, cathepsin B, falcipain, and an siRNA.

[0436] Embodiment 30. The method of any of the preceding embodiments, wherein said administration is effective to reduce said protein aggregates, thereby alleviating symptoms of said CAA.

[0437] Embodiment 31. The method of embodiment 30, wherein said administration reduces protein aggregates in the wall of cerebral blood vessel.

[0438] Embodiment 32. The method of anyone of the preceding embodiments, wherein said administration increases the dementia rating scale-2 (DRS-2) overall index total score of the subject by at least 10%, at least 20%, or at least 30% when compared to the DRS-2 overall index total score of the subject prior to administration.

[0439] Embodiment 33. The method of anyone of the preceding embodiments, wherein the dementia rating scale-2 (DRS-2) overall index total score of the subject does not change after said administration.

[0440] Embodiment 34. The method of any one of the preceding embodiments further comprising monitoring said patient for amyloid deposit levels.

[0441] Embodiment 35. A method for identifying a therapeutic agent which disrupts amyloid deposits in the brain and / or the walls of blood vessels, the method comprising

[0442] a) providing a population of cells which express a nucleic acid encoding a mutant A0 protein said mutant causing formation of amyloid protein aggregates;

[0443] b) providing a population of cells which express an A0 protein which lacks the mutation; c) contacting the cells of step a) and step b) with a test agent; and

[0444] d) detecting whether the test agent alters amyloid protein aggregate formation in the cells of step a) and the cells of step b), wherein the test agents that decrease amyloid protein aggregate formation in the cells of step a) relative to the cells of step b) are identified as therapeutic agents.

[0445] Embodiment 36. A method for identifying a therapeutic agent which disrupts amyloid deposits in the brain and / or the walls of blood vessels, the method comprisinga) providing a first population cells which express a nucleic acid encoding a mutant A0 protein said mutant causing formation of amyloid protein aggregates;

[0446] b) providing a second population cells which express a nucleic acid encoding a mutant Ap protein said mutant causing formation of amyloid protein aggregates;

[0447] c) contacting the cells of step a) with N-acetyl cysteine or a function derivative thereof; d) contacting the cells of step b) with a test agent:

[0448] e) detecting whether the test agent alters amyloid protein aggregate formation in the cells of step b), wherein the test agents that decrease amyloid protein aggregate formation in the cells of step b) equal to, or greater than the decrease of amyloid protein aggregate formation in the cells of step a) are identified as therapeutic agents.

[0449] Embodiment 37. The method of embodiment 35, wherein N-acetyl cysteine derivative is selected from NAC-amide, NAC-methyl ester, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt.

[0450] Embodiment 38. The method of any one of embodiments 35-37. wherein the mutation is selected from any one of the mutations in Table 1.

[0451] Embodiment 39. The method of anyone of embodiments 35-37, wherein the mutation is in the APP gene.

[0452] Embodiment 40. The method of anyone of embodiments 35-39, wherein the test agent decreases amyloid protein aggregation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0453] Embodiment 41. A method for identifying a therapeutic agent for the treatment of a first cerebral amyloid angiopathy (CAA), the method comprising

[0454] a) providing a population of cells from a patient with a second CAA;

[0455] b) contacting the cells of step a) a test agent; and

[0456] c) detecting whether the test agent alters levels of at least one CAA biomarker selected from fibronectin, collagen IV, vimentin, a-SMA, SMAD2 / 3, or WNT-1, wherein the test agents that alter the at least one CAA biomarker level in the cells of step a) are identified as therapeutic agents.Embodiment 42. The method of embodiment 41, wherein the first CAA is Alzheimer’s disease or a hereditary cerebral amyloid angiopathy.

[0457] Embodiment 43. The method of embodiment 42, wherein the hereditary cerebral amyloid angiopathy is selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF).

[0458] Embodiment 44. The method of any one of embodiments 41-43, wherein the second CAA is Alzheimer’s disease or a hereditary cerebral amyloid angiopathy.

[0459] Embodiment 45. The method of embodiment 44, wherein the hereditary cerebral amyloid angiopathy is selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF).

[0460] Embodiment 46. The method of any one of embodiments 41-45, wherein the test agents that decrease levels of at least one of fibronectin, collagen IV, vimentin, SMAD2 / 3, and WNT-1 in the cells of step a) are identified as therapeutic agents.

