A biomarker for cerebral amyloid angiopathy and amyloid related imaging abnormality risk

Abstract

The disclosed invention relates to the development of diagnostic methods for identifying patients having cerebral amyloid angiopathy ("CAA"), which methods afford earlier detection and, thus, earlier treatment of the disease to slow or potentially reverse disease progression. Kits and reagents for carrying out the disclosed methods are also described.

Description

A BIOMARKER FOR CEREBRAL AMYLOID ANGIOPATHY AND AMYLOID RELATED IMAGING ABNORMALITY RISK

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 672,061, filed July 16, 2025, which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under P01 AG060882, P30 AG066512 and U24 NS141774 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] The Sequence Listing is being submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 15, 2025, is named 147462_002571.xml and is 65,536 bytes in size. No new matter is being introduced.FIELD

[0004] The present invention relates to the development of diagnostic methods for identifying patients having cerebral amyloid angiopathy (“CAA”), which methods afford earlier detection and, thus, earlier treatment of the disease to slow or potentially reverse disease progression. Kits and reagents for carrying out the disclosed methods are also described.BACKGROUND

[0005] Cerebral amyloid angiopathy is characterized by amyloid beta (AP) deposition in the cerebrovasculature. It is not only prevalent in aging and in almost all Alzheimer’s disease (AD) patients but can also occur on its own, independent of other AD-related pathology (Charidimou et al., “Emerging Concepts in Sporadic Cerebral Amyloid Angiopathy,” Brain 140: 1829-1850 (2017); Greenberg et al., “Cerebral Amyloid Angiopathy and Alzheimer Disease — One Peptide, Two Pathways,” Nat. Rev. Neurol. 16:30-42 (2020); Greenberg et al., “Cerebral Amyloid Angiopathy and Alzheimer Disease — One Peptide, Two Pathways,” Nat. Rev. Neurol. 16:30-42 (2020); Koemans et al., “Progression of Cerebral Amyloid Angiopathy: A Pathophysiological Framework,” Lancet Neurol 22:632-642 (2023); and Weber et al., “Cerebral Amyloid Angiopathy: Diagnosis and Potential Therapies,” Expert Rev. Neurother. 18:503-513 (2018)). The presence and severity of CAA promotes the progression of AD-related clinical symptoms, and is also associated with more rapid cognitive decline in normal aged subjects (Bos et al., “Cerebrovascular and Amyloid Pathology in Predementia Stages: The Relationship with Neurodegeneration and Cognitive Decline,” Alzheimers Res. Ther. 9: 101 (2017); Boyle et al., “Cerebral Amyloid Angiopathy and Cognitive Outcomes in Community -Based Older Persons,”Neurology 85: 1930-1936 (2015); and Charidimou et al., “Emerging Concepts in Sporadic Cerebral Amyloid Angiopathy,” Brain 140: 1829-1850 (2017)). The development of CAA may contribute to cognitive decline directly by facilitating hypoxia and neuronal injury or indirectly by promoting tau pathology (Malek-Ahmadi et al., “Braak Stage, Cerebral Amyloid Angiopathy, and Cognitive Decline in Early Alzheimer’s Disease,” J. Alzheimers Dis. 74: 189-197 (2020); Rabin et al., “Cerebral Amyloid Angiopathy Interacts with Neuritic Amyloid Plaques to Promote Tau and Cognitive Decline,” Brain 145:2823-2833 (2022); and Weber et al., “Cerebral Amyloid Angiopathy: Diagnosis and Potential Therapies,” Expert Rev. Neurother. 18:503-513 (2018)). CAA is linked to cerebral hemorrhages and amyloid-related brain imaging (ARIA) abnormalities, a major complication of AD immunotherapeutics (Charidimou et al., “Emerging Concepts in Sporadic Cerebral Amyloid Angiopathy,” Brain 140: 1829-1850 (2017); Hampet et al., “Amyloid-Related Imaging Abnormalities (ARIA): Radiological, Biological and Clinical Characteristics,” Brain 1446(11):4414-4424 (2023); Herline et al., “Recent Advancements Toward Therapeutic Vaccines Against Alzheimer’s Disease,” Expert. Rev. Vaccines 17:707-721 (2018); Pfeifer et al., “Cerebral Amyloid Angiopathy and Cognitive Function: The HAAS Autopsy Study,” Neurology 58: 1629-1634 (2002); Pfeifer M et al., “Cerebral Hemorrhage after Passive Anti-Ap Immunotherapy,” Science 298: 1379 (2002); Piazza, W ., “Amyloid-Related Imaging Abnormalities (ARIA) in Immunotherapy Trials for Alzheimer’s Disease: Need for Prognostic Biomarkers?” J. Alzheimers Dis. 52:417-420 (2016); Sims et al., “Donanemab in Early Symptomatic Alzheimer Disease: the TRAILBLAZER-ALZ 2 Randomized Clinical Trial,” JAMA 330:512-527 (2023); Sweeney et al., “The Role of Brain Vasculature in Neurodegenerative Disorders,” Nat. Neurosci. 21 : 1318-1331 (2018); and van Dyck CH et al., “Lecanemab in Early Alzheimer’s Disease,” N. Engl. J. Med. 388:9-21 (2023)).

[0006] There are some mechanistic components of CAA development that have been elucidated, but the underlying pathogenesis is not well understood. Ap deposition in CAA usually occurs in a spiral-like fashion, distributed in a characteristic patchy pattern, often in an adventitial manner rather than medially in larger arterioles, and most commonly consists of the more soluble Ap40 over less soluble Ap42 (Charidimou et al., “Emerging Concepts in Sporadic Cerebral Amyloid Angiopathy,” Brain 140: 1829-1850 (2017)). It has been proposed that Ap expands from the basement membrane and replaces elements of the vessel wall with disease progression, potentially impacting neighboring cells including neurons, inflammatory cells, endothelial cells, and the integrity of the blood brain barrier (BBB) (Charidimou et al., “Emerging Concepts in Sporadic Cerebral Amyloid Angiopathy,” Brain 140: 1829-1850 (2017)). Increased BBB permeability has been observed in CAA patients and animal models, along withcerebral hypoperfusion that occurs before clinical dementia (Fisher et al., “Pathological Changes within the Cerebral Vasculature in Alzheimer’s Disease: New Perspectives,” Brain Pathol. 32:el3061 (2022); Magaki et al., “The Effects of Cerebral Amyloid Angiopathy on Integrity of the Blood-Brain Barrier,” NeurobioL Aging 70:70-77 (2018); and Situ et al., “Transcriptomic Profile of Blood-Brain Barrier Remodeling in Cerebral Amyloid Angiopathy,” Front. Cell Neurosci. 16:931247 (2022)). Previous proteomic studies aiming to identify CAA-associated proteins have largely relied on blood vessel extraction using density gradient approaches, which results in a mixture of CAA(+) and CAA(-) vessels, making it difficult to parse proteins selectively enriched in CAA(+) vessels (Ojo et al., “Molecular Pathobiology of the Cerebrovasculature in Aging and in Alzheimers Disease Cases with Cerebral Amyloid Angiopathy,” Front. Aging Neurosci. 13:658605 (2021); Wojtas et al., “Proteomic Changes in the Human Cerebrovasculature in Alzheimer’s Disease and Related Tauopathies Linked to Peripheral Biomarkers in Plasma and Cerebrospinal Fluid,” Alzheimers Dement. 20(6):4043- 4065(2024); and Zellner et al., “Proteomic Profiling in Cerebral Amyloid Angiopathy Reveals an Overlap with CADASIL Highlighting Accumulation of HTRA1 and its Substrates,” Acta Neuropathol. Commun. 10:6 (2022)).

[0007] An alternative approach has been to identify CAA-associated proteins in large tissue regions enriched in CAA (Hondius et al., “Proteomics Analysis Identifies New Markers Associated with Capillary Cerebral Amyloid Angiopathy in Alzheimer’s Disease,” Acta. Neuropathol. Commun. 6(1):46 (2018)). Only two studies to date have used laser capture microdissection (LCM) to selectively capture CAA(+) vessels from AD tissue for proteomic analyses (Handa et al., “Proteomics-Based Investigation of Cerebrovascular Molecular Mechanisms in Cerebral Amyloid Angiopathy by the FFPE-LMD-PCT-SWATH Method,” Fluids Barriers CNS 19(1):56 (2022) and Inoue et al., “Sushi Repeat-Containing Protein 1 : A Novel Disease-Associated Molecule in Cerebral Amyloid Angiopathy,” Acta. Neuropathol. 134(4):605— 617 (2017)). These previous studies have identified multiple CAA-associated proteins such as HTRA1, SMOC1, SMOC2, and SRPX1 (Handa et al., “Proteomics-Based Investigation of Cerebrovascular Molecular Mechanisms in Cerebral Amyloid Angiopathy by the FFPE-LMD-PCT-SWATH Method,” Fluids Barriers CNS 19(1):56 (2022); Hondius et al., “Proteomics Analysis Identifies New Markers Associated with Capillary Cerebral Amyloid Angiopathy in Alzheimer’s Disease,” Acta. Neuropathol. Commun. 6(1):46 (2018); Inoue et al., “Sushi Repeat-Containing Protein 1 : A Novel Disease- Associated Molecule in Cerebral Amyloid Angiopathy,” Acta. Neuropathol. 134(4):605— 617 (2017); Ojo et al., “Molecular Pathobiology of the Cerebrovasculature in Aging and in Alzheimers Disease Cases with Cerebral AmyloidAngiopathy,” Front. Aging Neurosci. 13:658605 (2021); Wojtas et al., “Proteomic Changes in the Human Cerebrovasculature in Alzheimer’s Disease and Related Tauopathies Linked to Peripheral Biomarkers in Plasma and Cerebrospinal Fluid,” Alzheimers Dement. 20(6):4043- 4065(2024); and Zellner et al., “Proteomic Profiling in Cerebral Amyloid Angiopathy Reveals an Overlap with CADASIL Highlighting Accumulation of HTRA1 and its Substrates,” Acta NeuropathoL Commun. 10:6 (2022)). However it is still not well understood which proteins are selectively enriched in CAA(+) vessels in comparison to neighboring CAA(-) vessels, whether the CAA proteome is the same in early and advanced AD, and whether the same proteins are enriched in CAA and amyloid plaques.

[0008] The present invention is directed to overcoming these and other deficiencies in the art.SUMMARY

[0009] A first aspect of the disclosure relates to an analytical method for diagnosing a patient having cognitive impairment with Cerebral Amyloid Angiopathy (CAA). This method involves the steps of: a) performing an assay on at least one biological sample from said patient to obtain quantifying data for said one or more than one first protein marker and optionally one or more than one second protein marker; b) generating a result of said assay, said result comprising (i) at least the presence or absence of an increase in the level of said one or more than one first protein marker in said sample based on a comparison of said quantifying data for said one or more than one first protein marker to a control or standard range for said one or more than one first protein marker, and optionally (ii) at least the presence or absence of a decrease in the level of said one or more than one second protein marker in said sample based on a comparison of said quantifying data for said one or more than one second protein marker to a control or standard range for said one or more than one second protein marker; and c) assigning the patient as having or not having CAA based on the result of said generating, where an increase in the level of said one or more than one first protein marker in the sample from the patient relative to the control or standard range indicates that the patient has CAA and / or where a decrease in the level of said one or more than one second protein marker in the sample from the patient relative to the control or standard range indicates that the patient has CAA.

[0010] A second aspect of the disclosure relates to an analytical method for detecting presence and quantity of a protein marker in a patient sample. This analytical method involves the steps of: providing at least one sample from a patient having cognitive impairment; and performing a quantitative assay on the at least one sample using either a mass spectrometer or abiological reagent that reacts specifically with one or more than one protein marker in said sample, thereby generating quantifying data for said one or more than one protein marker; and where the protein marker is one or more than one first protein marker identified in Table 1 (infra) as being elevated in individuals having CAA, the protein marker is one or more than one second protein marker identified in Table 2 (infra) as being decreased in individuals having CAA, or both.

[0011] A third aspect of the present disclosure relates to a method for diagnosing and treating a patient having cognitive impairment with CAA. This method includes the steps of performing the method according to the first or second aspect of the disclosure; and administering one or more therapeutic agents to the patient under conditions effective to reduce the risk of first-time or recurrent lobar intracerebral hemorrhages or cerebral microbleeds, manage hypertension, manage CAA-related inflammation, slow vascular deposition of A0 and / or remove insoluble A0 from vasculature.

[0012] A fourth aspect is directed to kits for carrying out the diagnostic methods according to the present disclosure. The kits according to this aspect of the disclosure includes one or more of(i) an antibody reagent, or antigen-binding fragment thereof, that binds specifically to one of the first protein markers; and / or(ii) an antibody reagent, or antigen-binding fragment thereof, that binds specifically to one of the second protein markers; and / or(iii) an antibody reagent, or antigen-binding fragment thereof, that binds specifically to one of the third protein markers; and / or(iv) protein extraction reagents for extraction of proteins from patient samples; and / or(v) an enzyme capable of cleaving the first and / or second protein markers; and / or(vi) a data acquisition template identifying polypeptide fragments detectable by the mass spectrometer for said first and / or second and / or third protein markers.

[0013] The Examples of the present disclosure leverage a localized proteomic approach to allow for distinct CAA(+) and CAA(-) vessel proteome characterization. The studies disclosed herein: (1) characterize the proteins selectively enriched in CAA(+) vessels, (2) compare the protein composition of CAA(+) vessels in early AD (mild cognitive impairment, or MCI) versus advanced AD, (3) characterize the proteome of neighboring CAA(-) vessels in MCI and AD, (4) compare the protein composition of CAA(-) vessels in MCI and AD, and (5) compare the CAA(+) vessel proteome to the amyloid-plaque proteome.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. l is a schematic approach overview for LCM of CAA(+) and CAA(-) vessels. Blood vessels (2 mm2) from the inferior temporal cortex were microdissected by LCM from FFPE autopsy brain tissue from control (n = 10), MCI (n = 4), and AD (n = 6) cases. Both CAA(+) (Ap positive) and neighboring CAA(-) (Ap negative) vessels were dissected separately, with only CAA(-) vessels from Control cases. Proteins were quantified by label-free quantitative mass spectrometry to identify protein differences. There were 2026 proteins detected in at least half of one group.

[0015] FIGS. 2A-2B show PCA of CAA(+) and CAA(-) vessels. FIG. 2A is a PCA graph showing the distribution of Control, MCI, and AD CAA(+) and CAA(-) vessel samples. FIG. 2B is a bar graph showing a significant segregation of CAA(+) vessel samples from CAA(-) vessel samples, particularly in PCA2 (p < 0.0001, unpaired t test), and some segregation was seen in PCA1 (p = 0.027). There was no segregation by disease group.

[0016] FIGS. 3 A-3F show markers of interest in dissected vessels. FIG. 3 A is a bar graph showing that the Ap peptide was enriched in the CAA(+) vessels from both MCI and AD cases. FIG. 3B is a bar graph showing that the endothelial cell marker PECAM1 (CD31) was detected in all samples and was not significantly different. FIGS. 3C-3E are bar graphs evaluating specific vessel type markers, among those detected, capillary markers BSG (FIG. 3C), SLC7A2 (FIG. 3D), and SLC3 A5 (FIG. 3E) were decreased in CAA(+) vessels. FIG. 3F is a bar graph showing that a marker for veins, PTGDS, was increased in CAA(+) vessels. Significant pairwise comparisons are indicated for those analyses that were performed, *p < 0.05, ** / ? < 0.01, *** / ? < 0.0001.

[0017] FIGS. 4A-4D provide CAA(+) vs CAA(-) vessel differential expression analysis and GO cell component terms in MCI and AD. FIG. 4A shows that when comparing MCI CAA(+) vessels to MCI CAA(-) vessels, there were 257 proteins differentially abundant (p < 0.05, fold change > 1.5), with 52 increased (red in a color version of FIG. 4A) and 205 decreased (blue in a color version of FIG. 4 A). The top 10 significantly increased and decreased are annotated by gene name, as well as Ap peptide when not among the top 10. FIG. 4B shows that when comparing AD CAA(+) vessels to AD CAA(-) vessels, there were 289 proteins differentially abundant, with 120 increased and 169 decreased. Blood vessel specific cell-type annotations are indicated for each protein. FIGS. 4C and 4D show that differentially abundant proteins were associated with the indicated increased (red in color versions of FIG. 4C and FIG. 4D) or decreased (blue in color versions of FIG. 4C and FIG. 4D) GO cell component (GOCC) terms atadj . p < 0.05 in MCI and AD. Number of proteins associated with a term are depicted by circle size.

[0018] FIGS. 5A-5C demonstrate that CAA(+) vs CAA(-) vessel protein differences positively correlate in MCI and AD. FIG. 5A is a Venn diagram showing that, of the differentially abundant proteins in CAA(+) vessels, when compared to CAA(-) vessels, 86 were shared in MCI and AD. Of the 86, 84 changed in the same fold-change direction. FIG. 5B is a graph showing that, of differentially abundant proteins in at least one disease group (460 proteins), there was a positive correlation of protein changes (p < 0.0001, R2= 0.62). There were 87% (400 / 460) of proteins changing in the same direction (purple) and 13.0% (60 / 460) proteins changing in the opposite direction (yellow in a color version of FIG. 5 A). The 84 proteins changing in the same fold-change direction and significant in both disease groups are indicated in blue. Proteins of interest are annotated by gene name. FIG. 5C is a heat map showing that, the 84 shared significant CAA(+) vessel proteins in MCI and AD were evaluated by unsupervised hierarchical clustering, and indicated clustering by tissue type as CAA(+) or CAA(-). Expression levels are depicted by z-score in the heatmap. The top cluster are proteins decreased in CAA(+) vessels and the bottom cluster are the proteins increased in CAA(+) vessels.

[0019] FIGS. 6A-6D provide non-CAA vessel differential expression analysis and GO cell component terms in MCI and AD. FIG. 6A shows that in MCI CAA(-) vessels when compared to Control CAA(-) vessels, there were 61 proteins differentially abundant (p < 0.05, fold change > 1.5), with 35 increased (red in a color version of FIG. 6A) and 26 decreased (blue in a color version of FIG. 6 A). The top ten increased and decreased are annotated by gene name. FIG. 6B shows that in AD CAA(-) vessels when compared to Control CAA(-) vessels, there were 112 proteins differentially abundant, with 44 increased and 68 decreased. FIG. 6C and FIG. 6D demonstrate that differentially abundant proteins were associated with the indicated increased (red in a color version of FIG. 6D) GOCC terms at adj . p < 0.05 in MCI and AD. The number of proteins associated with a term are depicted by circle size.