[0461] Embodiment 47. The method of any one of embodiments 41-46, wherein the test agents that increase levels a-SMA, in the cells of step a) are identified as therapeutic agents.

[0462] Embodiment 48. The method of any one of embodiments 41-47, wherein the cell is a vascular smooth muscle cell (VMSC), a pericyte, an endothelial cell, a brain microvascular endothelial cell, an adventitial fibroblast, a myofibroblast, a fibroblast, a dermal fibroblast, an astrocyte, a microglial cell / macrophage, or a neuron.

[0463] Embodiment 49. A method for reducing cerebral vascular basement membrane remodeling in a subject in need thereof, comprising administering to said subject, an effective amount of a composition comprising N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier, thereby reducing cerebral vascular basement membrane remodeling in the subject.Embodiment 50. A method for reducing extracellular matrix accumulation of one or more CAA extracellular matrix proteins in a subject in need thereof, comprising administering to said subject, an effective amount of a composition comprising N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier, thereby reducing extracellular matrix accumulation of the one or more CAA extracellular matrix proteins.

[0464] Embodiment 51. The method of embodiment 49, wherein the composition further reduces extracellular matrix accumulation of the one or more CAA extracellular matrix proteins.

[0465] Embodiment 52. The method of embodiment 50, wherein the composition further reduces cerebral vascular basement membrane remodeling.

[0466] Embodiment 53. The method of any one of embodiments 50-52, wherein the one or more CAA extracellular matrix proteins is selected from fibronectin (FN1) and collagen IV (COL IV).

[0467] Embodiment 54. The method of any one of embodiments 49-53. wherein the subject has a cerebral amyloid angiopathy (CAA).

[0468] Embodiment 55. The method of embodiment 54, wherein the treatment ameliorates symptoms of the CAA in the patient.

[0469] Embodiment 56. The method of any one of embodiments 54-55, wherein the CAA is Alzheimer's Disease or a hereditary cerebral amyloid angiopathy.

[0470] Embodiment 57. The method of any one of embodiments 54-56, wherein the CAA is Alzheimer’s Disease.

[0471] Embodiment 58. The method of embodiment 57, wherein the Alzheimer’s Disease is sporadic Alzheimer’s Disease.

[0472] Embodiment 59. The method of embodiment 56, wherein the hereditary cerebral amyloid angiopathy is selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF).Embodiment 60. The method of any one of embodiments 54-59, wherein N-acetyl cysteine derivative is selected from NAC-amide, NAC-methyl ester, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt.

[0473] Embodiment 61. The method of any one of embodiments 54-60, wherein the composition reduces accumulation of collagen IV and fibronectin within cerebral vessel walls.

[0474] Embodiment 62. The method of any one of embodiments 54-61, wherein the composition attenuates vascular fibrosis associated signaling.

[0475] Embodiment 63. The method of embodiment 62, wherein the vascular fibrosis associated signaling is selected from TGF-β signaling-associated vascular fibrosis and / or WNT signaling-associated vascular fibrosis.

[0476] Embodiment 64. The method of any one of embodiments 54-63, wherein the composition preserves vascular smooth muscle cell integrity and / or prevents phenotypic switching of vascular smooth muscle cells to fibroblast-like states.

[0477] Embodiment 65. The method of any one of embodiments 54-64, wherein the composition improves intramural periarterial drainage (IPAD) or reduces protein elimination failure angiopathy (PEFA).

[0478] Embodiment 66. The method of any one of the preceding embodiments, wherein the subject exhibits increased levels vascular collagen IV or fibronectin prior to administration of the composition.

[0479] Embodiment 67. The method of any one of the preceding embodiments, wherein the composition reduces extracellular matrix expansion within one or more layers of a cerebral vessel wall.

[0480] Embodiment 68. The method of any one of the preceding embodiments, wherein the composition attenuates profibrotic signaling in cerebral vasculature.

[0481] Embodiment 69. The method of embodiment 68, wherein the profibrotic signaling comprises transforming growth factor-β (TGF-β) signaling and / or WNT signaling.Embodiment 70. The method of any one of the preceding embodiments wherein administration of the composition reduces activation of SMAD2 / 3, phosphorylated SMAD2 / 3, WNT-1, or combinations thereof.