[0020] FIGS. 7A-7C demonstrate that non-CAA vessel protein differences have a mild positive correlation in MCI and AD. FIG. 7A is a Venn diagram showing that of the differentially abundant proteins in MCI or AD CAA(-) vessels when compared to Control CAA(-) vessels, 22 were shared and all changed in the same fold-change direction. FIG. 7B is a plot showing that of differentially abundant proteins in at least one disease group (151 proteins), there was a mild positive correlation of protein changes (p < 0.0001, R2= 0.18). There were 85% (129 / 151) of proteins changing in the same direction (purple) and 14.6% (22 / 151) proteinschanging in the opposite direction (yellow). The 22 proteins changing in the same fold-change direction and significant in both disease groups are indicated in blue. Proteins of interest are annotated by gene name. FIG. 7C is a head map showing the evaluation of the 22 shared significant CAA(-) vessel proteins in MCI and AD by unsupervised hierarchical clustering, which indicated clustering of Control cases from MCI and AD cases. Expression levels are depicted by z-score in the heatmap. The top cluster are proteins decreased in MCI and AD CAA(-) vessels, and the bottom cluster are the proteins increased in MCI and AD CAA(-) vessels.

[0021] FIGS. 8A-8C show COL6A2 histologic characterization. FIG. 8A is a bar graph showing proteomics quantification of COL6A2 showed enrichment in CAA(-) vessels in both MCI and AD, as well as in CAA(+) vessels from MCI. FIG. 8B is a bar graph showing quantification of COL6A2 immunoreactive area in CAA(+) and CAA(-) vessels in temporal cortex sections from progressive stages of disease, Control (n = 12), Preclinical AD (n = 10), MCI (n = 12), and AD (n = 12), showed enrichment in preclinical AD, MCI, and AD CAA(+) vessels. FIG. 8C are representative images show COL6A2 immunoreactivity in CAA at progressive stages of disease. Significant pairwise comparisons are indicated for those analyses that were performed, ** / ? < 0.01, *** / ? < 0.0001. Scale bar = 50 pm.

[0022] FIGS. 9A-9C show SMOC1 histologic characterization. FIG. 9A is a bar graph showing proteomics quantification of SMOC1 showed enrichment in CAA(+) vessels in both MCI and AD. FIG. 9B is a bar graphs showing quantification of SMOC1 immunoreactive area in CAA(+) and CAA(-) vessels in temporal cortex sections from progressive stages of disease, control (n = 12), preclinical AD (n = 10), MCI (n = 12), and AD (n = 12), showed enrichment in preclinical AD, MCI, and AD CAA(+) vessels, similar to proteomic quantification. FIG. 9C are representative images show SMOC1 immunoreactivity in CAA at progressive stages of disease. Significant pairwise comparisons are indicated for those analyses that were performed, *p < 0.05, *** / ? < 0.0001. Scale bar = 25 pm.

[0023] FIGS. 10A-10B shows potential CAA and Ap plaque protein markers. FIG. 10A is a schema highlighting key proteins detected as enriched in CAA(+) vessels compared to plaque- enriched proteins from Drummond (Drummond et al., “Proteomic Differences in Amyloid Plaques in Rapidly Progressive and Sporadic Alzheimer’s Disease,” Acta NeuropathoL 133(6):933-954 (2017), which is hereby incorporated by reference in its entirety) and Xiong (Xiong et al., “Quantitative Proteomics Reveals Distinct Composition of Amyloid Plaques in Alzheimer’s Disease,” Alzheimers Dement. 15(3):429-440 (2019), which is hereby incorporated by reference in its entirety), which may act as markers for these pathologies. Venn diagramshows the overlap between the two pathologies. Boxes show examples of high confidence proteins in each group. Representative images of CAA and plaques: blue = nuclei (Hoescht), green = SMOC1 (example extracellular matrix protein), and red = Ap. FIG. 10B shows the top 10 GOCC terms for each subgroup (potential CAA marker, potential plaque marker, and common proteins). GOCC terms have been simplified to reduce similar terms. Red = CAA enriched, blue = plaque enriched, and magenta = common proteins.

[0024] FIGS. 11A-1 ID SEMA3G histologic characterization. FIG. 11A is a bar graph showing proteomics quantification of SEMA3G showed enrichment in CAA(+) vessels in both MCI and AD. N.D. not detected. FIG. 1 IB is a bar graph showing quantification of SEMA3G immunoreactive area in CAA(+) and CAA(-) vessels in temporal cortex sections from progressive stages of disease, Control (n = 12), Preclinical AD (n = 10), MCI (n = 12), and AD (n = 12), showed enrichment in preclinical AD, trend in MCI, and enrichment in AD CAA(+) vessels. FIG. 11C shows histologic quantification of SEMA3G in CAA(+) vessels correlated to CAA levels (p < 0.0001; R2= 0.90). FIG. 1 ID are representative images show SEMA3G immunoreactivity in CAA at progressive stages of disease. Significant pairwise comparisons are indicated for those analyses that were performed, *p < 0.05, **p < 0.01. Scale bar = 50 pm.

[0025] FIGS. 12A-12M are bar graphs showing all detected collagen proteins: COL1 Al (FIG. 12A), COL1A2 (FIG. 12B), COL4A1 (FIG. 12C), COL4A2 (FIG. 12D), COL4A3 (FIG. 12E), COL6A1 (FIG. 12F), COL6A2 (FIG. 12G), COL6A3 (FIG. 12H), COL8A1 (FIG. 121), COL12A1 (FIG. 12J), COL14A1 (FIG. 12K), COL18A1 (FIG. 12L), and COL25A1 (FIG. 12M). Significant pairwise comparisons are indicated for those analyses that were performed, * p < 0.05, ** p < 0.01, *** p < 0.0001.

[0026] FIGS. 13A-13C show proteins important for blood brain barrier function. FIG. 13A and FIG. 13B are bar graphs showing Occludin (OCLN) (FIG. 13A) and TJP1 (ZO-1) (FIG. 13B) were decreased or not detected (N.D.) in CAA(+) vessels when compared to CAA(-) vessels. FIG. 13C is a bar graph showing that SLC2A1 (GLUT1), important to the function of the BBB, was decreased in AD CAA(+) vessels, with a similar trend seen in MCI. Significant pairwise comparisons are indicated for those analyses that were performed, * p < 0.05, ** p < 0.01, *** p < 0.0001.DETAILED DESCRIPTION

[0027] Neuropathological studies have demonstrated that microinfarcts occur in brain regions with increased amyloid-P burden and are associated with vessels with significant amyloid-P deposition (Van Veluw et al., “Histopathology of Diffusion Imaging Abnormalities inCerebral Amyloid Angiopathy,” Neurology 92:E933-E943 (2019), which is hereby incorporated by reference in its entirety). In addition to microinfarcts, white matter hyperintensities and atrophy and structural network alterations have been shown to contribute to cognitive impairment in patients with CAA (Reijmer et al., “Structural Network Alterations and Neurological Dysfunction in Cerebral Amyloid Angiopathy,” Brain 138: 179-188 (2015);Fotiadis et al., “White Matter Atrophy in Cerebral Amyloid Angiopathy,” Neurology 95(5):e554- e562 (2020); Smith et al., “White Matter Lesions, Cognition, and Recurrent Hemorrhage in Lobar Intracerebral Hemorrhage,” Neurology 63: 1606-1612 (2004), each of which is hereby incorporated by reference in its entirety).

[0028] Described herein are analytical methods and kits for diagnosing a patient with cognitive impairment as having Cerebral Amyloid Angiopathy (CAA). This will facilitate earlier treatment intervention, likely maintaining quality of life for a longer duration than if the disease progressed without intervention.Definitions

[0029] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.

[0030] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods and / or steps of the type described herein and / or which will become apparent to a person of ordinary skill in the art upon reading this disclosure. In another example, reference to “a compound” includes both a single compound and a plurality of different compounds.

[0031] The term “and / or” as used herein means that the listed features are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed features is used or present.

[0032] As will be understood by a person of ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof, as well as any value within a range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so on. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and so on. As will also be understood by aperson of ordinary skill in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges or specific values therein as discussed above. Finally, as will be understood by a person of ordinary skill in the art, and as discussed above, a range includes each individual value.

[0033] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open-ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps. In embodiments or claims where the term comprising (or the like) is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of’ or “consisting essentially of.” The methods, kits, systems, and / or compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, some embodiments of the methods and materials are now described.

[0035] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.Analytical Methods

[0036] To allow for earlier intervention and treatment of CAA patients, the invention described herein affords an approach for diagnosing CAA in a patient using a sample obtained from the patient. In certain embodiments, the patient may exhibit some signs of cognitive decline (e.g., memory loss, trouble with language, and displaying poor judgment), but has not undergone some form of cognitive screening. In other embodiments, the patient may exhibit some signs of cognitive decline and has undergone some form of cognitive screening with scores suggestive of mild cognitive impairment or normal but approaching the cutoff for mild cognitive impairment. Cognitive screening may include any one or more well known screening exams. Exemplary screening exams and scores indicative of mild cognitive impairment include, without limitation, a Mini-Mental State Exam (“MMSE”) score of 25 or less (Albert, “MMSE 2.0: A New Approach to an Old Measure,” Neuroepidemiology 43(l):26-7 (2014), which is hereby incorporated by reference in its entirety); a Clinical Dementia Rating (“CDR”) of 0.5 or higher (Morris, “The Clinical Dementia Rating (CDR): Current Version and Scoring Rules,” Neurology 43(11):2412— 2414 (1993); Lee et al., “Online Clinical Consensus Diagnosis of Dementia: Development and Validation,” J Am Geriatr Soc. 68(Suppl 3):S54-S59 (2020), each of which is hereby incorporated by reference in its entirety); a Montreal Cognitive Assessment (“MoCA”) score of less than 26 (Nasreddine et al., “The Montreal Cognitive Assessment, MoCA: A Brief Screening Tool for Mild Cognitive Impairment,” J Am Geriatr Soc. 53(4):695-9 (2005), which is hereby incorporated by reference in its entirety); or a Saint Louis University Mental Status Examination (“SLUMS”) score of 24 or less (Merz et al., “Clinical Utility of the Saint Louis University Mental Status Examination (SLUMS) in a Mixed Neurological Sample: Proposed Revised Cutoff Scores for Normal Cognition, Mild Cognitive Impairment, and Dementia,” Appl Neuropsychol Adult 31(5): 1024-1031 (2024), which is hereby incorporated by reference in its entirety). The patient may or may not have a family history of dementia such as vascular dementia, frontotemporal dementia, Alzheimer’s disease, Parkinson’s disease with dementia, or Lewy body disease, all of which frequently coexist CAA.

[0037] According to one embodiment, the analytical method for diagnosing a patient having cognitive impairment with CAA includes the steps of: a) performing an assay on at least one biological sample from the patient to obtain quantifying data for one or more than one first protein marker and optionally one or more than one second protein marker; b) generating a result of said assay, said result comprising (i) at least the presence or absence of an increase in the level of said one or more than one first protein marker in saidsample based on a comparison of said quantifying data for said one or more than one first protein marker to a control or standard range for said one or more than one first protein marker, or (ii) at least the presence or absence of a decrease in the level of said one or more than one second protein marker in said sample based on a comparison of said quantifying data for said one or more than one second protein marker to a control or standard range for said one or more than one second protein marker, or (iii) both (i) and (ii); and c) assigning the patient as having or not having CAA based on the result of said generating, wherein an increase in the level of said one or more than one first protein marker in the sample from the patient relative to the control or standard range indicates that the patient has CAA and / or wherein a decrease in the level of said one or more than one second protein marker in the sample from the patient relative to the control or standard range indicates that the patient has CAA.

[0038] According to another embodiment, the analytical method is directed to detecting the presence and quantity of a protein marker in a patient sample, comprising the steps of: providing at least one sample from a patient having cognitive impairment; and performing a quantitative assay on the at least one sample using either a mass spectrometer or a biological reagent that reacts specifically with one or more than one protein marker in said sample, thereby generating quantifying data for said one or more than one protein marker; and wherein the protein marker is one or more than one first protein marker identified in Table 1 as being elevated in individuals having CAA, the protein marker is one or more than one second protein marker identified in Table 2 as being decreased in individuals having CAA, or both.

[0039] The diagnostic methods of the disclosure are minimally invasive and are indicative of CAA. The diagnostic methods allow for the detection of CAA at earlier stages of the disease and, therefore, allow for earlier intervention for treatment of CAA. Translation of the disclosed methods into a clinical assay compatible with current clinical chemistry laboratory hardware is commercially acceptable and effective. Furthermore, the methods disclosed herein do not require highly trained personnel to perform and interpret the test.

[0040] The biological samples may originate from anywhere within the body, but preferably are blood (serum / plasma) or cerebral spinal fluid (“CSF”). Solid tissue samples, for example, those obtained during pre-surgical biopsy or following surgical resection of tissue can also be used. While the term “serum” is used herein, those skilled in the art will recognize that plasma or whole blood or a sub-fraction of whole blood may be used.

[0041] When a blood sample is drawn from a patient there are several ways in which the sample can be processed. The range of processing can be as little as none (i.e. frozen whole blood) or isolation of fractions from the whole blood, such as serum or plasma. The most common and routine procedures involve the preparation of either serum or plasma from whole blood. All blood sample processing methods, including spotting of blood samples onto solidphase supports, such as filter paper or other immobile substrate materials, are also contemplated by this disclosure.

[0042] The processed blood sample described above may be further processed to make it compatible with the methodical analysis technique to be employed in the detection and measurement of the first and / or second protein markers within the processed sample. The types of processing can range from as little as minimal processing (i.e., separating plasma from blood) to precipitation and / or purification of proteins prior to detection.

[0043] Protein solutions are generally dilute when they undergo analysis. Thus, it may be desirable to perform an enrichment process, such as concentration or precipitation. The traditional techniques of salting out and heat denaturation have the advantage of being very simple. In addition, the precipitated proteins are very stable, meaning that this process may be used as a means of increasing the shelf life of a protein. Salting out is a method of lowering the solubility of proteins through competing solubility in water using salts that are more soluble in water, such as ammonium sulfate. Since proteins precipitate at a specific concentration of salt, this procedure also has the advantage that the desired proteins can be separated from other proteins and precipitated.

[0044] Instead of using salts, it may be desirable to use isoelectric precipitation by lowering the pH. Isoelectric precipitation can be performed when the pH reaches at isoelectric point (pl) of the target protein. Each protein has a different pl value, meaning that isoelectric precipitation can be used as a fractionation method as well. Generally, this method is also very simple since a mineral acid or trichloroacetic acid is titrated until the target protein is obtained through precipitation. When changing the pH or salting out is not preferable, polymers such as polyethylene glycol or organic solvents such as methanol or acetone can be used to promote precipitation. If required, a cocktail of precipitating reagents, e.g., a mixture of acetone and trichloroacetic acid, can be developed.

[0045] However, the isolation technique should be chosen with the method of analysis in mind, as the isolation step may cause denaturation of the protein or require additional steps such as salt removal. The centrifugal membrane concentration method is a useful single-step method of concentrating a protein. However, it is important to keep in mind that this method may exhibitproblems involving clogging when used with highly concentrated proteins, and such problems decrease the efficiency of the concentration procedure and increase the loss of the protein.

[0046] After enrichment, purification is often desirable. Proteins for analytic experiments should be pure enough to have a high signal -to-noise ratio. Methods for purifying target proteins from dirty mixtures vary widely, but preparation-grade purification is most commonly achieved using chromatography (Burgess et al., Methods in Enzymology: Guide to Protein Purification, 2nd ed., Vol. 436, San Diego: Academic Press (2009); Janson, Protein Purification: Principles, High Resolution Methods, and Applications, Hoboken: John Wiley & Sons (2011), each of which is hereby incorporated by reference in its entirety). The type of chromatography to be used depends entirely on the physical and chemical properties of the target protein. Proteins are usually purified by high-performance liquid chromatography (HP-LC) for quantitative analysis. The LC steps can be carried out using one or more of hydrophobic interaction column chromatography, size exclusion chromatography, ion exchange column chromatography, and affinity chromatography.

[0047] Electrophoresis may also be used to separate proteins according to their molecular size. If the molecular weight of the target protein is known, one or more approximate band positions on the gel can be cut and be used for further analysis like mass spectrometry. In general, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) provides 1- dimensional (ID) results when analyzing proteins by size. Instead of directly loading the protein sample when performing SDS-PAGE, it is possible to perform 2-dimensional (2D) electrophoresis by performing isoelectric focusing and loading the resultant gel tube with proteins separated according to their pl values. Gels developed in 2D have much better resolution than gels developed in ID, making this a favorite technique in proteomics.

[0048] As is well known in the art, for certain immuno-detection procedures, purification is not required because complex protein mixtures can be analyzed to determine the presence or absence of one or more of the protein markers identified herein.

[0049] Where mass spectrometry (“MS”) is used for protein identification, then protein bands must first be excised from the gel and destained, and then the proteins in excised gel plugs are reduced, alkylated and digested. The peptides are then extracted from the gel matrix and prepared for MS analysis.

[0050] The decision to perform in-solution or in-gel protein digestion depends on a number of factors, including the sample amount and complexity. In-solution digestion is useful when the sample amount is small because peptide extraction from the gel matrix after in-gel digestion can result in significant peptide loss. In-solution digestion is also good for samples with low-to-moderate complexity and when detergents would negatively affect the sample. The benefit of ingel digestion is that SDS-PAGE combines protein denaturation with separation, which visually indicates the relative abundance of proteins in the sample. Also, peptide extraction inherently removes much of the detergents and salts, although peptide recovery is affected. Regarding processing time, in-solution digestion can be performed more rapidly because SDS-PAGE is not required. It also has greater high-throughput potential because of automation of the entire process (although automated systems have also been developed for in-gel digestion and extraction).

[0051] Purified peptide samples are then ready for the final preparation for MS analysis, which varies based on the type of analysis. For LC-MS or LC-MS / MS analysis, the correct choice of mobile phases and ion-pairing reagents is well known to achieve good LC resolution and analytical results.

[0052] One embodiment of the disclosed invention detects and measures one or more protein markers that were found to have statistically significantly differential abundances in samples obtained from CAA patients when compared to normal, non-CAA patients. These include protein markers that are elevated in CAA patient samples and / or protein markers that are reduced in CAA patient samples.Table 1. Proteins Enriched in CAA(+) VesselsEach of the above-identified Uniprot Accessions is hereby incorporated by reference in its entirety.Based on all / nothing. t Statistical calculations are reported in Supplemental Materials for Leitner et al., "Differences in the Cerebral Amyloid Angiopathy Proteome in Alzheimer's Disease and Mild Cognitive Impairment,” Acta Neuropathol 48(1 ):9 (2024), doi:10.1007 / s00401 -024-02767-1, which is hereby incorporated by reference in its entirety.Table 2. Proteins Depleted in CAA(+) VesselsEach of the above-identified Uniprot Accessions is hereby incorporated by reference in its entirety. t Statistical calculations are reported in Supplemental Materials for Leitner et al., "Differences in the Cerebral Amyloid Angiopathy Proteome in Alzheimer's Disease and Mild Cognitive Impairment,” Acta Neuropathol 48(1 ):9 (2024), doi:10.1007 / s00401 -024-02767-1 , which is hereby incorporated by reference in its entirety.