[0482] Embodiment 71. The method of any one of the preceding embodiments, wherein the composition preserves vascular smooth muscle cell integrity.

[0483] Embodiment 72. The method of any one of the preceding embodiments, wherein the composition inhibits phenotypic switching of vascular smooth muscle cells to fibroblast-like or myofibroblast- like cells.

[0484] Embodiment 73. The method of embodiment 72, wherein the phenotypic switching is associated with expression of a-smooth muscle actin and vimentin.

[0485] Embodiment 74. The method of any one of the preceding embodiments, wherein the composition improves perivascular or intramural periarterial drainage of proteins from the brain.

[0486] Embodiment 75. The method of any one of the preceding embodiments, wherein the composition reduces protein elimination failure angiopathy (PEFA).

[0487] Embodiment 76. The method of any one of the preceding embodiments, wherein the composition reduces vascular retention of amyloidogenic proteins.

[0488] Embodiment 77. The method of embodiment 76, wherein the amyloidogenic proteins comprise one or more of amyloid-β, cystatin C, or medin.

[0489] Embodiment 78. The method of embodiment 77, wherein at least two of the amyloidegenic proteins are present.

[0490] Embodiment 79. The method of anyone of the preceding embodiments, wherein the composition reduces or prevents neuronal proteinopathy associated with Alzheimer’s disease.

[0491] Embodiment 80. The method of embodiment 79, wherein the neuronal proteinopathy comprises pathological aggregation or mislocalization of TDP-43.Embodiment 81. The method of any one of embodiments 49-80, wherein composition is administered orally, intravenously, or by another systemic route.

[0492] Embodiment 82. The method of any one of embodiments 49-81, wherein composition is administered chronically.

[0493] Embodiment 83. The method of any one of embodiment 49-82, further comprising assessing the subject for one or more of:

[0494] a) a reduction in cerebral vascular basement membrane remodeling;

[0495] b) a reduction in extracellular matrix accumulation of one or more CAA extracellular matrix protein;

[0496] c) an increase perivascular clearance of amyloidogenic proteins;

[0497] d) a reduction in accumulation of collagen IV and fibronectin within cerebral vessel walls;

[0498] e) attenuated TGF-β and / or WNT signaling-associated vascular fibrosis;

[0499] f) preservation of vascular smooth muscle cell integrity;

[0500] g) an increase in intramural periarterial drainage (IPAD) or reduces protein elimination failure angiopathy (PEFA);

[0501] h) a reduction in aggregation or vascular retention of one or more amyloidogenic proteins selected from cystatin C, amyloid-β, and medin; or

[0502] i) reduces neuronal TDP-43 pathology associated with Alzheimer’s disease; wherein each of these criteria are compared to an untreated control.

[0503] While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. It will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the scope of the present invention, as set forth in the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for reducing cerebral vascular basement membrane remodeling in a subject in need thereof, comprising administering to said subject, an effective amount of a composition comprising N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier, thereby reducing cerebral vascular basement membrane remodeling in the subject.

2. A method for reducing extracellular matrix accumulation of one or more CAA extracellular matrix proteins in a subject in need thereof, comprising administering to said subject, an effective amount of a composition comprising N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier, thereby reducing extracellular matrix accumulation of the one or more CAA extracellular matrix proteins.

3. The method of claim 1, wherein the composition further reduces extracellular matrix accumulation of the one or more CAA extracellular matrix proteins.

4. The method of claim 2, wherein the composition further reduces cerebral vascular basement membrane remodeling.

5. The method of claim 3, wherein the one or more CAA extracellular matrix proteins is selected from fibronectin (FN1) and collagen IV (COL IV).

6. The method of claim 1, wherein the subject has a cerebral amyloid angiopathy (CAA).

7. The method of claim 6, wherein the treatment ameliorates symptoms of the CAA in the patient.

8. The method of claim 6, wherein the CAA is Alzheimer's Disease or a hereditary cerebral amyloid angiopathy.

9. The method of claim 6, wherein the CAA is Alzheimer’s Disease.

10. The method of claim 9, wherein the Alzheimer’s Disease is sporadic Alzheimer’s Disease.

11. The method of claim 8, wherein the hereditary cerebral amyloid angiopathy is selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Priondisease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF).