[0053] In certain embodiments, the one or more than one first protein markers excludes NDP, CLU, APOE, HTRA1, APCS, COL6A2 and COL6A3. See Hondius et al., “Proteomics Analysis Identifies New Markers Associated with Capillary Cerebral Amyloid Angiopathy in Alzheimer’s Disease,” Acta. NeuropathoL Commun. 6(1):46 (2018), which is hereby incorporated by reference in its entirety. In alternative embodiments, one or more of NDP, CLU, APOE, HTRA1, APCS, COL6A2 and COL6A3 can be used in combination with other first protein markers identified in Table 1.

[0054] Semaphorin 3G (Sema3G) is particularly preferred as one of the first protein markers, and is demonstrated in the accompanying examples to correlate with a diagnosis of CAA. Thus, in certain embodiments, detection of Sema3G alone or in combination with detection of A0 (such as Ap40 and / or Ap42) can be used to diagnose CAA. Due to the association of tau in plaque formations, detection of one or more of total-tau, pTau-181, or pTau-217 is also contemplated.

[0055] In certain other embodiments, the first protein marker is one that is associated with collagen-containing extracellular matrix including, without limitation, COL6A proteins such as COL6A1, COL6A2, and COL6A3; COL4A proteins such as COL4A1 and COL4A2; COL1 Aproteins such as COL1A1 and COL1A2; COL12 proteins such as COL 12A1; COL18 proteins such as COL18A1; and COL25 proteins such as COL25A1. These markers are demonstrated in the examples to be up-regulated in CAA patients. In certain embodiments, any one or more of these collagen markers can be detected in combination with Sema3G, or both Sema3G and Ap. By way of example, a regression analysis using data for the combination of COL6A2, SEMA3G, and A0 together (z.e., considering a value of 0 for those with no detected SEMA3G) results in an AUC 0.92 and p = 0.0020, indicating that this combination has a high predictive value.

[0056] In certain other embodiments, the first protein marker includes one or more of APP, APOE, SMOC1, SPON1, GPNMB, C4A, HTRA1, OLFML3, NRXN1, and combinations thereof. These markers are demonstrated to be up-regulated in CAA or AD patients. In certain embodiments, any one or more of these first protein markers can be detected in combination with Sema3G, or both Sema3G and A0 (such as Ap40 and / or Ap42).

[0057] In certain other embodiments, the first protein marker includes one or more of hFGF, hPLGF, hAng-2, hHB-EGF, hVEGF, hHGF, and combinations thereof. These markers are demonstrated to be up-regulated in CAA or AD patients. In certain embodiments, any one or more of these first protein markers can be detected in combination with Sema3G, or both Sema3G and AP (such as AP40 and / or AP42).

[0058] In certain embodiments, the second protein marker includes one or more proteins associated with disruption of the blood brain barrier. Exemplary proteins associated with disruption of the blood brain barrier include, OCLN, TJP1, and SLC2A1 (GLUT1), which are demonstrated in the examples to be down-regulated in CAA patients. In certain embodiments, any one or more of these down-regulated blood brain barrier markers can be detected in combination with Sema3G, or both Sema3G and Ap.

[0059] In certain embodiments, the second protein marker includes one or more proteins associated with ribonucleoprotein complex. Exemplary proteins associated with ribonucleoprotein complex include, among others, HNRNPA1, which is demonstrated in the examples to be down-regulated in CAA patients. In certain embodiments, any one or more of these down-regulated ribonucleoprotein complex markers can be detected in combination with Sema3G, or both Sema3G and Ap.

[0060] In certain embodiments, the second protein marker includes DNA2 or WIPF3, or a combination thereof. Both DNA2 and WIPF3 are demonstrated in the examples to be down- regulated in CAA patients having mild cognitive impairment. In certain embodiments, any one or both of these markers can be detected in combination with Sema3G, or both Sema3G and Ap.

[0061] The step of analyzing the sample may comprise analyzing the sample using a mass spectrometer (MS). MS is suitable for use in protein detection, identification and quantitation. The accuracy, sensitivity and flexibility of MS instruments have enabled new applications in biological research, biopharmaceutical characterization and diagnostic detection. With its many ionization and measurement options, MS can be used to analyze proteins and peptides ranging in mass from 50 to 300 kDa and in attomole to nanomole quantities.

[0062] MS is a well-established technology for analyzing for the presence and concentration (or amount) of a wide variety of chemical constituents with high sensitivity. One area of research and study for which mass spectrometry has proven to be particularly useful is the field of proteomics. Proteomics studies frequently take the form of simultaneous quantitative analysis of a plurality of target proteins through surrogate peptides generated by enzymatic digestion in biological samples. Such studies typically employ the technique of tandem mass spectrometry, often referred to as MS / MS or MS-2 mass spectrometry, in which particular peptide precursor ions are selected, isolated and fragmented (e.g., in a collision cell), and the resulting fragment (product) ions are mass analyzed in a mass analyzer. The mass analysis generates a record of the intensities of the various fragment ion species as a function of their mass-to-charge ratio (m / z) values. Calibration of the intensity scale, e.g., by comparison to internal standard compounds (synthetic isotopically labeled forms of surrogate peptides) introduced into the sample in known amounts, provides a measure of the concentration, within the sample, of the target protein from which the peptide ion species were generated. The MS-2 method can be extended by further fragmentation of selected and isolated fragment ion species, and so on, with possible mass analysis of the resulting fragments for each generation. Such extensions of the tandem mass spectrometry technique are typically referred to an MS-n spectrometry, with n indicating the number of steps of mass analysis and the number of generations of ions.

[0063] Exemplary mass spectrometers include, without limitation, the Fourier-Transform (FTMS), orbitrap, time of flight (TOF), and quadrupole types of mass spectrometers. Alternatively, the mass spectrometer could be equipped with an additional pre-detector mass filter. For example, and without wishing to be limiting such instruments are commonly referred to as quadrupole-FTMS (Q-FTMS), quadrupole -TOF (Q-TOF) or triple quadrupole (TQ or QQQ). Alternatively, the sample could be introduced into the mass spectrometer using a liquid or gas chromatographic system or by direct injection.

[0064] The extracted samples may be analyzed using any suitable method known in the art. For example, and without wishing to be limiting in any manner, extracts of biological samples are amenable to analysis on essentially any mass spectrometry platform, either by direct injectionor preferably following chromatographic separation. Typical mass spectrometers are comprised of a source which ionizes molecules within the sample, and a detector for detecting the ionized molecules or fragments of molecules. Non-limiting examples of common sources include electron impact, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photo ionization (APPI), matrix assisted laser desorption ionization (MALDI), surface enhanced laser desorption ionization (SELDI), and derivations thereof. Common mass separation and detection systems can include quadrupole, quadrupole ion trap, linear ion trap, time-of-flight (TOF), magnetic sector, ion cyclotron (FTMS), Orbitrap, and derivations and combinations thereof. The advantage of FTMS over other MS-based platforms is its high resolving capability that allows for the separation of fragments of molecules differing by only hundredths of a Dalton, many which would be missed by lower resolution instruments.

[0065] Data is collected during analysis and quantifying data for one or more than one protein marker is obtained. “Quantifying data” is obtained by measuring the levels or intensities of the peptide ion species that were generated, thus quantifying the one or more protein markers.

[0066] The quantifying data is compared to corresponding data from one or more than one reference sample. The “reference sample” is any suitable reference sample for non-CAA individuals. For example, and without wishing to be limiting in any manner, in the present invention the reference sample may be a sample from an age-matched or non-age-matched, non- CAA control individual, i.e., a person not suffering from CAA (also referred to herein as “a ‘normal’ counterpart”), which is a person exhibiting no signs of cognitive decline and / or negative for CAA by MRI or PET imaging. As would be understood by a person of skill in the art, more than one reference sample may be used for comparison to the quantifying data.

[0067] In another embodiment, the presence or absence of one or more first proteins and / or one or more than one second proteins can also be quantifiably detected using one or more label- free detection methods. Label-free biosensors directly measure the interaction of an analyte with a transducer (Samuel and Rao, “A Review on Label Free Biosensors,” Biosens Bioelectron A l l : 100216 (2022), which is hereby incorporated by reference in its entirety). The field of label-free optical biosensors includes surface plasmon resonance (Liedberg et al., “Surface Plasmon Resonance for Gas Detection and Biosensing,” Sens. Actuators 4:299- 304 (1983), which is hereby incorporated by reference in its entirety), Raman spectroscopy (Serebrennikova et al., Biosensors 11 (12): 512 (2021), which is hereby incorporated by reference in its entirety), integrated photonic devices (Ramirez et al., “Current Trends in Photonic Biosensors: Advances Towards Multiplexed Integration,” Chemosensors 10:398 (2022), which is hereby incorporated by reference in its entirety), and reflectometry (Fechner et al., “Through theLooking-glass — Recent Developments in Reflectometry Open New Possibilities for Biosensor Applications,” TrAC, Trends Anal. Chem. 156: 116708 (2022), which is hereby incorporated by reference in its entirety). Reflectometry is a group of techniques that measure the optical thickness of a functionalized thin film by illuminating the substrate and evaluating the properties of the reflected light. As light encounters a thin-film layer stack, reflections at the layer boundaries interfere with each other, encoding information about layer properties into the reflected light. Typically, biological binding events, such as a protein binding to its receptor or an antibody binding to its antigen, cause Angstrom-level increases in film thickness as bound and unbound probe sites are averaged by the optical measurement.