12. The method of claim 1, wherein N-acetyl cysteine derivative is selected from NAC-amide, NAC-methyl ester, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt.

13. The method of a claim 1, wherein the composition reduces accumulation of collagen IV and fibronectin within cerebral vessel walls.

14. The method of any one of claims 1, wherein the composition attenuates vascular fibrosis associated signaling.

15. The method of claim 14, wherein the vascular fibrosis associated signaling is selected from TGF-β signaling-associated vascular fibrosis and / or WNT signaling-associated vascular fibrosis.

16. The method of claim 1, wherein the composition preserves vascular smooth muscle cell integrity and / or prevents phenotypic switching of vascular smooth muscle cells to fibroblast-like states.

17. The method of any one of claims 54-64, wherein the composition improves intramural periarterial drainage (IPAD) or reduces protein elimination failure angiopathy (PEFA).

18. The method of claim 1, wherein the subject exhibits increased levels vascular collagen IV or fibronectin prior to administration of the composition.

19. The method of claim 1, wherein the composition reduces extracellular matrix expansion within one or more layers of a cerebral vessel wall.

20. The method of claim 1, wherein the composition attenuates profibrotic signaling in cerebral vasculature.

21. The method of claim 20, wherein the profibrotic signaling comprises transforming growth factor-β (TGF-β) signaling and / or WNT signaling.

22. The method of claim 1 wherein administration of the composition reduces activation of SMAD2 / 3, phosphorylated SMAD2 / 3, WNT-1, or combinations thereof.

23. The method of claim 1, wherein the composition preserves vascular smooth muscle cell integrity.

24. The method of claim 1, wherein the composition inhibits phenotypic switching of vascular smooth muscle cells to fibroblast-like or myofibroblast-like cells.

25. The method of claim 24, wherein the phenotypic switching is associated with expression of a-smooth muscle actin and vimentin.

26. The method of claim 1, wherein the composition improves perivascular or intramural periarterial drainage of proteins from the brain.

27. The method of claim 1, wherein the composition reduces protein elimination failure angiopathy (PEFA).

28. The method of claim 1, wherein the composition reduces vascular retention of amyloidogenic proteins.

29. The method of claim 28, wherein the amyloidogenic proteins comprise one or more of amyloid-β, cystatin C, or medin.

30. The method of claim 29, wherein at least two of the amyloidegenic proteins are present.

31. The method of claim 1, wherein the composition reduces or prevents neuronal proteinopathy associated with Alzheimer’s disease.

32. The method of claim 31, wherein the neuronal proteinopathy comprises pathological aggregation or mislocalization of TDP-43.

33. The method of claim 1, wherein composition is administered orally, intravenously, or by another systemic route.

34. The method of claim 1, wherein composition is administered chronically.

35. The method of claim 1, further comprising assessing the subject for one or more of:a) a reduction in cerebral vascular basement membrane remodeling;b) a reduction in extracellular matrix accumulation of one or more CAA extracellular matrix protein;c) an increase perivascular clearance of amyloidogenic proteins;d) a reduction in accumulation of collagen IV and fibronectin within cerebral vessel walls;e) attenuated TGF-β and / or WNT signaling-associated vascular fibrosis;f) preservation of vascular smooth muscle cell integrity;g) an increase in intramural periarterial drainage (IPAD) or reduces protein elimination failure angiopathy (PEFA);h) a reduction in aggregation or vascular retention of one or more amyloidogenic proteins selected from cystatin C, amyloid-β, and medin; ori) reduces neuronal TDP-43 pathology associated with Alzheimer’s disease; wherein each of these criteria are compared to an untreated control.

36. A method for treating a cerebral amyloid angiopathy (CAA) in a subject in need thereof, comprising administering to said subject, an effective amount of a composition comprising an agent that disrupts both hydrogen and disulfide bonds between amyloid dimers and / or oligomers, thereby disrupting amyloid deposition in the brain and / or the walls of blood vessels.