[0068] Reflectometric biosensors detect binding events by observing the characteristics of the reflected light, such as the polarization state, spectral interference patterns, or reflectivity at a fixed angle or wavelength. Ellipsometry measures change in the polarization of reflected light (Tompkins and Irene, Handbook of Ellipsometry , William Andrew Publishing, Norwich, NY (2005), which is hereby incorporated by reference in its entirety). Oblique-Incidence Reflectivity Difference (OI-RD) microscopy measures the reflectivity difference between p- polarized and s-polarized light (Zhu et al., “Oblique-incidence Reflectivity Difference Microscope for Label -free High-throughput Detection of Biochemical Reactions in a Microarray Format,” AppL Opt 46(10): 1890- 1895 (2007), which is hereby incorporated by reference in its entirety). Reflectometric Interference Spectroscopy (RIfS) and Biolayer Interferometry (BLI) illuminate a thin-film stack at normal or near-normal incidence and evaluate changes in the interference pattern of reflected light as a function of wavelength (Gauglitz and Nahm, “Observation of Spectral Interferences for the Determination of Volume and Surface Effects of Thin-films,” Fresenius ’ J. Anal. Chem 341 :279- 283 (1991); Do et al., “A Rapid Method for Determining Dynamic Binding Capacity of Resins for the Purification of Proteins,” Protein Expr. Purif 60(2): 147-150 (2008), each of which is hereby incorporated by reference in its entirety). Other techniques measure a change in the reflected light intensity at a single or a few discrete wavelengths at a fixed angle, including Spectral Reflectance Imaging Biosensor (SRIB / IRIS) (Ozkumur et al., “Label-free and Dynamic Detection of Biomolecular Interactions for High-throughput Microarray Applications,” roc. Natl. Acad. Sci. U.S.A 105(23):7988- 7992), which is hereby incorporated by reference in its entirety), Ik Reflectometry (Frank et al., “A Label -free Detection Method of Biochemical Interactions with Low-Cost Plastic and Other Transparent Transducers,” In Opto-Ireland 2005: Optical Sensing and Spectroscopy, SPIE, Bellingham, WA, Vol. 5826:551-560 (2005), which is hereby incorporated by reference in its entirety), and Arrayed Imaging Reflectometry (AIR) (Kosoy and Miller, “Two Decades ofArrayed Imaging Reflectometry for Sensitive, High-Throughput Biosensing,” Biosensors 13(9):870 (2023), which is hereby incorporated by reference in its entirety). AIR employs a single wavelength and angle of incidence (AO I) to measure analyte binding via increased reflectance from an antireflective condition. AIR is unique among reflectometry techniques because it leverages the steep drop in reflectivity under absolute antireflective conditions.

[0069] In these label-free sensing applications, an antibody or antigen-binding fragment thereof that binds specifically to one of the first or second protein markers is bound to a substrate. Multiple antibodies or antigen-binding fragments thereof can be bound to the substrate in an array. Upon binding of one or more of the first and / or second protein markers at a particular spot on the array, the binding event can be quantifiably detected where the signal output is related to the quantity of the first and / or second protein marker present in the sample. In this embodiment, antibody or antigen-binding fragments for any two or more of the first protein markers (from Table 1 above) and / or antibody or antigen-binding fragments for any two or more of the second protein markers (from Table 2 above) can be utilized in combination. Indeed, antibody or antigen-binding fragments for any three or more of the first protein markers (from Table 1 above) and / or antibody or antigen-binding fragments for any three or more of the second protein markers (from Table 2 above) can be utilized in combination.

[0070] With multiple antibodies or antigen-binding fragment thereof bound to a substrate used for such label-free sensing platforms, the antibodies or antigen-binding fragments are assigned to one or more specific locations in an array on the substrate. Different concentrations of the same antibody can be used at different locations on the array. This allows for discrimination of binding events with the first and / or second protein markers.

[0071] In another embodiment, the presence or absence of one or more than one first proteins and / or one or more than one second proteins can also be quantifiably detected using any of a variety of immunological detection assays such as Western blot, immunoblot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, and immunohistochemical analysis.

[0072] In one exemplary ELISA format, a sandwich immunoassay is carried using two antibodies, which bind to different sites on the antigen or ligand (i.e., any of the first protein markers or any of the second protein markers). The capture antibody, which is highly specific for the antigen, is attached to a solid surface. The sample (or diluted sample) is then added, followed by addition of a detection antibody. The detection antibody binds the antigen at a different epitope than the capture antibody. As a result, the antigen is ‘sandwiched’ between the two antibodies. The antibody binding affinity for the antigen is usually the main determinant ofimmunoassay sensitivity. As the antigen concentration increases, the amount of detection antibody increases, leading to a higher measured response. The standard curve of a sandwichbinding assay has a positive slope. To quantify the extent of binding, different reporters can be used. These reporters (i.e. enzyme, fluorophore, or biotin) can be directly attached to the detection antibody or to a secondary antibody which binds the detection antibody (i.e. goat, antimouse IgG - HRP). In this latter case, the capture antibody and the detection antibody must be from different species (i.e. if the capture antibody is a rabbit antibody, the detection antibody would be from goat, chicken, etc., but not rabbit). If the detection antibody is directly labeled, then the capture and detection antibodies can be from the same species. Polyclonal antibodies often contain multiple epitopes and the same affinity purified polyclonal can be used as the capture and labeled detection antibody. The substrate for the enzyme is added to the reaction that forms a colorimetric readout as the detection signal. The signal generated is proportional to the amount of target antigen present in the sample.

[0073] In another format, a single antibody ELISA format can be used when there is only one antibody available that recognizes the analyte. The assay is configured using the same antibody as both capture and detection. The antibody is biotinylated for use as the detection antibody. The method also utilizes two plates. The basic concept is to capture the antibody to an ELISA plate and allow the analyte of interest to bind to the capture antibody. Unbound material is removed by washing the plate and then adding an acid solution to elute the analyte from the capture antibody. The eluted analyte is then transferred to another ELISA plate containing the neutralization solution. The eluted analyte is then allowed to bind to the second ELISA plate. The unbound material is removed and the plate is blocked followed by a wash step. The detection antibody, which is biotinylated, is then added to the plate, followed by an incubation period. Another wash step is performed to remove excess detection antibody, followed by addition of a streptavidin reporter. The last wash step is performed to remove the excess reporter, followed by addition of the substrate.

[0074] Many antibodies suitable for detecting the proteins in Tables 1 and 2 are commercially available. In some circumstances, commercial antibodies may be polyclonal antiserum or monoclonal antibodies, and these antibodies can be specific for human proteins or capable of recognizing proteins from multiple species. Of these, monoclonal antibodies are preferred, and monoclonal antibodies specific for the human proteins are most preferred.

[0075] For proteins in Tables 1 and 2 where commercial antibodies are not available, commercial services can be used to prepare those antibodies or antibodies can be generated using routine procedures.

[0076] Monoclonal antibody production may be carried out by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. A practical methodology of fusing such cells is set forth in Kohler et al., Nature 256:495 (1975), which is hereby incorporated by reference in its entirety.

[0077] Mammalian lymphocytes are formed by in vivo immunization of the animal (e.g., a mouse) with the desired protein or polypeptide. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.

[0078] Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol (“PEG”) or other fusing agents (Milstein et al., Eur. J. Immunol. 6:511 (1976), which is hereby incorporated by reference in its entirety). This immortal cell line, which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats, rabbits, and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.

[0079] Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering a target protein or polypeptide subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 pl per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. This and other procedures for raising polyclonalantibodies are disclosed in Harlow et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby incorporated by reference in its entirety.

[0080] In addition to utilizing whole antibodies, the present invention may employ the use of antigen binding portions of such antibodies. Such binding portions include Fab fragments, F(ab’)2 fragments, and Fv fragments. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, Monoclonal Antibodies: Principles and Practice , pp. 98-118 (N.Y. Academic Press, 1983), which is hereby incorporated by reference in its entirety. Single chain antibodies (scFv) are also contemplated (Bird et al, “Single-Chain Antigen-Binding Proteins,” Science 242:423-426 (1988), which is hereby incorporated by reference in its entirety) or VHH camelid antibodies.

[0081] Regardless of the source of antibodies used, the above-described assay formats are compatible for use with multi-well, high throughput assays.

[0082] Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.

[0083] In coating a substrate with one or more of the first or second protein markers described above, or antibody that selectively binds to the same, one can generally incubate the wells of the plate with a solution of the protein or antibody, either overnight or for a specified period of hours. In certain aspects, the plate can be blocked using any suitable blocking buffer (e.g., 1% BSA, TBS, 0.1% Tween-20). The wells of the plate can then be washed to remove incompletely adsorbed material using any suitable wash solution (e.g., TBS, 0.1% Tween-20). Any remaining available surfaces of the substrate can then be coated with a non-specific protein that is antigenically neutral with regard to the antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of detection antibodies onto the surface.

[0084] Under conditions effective to allow immune complex (antigen / antibody) formation means that the conditions can include diluting the antigens and / or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween. These added agents can assist in the reduction of nonspecific background.