37. The method of claim 36, wherein the blood vessels are cerebral blood vessels.

38. The method of claim 36, wherein the treatment ameliorates symptoms of the CAA in said patient.

39. The method of claim 36, wherein the CAA is Alzheimer's Disease or a hereditary cerebral amyloid angiopathy.

40. The method of claim 39, wherein the hereditary cerebral amyloid angiopathy is selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF).

41. The method of claim 36, wherein the agent is N-acetyl cysteine or a function derivative thereof in a pharmaceutically acceptable carrier.

42. The method of claim 41, wherein N-acetyl cysteine derivative is selected from NAC-amide, NAC-methyl ester, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt.

43. A method for treating a cerebral amyloid angiopathy (CAA) comprising administering an effective amount of a composition comprising N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier, to a patient presenting with the CAA, said agent disrupting amyloid deposits in the brain and / or the walls of blood vessels caused by said CAA, thereby alleviating disease symptoms.

44. The method of claim 43, wherein said CAA is a hereditary cerebral amyloid angiopathy.

45. The method of claim 44, wherein said hereditary cerebral amyloid angiopathy is selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF).

46. The method of claim 45, wherein the CAA is a subtype of HCHWA.

47. The method of claim 46, wherein the HCHWA is the Dutch subtype (HCHWA-D), the Italian subtype (HCHWA-It), the Iowa subtype (HCHWA-Io). the Flemish subtype (HCHWA-F), the Piedmont subtype (HCHWA-P), or the Arctic subtype (HCHWA-A).

48. The method of claim 47, wherein the subtype is HCHWA-D and the mutation is a E639Q mutation residing in the A0 domain of the amyloid precursor protein (APP).

49. The method of claim 47, wherein the subtype is HCHW-It and the mutation is a E639K or A713T mutation residing in the Ap domain of APP.

50. The method of claim 47, wherein the subtype is HCHWA-Io and the mutation is a D694N mutation residing in the A domain of APP.

51. The method of claim 47, wherein the subtype is HCHWA-F and the mutation is a A692G mutation residing in the Ap domain of APP.

52. The method of claim 47, wherein the subtype is HCHWA-P and the mutation is a L705V mutation residing in the Ap domain of APP.

53. The method of claim 47, wherein the subtype is HCHWA-A and the mutation is a E693G mutation residing in the Ap domain of APP.

54. The method of claim 45, wherein the FBD is caused by a mutation in the BRI2 / ITM2B gene.

55. The method of claim 54, wherein the mutation is a mutation in the stop codon.

56. The method of claim 45, wherein the TRR is caused by a mutation in transthyretin selected from L12P, D18G, A25T, V30G, V30M, T49P, L58R, F64S, Y69H. Y114C, G53R, G53E, G53A, V122I, or E89Q.

57. The method of claim 45, wherein the FDD is caused by a mutation in the BRI2 / ITM2B gene.

58. The method of claim 57, wherein the mutation is a duplication mutation that disrupts the stop codon.

59. The method of claim 1, wherein N-acetyl cysteine derivative is selected from NAC-amide, NAC-methyl ester, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt.

60. The method of any of the preceding claims further comprisingi) performing a skin biopsy on said subject following treatment to assess reduction in amyloid protein aggregates in skin; and / orii) performing an MRI, CAT scan, or PET scan on said subject following treatment to assess reduction in protein aggregates in the cerebral blood vessels.

61. The method of any of the preceding claims further comprising administration of an ionophore.

62. The method of any of the preceding claims further comprising administration of an antiinflammatory agent.

63. The method of claim 62, wherein said anti-inflammatory agent is selected from the group consisting of one or more of corticosteroids, aspirin, celecoxib, diclofenac, diflunisal, etodolac.ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac, tolmetin, donepezil, fasoracetam, interleukin (IL)-l receptor antagonist, IL-4, IL-6, IL-10, IL-11, IL-13, cytokine receptors for IL-1, tumor necrosis factor-alpha, IL-18 and derivatives and biosimilars thereof.

64. The method of claim 1. further comprising administration of one or more of glutathione, monensin, papain, cathepsin B, falcipain, and an siRNA.

65. The method of any of the preceding claims, wherein said administration is effective to reduce said protein aggregates, thereby alleviating symptoms of said CAA.