[0085] The suitable conditions can also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps can be from about 1 to about 2 hours, or up to about 4 to about 6 hours, or even up to about 24 hours or even about 48 hours, at temperatures on the order of about 20°C to about 37°C. In some embodiments, about21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or about 37°C, or may be overnight at about 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or so.

[0086] Following incubation steps in an ELISA, the contacted surface can be washed so as to remove non-complexed material. In some embodiments, the washing procedure includes washing with a solution such as PBS / Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

[0087] Detection may utilize an enzyme that can generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one can desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody or ligand for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 hours at room temperature in a PBS- containing solution such as PBS-Tween).

[0088] After incubation with a labeled antibody, and subsequent to washing to remove unbound material, the amount of label can be quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label.

[0089] Quantification can be achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.

[0090] As demonstrated in the accompanying examples, there is a diagnostic relationship between an overexpression of certain first protein markers in CAA patients as well as a reduced expression of certain second protein markers in CAA patients. Since patients with CAA often develop vascular dementia, Alzheimer’s disease, Parkinson’s disease with dementia, or Lewy body disease, the disclosed diagnostic methods provide a method of diagnosing an elevated risk of developing dementia by measuring the levels of one or more than one first protein markers and / or one or more than one second protein markers, as identified above, in a serum sample taken from a subject of unknown disease status and comparing the levels to “normal” or age- related deficiency reference levels and through this comparison arriving at a determination of elevated risk (or not). The samples and the diagnostic methods according to this aspect of the invention are as described in detail above.

[0091] It is contemplated that the diagnostic methods disclosed herein can be used in combination with other minimally invasive approaches for diagnosing CAA. For example, MRIor PET scans can be used for imaging the brain of a patient to assess the presence of cortical hemorrhagic lesions including lobar intracerebral hemorrhage (ICH), cortical cerebral microbleeds (CMBs) and cortical superficial siderosis (cSS). As reported by Knudsen, the presence of these cortical hemorrhagic lesions without the presence of deeper hemorrhages in patients over 55 years of age is highly sensitive and specific for CAA and can be detected via MRI (Knudsen et al., “Clinical Diagnosis of Cerebral Amyloid Angiopathy: Validation of the Boston Criteria,” Neurology 56:537-539 (2001), which is hereby incorporated by reference in its entirety). As reviewed by Charidimou, amyloid-PET appears to have moderate to good diagnostic accuracy in differentiating patients with probable CAA from cognitively normal healthy controls or patients with deep ICH (Charidimou et al., “Amyloid-PET in Sporadic Cerebral Amyloid Angiopathy: A Diagnostic Accuracy Meta-analysis,” Neurology 89(14): 1490- 1498 (2017), which is hereby incorporated by reference in its entirety). See also Tsai et al., “Superficial Cerebellar Microbleeds and Cerebral Amyloid Angiopathy: A Magnetic Resonance Imaging / Positron Emission Tomography Study,” Stroke 51(l):202-208 (2020), which is hereby incorporated by reference in its entirety.

[0092] A further aspect of the present disclosure relates to a method for diagnosing and treating a patient having cognitive impairment with CAA. Having diagnosed a patient as having CAA using the procedures described above, the administering of one or more therapeutic agents to the patient under conditions effective to treat the CAA is contemplated. Treatments for CAA include both lifestyle choices and therapeutic intervention designed to reducing the risk of firsttime or recurrent lobar intracerebral hemorrhages (“ICH”) as well as smaller regions of bleeding including cerebral microbleeds (“CMBs”), management of hypertension, management of CAA- related inflammation, slowing vascular deposition of A0 and / or removal of insoluble A0 from vasculature.

[0093] Lifestyle choices typically involve adopting healthy dietary regimen based on fruits, vegetables, and whole grains, and limiting intake of sugar and saturated fats as well as alcohol; maintaining proper hydration; getting sufficient sleep; adopting or maintaining an exercise regimen; and engaging in new and mentally stimulating activities.

[0094] One treatment involves management of hypertension using both diet and lifestyle, as well as therapeutic agents selected from angiotensin-converting enzyme (ACE) inhibitors, alphablockers, beta-blockers, calcium channel blockers, central agonists, angiotensin II receptor blockers, and diuretics.

[0095] Exemplary ACE inhibitors include, without limitation, benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandopril.

[0096] Exemplary alpha-blockers include, without limitation, tamsulosin, silodosin, alfuzosin, doxazosin, and terazosin.

[0097] Exemplary beta-blockers (selective or non-selective) include, without limitation, metoprolol, carvedilol, atenolol, propranolol, labetalol, bisoprolol, timolol, nadolol, betaxolol, nebivolol, penbutolol, and acebutolol.

[0098] Exemplary calcium channel blockers include, without limitation, amlodipine, diltiazem, verapamil, nifedipine, felodipine, nisoldipine, nimodipine, isradipine, nicardipine, levamlodipine, and clevidipine.

[0099] Exemplary central agonists include, without limitation, clonidine, methyldopa, guanfacine, guanabenz, lofexidine, aldomet, lucemyra, catapres, kapvay, and nexiclon.

[0100] Exemplary angiotensin II receptor blockers include, without limitation, azilsartan, candesartan, irbesartan, losartan, olmesartan, valsartan, telmisartan, and eprosartan.

[0101] Exemplary diuretics include, without limitation, bendroflumethiazide, chlorothiazide, chlorthalidone, hydrochlorothiazide, indapamide, and metolazone.

[0102] Another treatment involves lipid management using both diet and lifestyle, as well as therapeutic agents designed to reduce the risk of ischemic stroke. Typically, this involves administration of statins. Exemplary statins include, without limitation, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

[0103] Another treatment involves glucose management using both diet and lifestyle, as well as therapeutic agents designed to reduce the risk of intracerebral hemorrhage. Both low (<4 mmol / L) and high (>7 mmol / L) fasting blood glucose levels are associated with increased ICH risk. Examples of glucose management drugs include, without limitation, insulin and insulin derivatives, insulin sensitizers such as biguanides (e.g., metformin) and thiazolidinediones (e.g., rosiglitazone, pioglitazone), insulin release secretagogues such as sulfonylureas (e.g., glipizide, glyburide, glimepiride) and meglitinides (e.g., repaglinide, nateglinide), DPP-4 inhibitors (e.g., sitagliptin, linagliptin, saxagliptin), SGLT2 inhibitors (e.g., canagliflozin, dapagliflozin, empagliflozin), GLP-1 Receptor Agonists (e.g., liraglutide, exenatide, liraglutide, semaglutide), alpha-glucosidase inhibitors (e.g., acarbose, miglitol), amylin analogs (e.g., pramlintide), and non-sulfonylureas (metformin).

[0104] A subset of patients with CAA develop episodes of spontaneous inflammation. The most common presenting symptoms of CAA-related inflammation are headache, seizures, focal neurological deficits, and subacute to acute cognitive decline. MRI features typically include 1) asymmetric WMH which extend subcortically, 2) hemorrhagic lesions including CMB and cSS and / or 3) post-contrast leptomeningeal enhancement. CSF profiles are generally inflammatory,with a lymphocytic pleocytosis and elevated protein, but these findings are not always present. Interestingly, during acute episodes of CAA-RI anti-amyloid-P autoantibodies are found in the CSF, suggesting a spontaneous immune-mediated response to vascular amyloid-P in the brain. These imaging and clinical findings resemble amyloid-related imaging abnormalities (“ARIA”) in the context of anti-amyloid immunotherapy trials. The treatment of CAA-RI relies on intensive immunosuppression, predominantly with steroids (Regenhardt et al., “Association Between Immunosuppressive Treatment and Outcomes of Cerebral Amyloid Angiopathy- Related Inflammation,” JAMA Neurol. 1-9 (2020), which is hereby incorporated by reference in its entirety). A typical treatment course involves a high-dose intravenous steroid pulse followed by a prolonged steroid taper (at least six months). Some providers use cyclophosphamide in addition to steroids.

[0105] Another treatment involves disrupting the deposition of amyloid-P within vessels in brain and / or dissolving amyloid-P within vessel walls. These are achieved using antibody- mediated therapeutic agents that function to remove aggregated or soluble forms of amyloid-P . Examples of anti-Ap therapies include, without limitation, daratumumab, elotuzumab, donanemab, gantenerumab, lecanemab, and solanezumab. When anti-Ap therapies are administered, it is desirable to monitor, via MRI or PET scan, for development of ARIA, which is swelling in the patient’s brain that usually goes away on its own, but it may also include small spots of bleeding in the patient’s brain.

[0106] As alternatives to antibody-mediated therapies, alternative therapies include reducing amyloid-P production, and nonpharmacological strategies to enhance amyloid-P clearance through perivascular drainage pathways.

[0107] For all of the above-identified therapeutic or prophylactic interventions, it should be appreciated by the skilled person that the administration of active ingredients may be continuous or intermittent, and that the mode of administration may also be determined by the timing and frequency of administration, but is also dependent on the severity of the CAA condition at the time of diagnosis. Thus, active ingredients of the types described above may be administered at varying therapeutically effective doses to a CAA patient in need thereof. A medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient, depending on the severity of CAA and the route of administration. For authorized drug products, the preferred dosage or dosage range is identified in prescribing information for such drug products.

[0108] In any event, the dose administered to a patient, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the patient over areasonable timeframe (as described hereinbefore). One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the CAA being treated, and the physical condition and mental acuity of the patient, as well as the age, condition, body weight, sex and response of the patient to be treated, and the stage / severity of the CAA, as well as genetic differences between patients.