66. The method of claim 65, wherein said administration reduces protein aggregates in the wall of cerebral blood vessel.

67. The method of anyone of the preceding claims, wherein said administration increases the dementia rating scale-2 (DRS-2) overall index total score of the subject by at least 10%, at least 20%, or at least 30% when compared to the DRS-2 overall index total score of the subject prior to administration.

68. The method of anyone of the preceding claims, wherein the dementia rating scale-2 (DRS-2) overall index total score of the subject does not change after said administration.

69. The method of any one of the preceding claims further comprising monitoring said patient for amyloid deposit levels.

70. A method for identifying a therapeutic agent which disrupts amyloid deposits in the brain and / or the walls of blood vessels, the method comprisinga) providing a population of cells which express a nucleic acid encoding a mutant Ap protein said mutant causing formation of amyloid protein aggregates;b) providing a population of cells which express an Ap protein which lacks the mutation; c) contacting the cells of step a) and step b) with a test agent; andd) detecting whether the test agent alters amyloid protein aggregate formation in the cells of step a) and the cells of step b), wherein the test agents that decrease amyloid protein aggregate formation in the cells of step a) relative to the cells of step b) are identified as therapeutic agents.

71. A method for identifying a therapeutic agent which disrupts amyloid deposits in the brain and / or the walls of blood vessels, the method comprisinga) providing a first population cells which express a nucleic acid encoding a mutant A0 protein said mutant causing formation of amyloid protein aggregates;b) providing a second population cells which express a nucleic acid encoding a mutant Ap protein said mutant causing formation of amyloid protein aggregates;c) contacting the cells of step a) with N-acetyl cysteine or a function derivative thereof; d) contacting the cells of step b) with a test agent:e) detecting whether the test agent alters amyloid protein aggregate formation in the cells of step b), wherein the test agents that decrease amyloid protein aggregate formation in the cells of step b) equal to, or greater than the decrease of amyloid protein aggregate formation in the cells of step a) are identified as therapeutic agents.

72. The method of claim 70, wherein N-acetyl cysteine derivative is selected from NAC-amide, NAC-methyl ester, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt.

73. The method of any one of claims 70-72, wherein the mutation is selected from any one of the mutations in Table 1.

74. The method of anyone of claims 70-72, wherein the mutation is in the APP gene.

75. The method of anyone of claims 70-74, wherein the test agent decreases amyloid protein aggregation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

76. A method for identifying a therapeutic agent for the treatment of a first cerebral amyloid angiopathy (CAA), the method comprisinga) providing a population of cells from a patient with a second CAA;b) contacting the cells of step a) a test agent; andc) detecting whether the test agent alters levels of at least one CAA biomarker selected from fibronectin, collagen IV, vimentin, a-SMA, SMAD2 / 3, or WNT-1, wherein the test agentsthat alter the at least one CAA biomarker level in the cells of step a) are identified as therapeutic agents.

77. The method of claim 76, wherein the first CAA is Alzheimer’s disease or a hereditary cerebral amyloid angiopathy.

78. The method of claim 77, wherein the hereditary cerebral amyloid angiopathy is selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF).

79. The method of any one of claims 76-78, wherein the second CAA is Alzheimer’s disease or a hereditary cerebral amyloid angiopathy.

80. The method of claim 79, wherein the hereditary cerebral amyloid angiopathy is selected from hereditary cerebral hemorrhage with amyloidosis (HCHWA), TTR amyloidosis, Genetic Prion disease, Familial British dementia (FBD), Familial Danish dementia (FDD), Familial amyloidosis-Finish type (FAF).

81. The method of any one of claims 76-80, wherein the test agents that decrease levels of at least one of fibronectin, collagen IV, vimentin, SMAD2 / 3, and WNT-1 in the cells of step a) are identified as therapeutic agents.

82. The method of any one of claims 76-81, wherein the test agents that increase levels a-SMA, in the cells of step a) are identified as therapeutic agents.

83. The method of any one of claims 76-82, wherein the cell is a vascular smooth muscle cell (VMSC), a pericyte, an endothelial cell, a brain microvascular endothelial cell, an adventitial fibroblast, a myofibroblast, a fibroblast, a dermal fibroblast, an astrocyte, a microglial cell / macrophage, or a neuron.

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