[0109] When using any of the above-identified therapies, in addition to monitoring progress via MRI or PET scan imaging techniques, it may also be desirable to continue monitoring patient samples for the one or more than one first protein markers identified above and / or one or more than one of the second protein markers identified above (see Tables 1 and 2). In addition, it may also be desirable to monitor one or more than one third protein marker associated with neuroinflammation or neuronal damage. Exemplary third protein markers include, without limitation, neurofilament light chain protein (NfL), glial fibrillary acidic protein (GFAP), interferon gamma (IFN-y), interleukins- 1, -4, -5, -6, -8, -10, -12, and -22 (IL-1, IL-4, IL-5-IL-6, IL-8, IL-10, IL-12, and IL-22), p70 (IL-2-R-beta), and tumor necrosis factor-alpha (TNFa). If inflammation is elevated, then it is proper to adjust the dose or schedule for therapeutic agents that can promote inflammation, such as amyloid-targeting therapeutic agents. Such adjustments may include administering a different (lower) dose, decreasing the frequency of administration, as well as discontinuing administration of the therapeutic agent.

[0110] As indicated above and demonstrated in the accompanying Examples, Sema3G levels are shown to be elevated in CAA patient populations. Thus, another aspect of the invention relates to monitoring longitudinal biofluid Sema3G levels over the course of time, which may inform on different treatment options in patients with risk for developing ARIA and intracranial hemorrhage. Increased Sema3G biofluid levels may be an indication for reducing anti -amyloid immunotherapy dose particularly in patients with severe CAA, reduction or alteration of long term anti-coagulation therapies in patients with history of atrial fibrillation with or without dementia, and may aide in selecting and matching appropriate patients for clinical trials for example that may include CAA-specific therapies described below. In addition to an association with CAA severity and ARIA, high Sema3G biofluid levels may indicate increased risk for cerebral hemorrhage in patients receiving anti-coagulation therapy after atrial fibrillation (AF). Anti -coagulation therapy may reduce risk of stroke but includes risk of bleeding, i.e. intracranial hemorrhage (Lin et al., “Post-Intracranial Hemorrhage Antithrombotic Therapy in Patients With Atrial Fibrillation,” J Am Heart Assoc. 1 l(6):e022849 (2022); Thiankhaw et al., “Left Atrial Appendage Occlusion in Patients with Atrial Fibrillation and Intracerebral HaemorrhageAssociated with Cerebral Amyloid Angiopathy: A Multicentre Observational Study and Pooled Analysis of Published Studies,” J Neurol Neurosurg Psychiatry 96(6):528-536 (2025), each of which is hereby incorporated by reference in its entirety). Approximately 5% of elderly individuals develop AF (hence it is very common); AF is most commonly treated with long term anti coagulation. Among individuals with CAA, this anti coagulation carries the significant risk of intracerebral hemorrhage that can lead to a stroke, vascular dementia and / or death. Currently, there are no biofluid biomarkers for CAA. Such biomarkers could help identify subjects who are at highest risk of ICH with anti coagulation for AF, and thereby allow for alternative therapeutic interventions to be used with this high risk population. Alternative therapeutic approaches for this population includes ablation or a Watchman device, both of which would avoid the need for long term anti-coagulation.[oni] CAA specific markers and signaling pathways identified in Tables 1 and 2 as well as the accompanying examples (see also Leitner et al., “Differences in the Cerebral Amyloid Angiopathy Proteome in Alzheimer's Disease and Mild Cognitive Impairment,” Acta Neuropathol .148(1):9 (2024), doi: 10.1007 / s00401-024-02767-l, which is hereby incorporated by reference in its entirety), including Sema3G and the related signaling pathways (Tan et al., “Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2 / PlexinA4,” Neuron 101(5):920-37 (2019); Mohammed et al., “Targeting SETD7 Rescues Diabetes-induced Impairment of Angiogenic Response by Transcriptional Repression of Semaphorin 3G,” Diabetes 74(6):969-982 (2025); Liu et al., “Mechanism of SEMA3G Knockdown-mediated Attenuation of High-fat Diet-induced Obesity,” J Endocrinol. 244(l):223-36 (2020) Luo et al., “Sema3G Activates YAP and Promotes VSMCs Proliferation and Migration via Nrp2 / PlexinAl,” Cell Signal. 105: 110613 (2023); Taniguchi et al., “Identification and Characterization of a Novel Member of Murine Semaphorin Family,” Genes Cells 10(8):785-92 (2005); Shoda et al., “Semaphorin 3G Exacerbates Joint Inflammation Through the Accumulation and Proliferation of Macrophages in the Synovium,” Arthritis Res Ther. 24(1): 134 (2022), each of which is hereby incorporated by reference in its entirety), inform on novel therapeutic targets for CAA. Increased Sema3G for example is associated with memory and plasticity in a young mouse model (Tan et al., “Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2 / PlexinA4,” Neuron 101 (5):920-37 (2019), which is hereby incorporated by reference in its entirety), increased macrophage migration and inflammation related to Sema3G receptor expression on macrophages in joint synovium from patients with rheumatoid arthritis and in an animal model (Shoda et al., “Semaphorin 3G Exacerbates Joint Inflammation Through the Accumulation and Proliferation ofMacrophages in the Synovium,” Arthritis Res Ther. 24(1): 134 (2022), which is hereby incorporated by reference in its entirety), increased in obesity and diabetes (Liu et al., “Mechanism of SEMA3G Knockdown-mediated Attenuation of High-fat Diet-induced Obesity,” J Endocrinol. 244(l):223-36 (2020); Luo et al., “Sema3G Activates YAP and Promotes VSMCs Proliferation and Migration via Nrp2 / PlexinAl,” Cell Signal. 105: 110613 (2023), each of which is hereby incorporated by reference in its entirety), knockdown in animal models decreases weight gain and diabetes measures (Liu et al., “Mechanism of SEMA3G Knockdown-mediated Attenuation of High-fat Diet-induced Obesity,” J Endocrinol. 244(l):223-36 (2020), which is hereby incorporated by reference in its entirety), modulation of signaling pathway to decrease SEMA3G facilitates angiogenesis in a model of diabetes and peripheral artery disease (Mohammed et al., “Targeting SETD7 Rescues Diabetes-induced Impairment of Angiogenic Response by Transcriptional Repression of Semaphorin 3G,” Diabetes 74(6):969-982 (2025), which is hereby incorporated by reference in its entirety), modulation may impact cell migration and outcomes for tumor development (Zhou et al., “Effects of SEMA3G on Migration and Invasion of Glioma Cells,” Oncol Rep 28(l):269-75 (2012); Zhang et al., A Pan-cancer Study of Class-3 Semaphorins as Therapeutic Targets in Cancer,” BMC Med Genomics 13(Suppl 5):45 (2020); Yuan et al., “SEMA3G, Downregulated by ncRNAs, Correlates with Favorable Prognosis and Tumor Immune Infiltration in Kidney Renal Clear Cell Carcinoma,” Aging (Albany, NY) 15(23): 13944-60 (2023), each of which is hereby incorporated by reference in its entirety). Therefore, novel therapies targeting SEMA3G and / or other proteins identified herein, z.e., antisense oligonucleotides (ASOs) that knockout or at least reduce expression of proteins, etc., may reduce CAA pathology and related complications. In particular, it is contemplated that reduction in the expression of proteins that are elevated in CAA patients (see Table 1, infra) via antisense oligonucleotides can be used to disrupt expression thereof.Kits

[0112] Also disclosed are kits for carrying out the methods according to the present disclosure. Such kits include one or more of:(i) an antibody reagent, or antigen-binding fragment thereof, that binds specifically to one of the first protein markers; and / or(ii) an antibody reagent, or antigen-binding fragment thereof, that binds specifically to one of the second protein markers; and / or(iii) an antibody reagent, or antigen-binding fragment thereof, that binds specifically to one of the third protein markers; and / or(iv) protein extraction reagents for extraction of proteins from patient samples; and / or(v) an enzyme capable of cleaving the first and / or second protein markers; and / or(vi) a data acquisition template identifying polypeptide fragments detectable by the mass spectrometer for said first and / or second and / or third protein markers.

[0113] Where a panel of first protein markers (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) are intended for use together, then for each of the first protein markers, the kit includes at least one antibody reagent, or antigen-binding fragment thereof, that binds specifically to such first protein markers. In certain embodiments, such as for sandwich ELISA assays, then for each of the first protein markers, the kit includes at least two antibody reagents, or antigen-binding fragments thereof, that bind specifically to such first protein markers at different epitopes.

[0114] Where a panel of second protein markers (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) are intended for use together, then for each of the second protein markers, the kit includes at least one antibody reagent, or antigen-binding fragment thereof, that binds specifically to such second protein markers. In certain embodiments, such as for sandwich ELISA assays, then for each of the second protein markers, the kit includes at least two antibody reagents, or antigen-binding fragments thereof, that bind specifically to such second protein markers at different epitopes.

[0115] Where a panel of third protein markers (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) are intended for use together, then for each of the third protein markers, the kit includes at least one antibody reagent, or antigen-binding fragment thereof, that binds specifically to such third protein markers. In certain embodiments, such as for sandwich ELISA assays, then for each of the third protein markers, the kit includes at least two antibody reagents, or antigenbinding fragments thereof, that bind specifically to such third protein markers at different epitopes.

[0116] Where mass spectrometry-based detection protocols are employed, then it may be desirable to use at least two or more enzymes capable of cleaving the first and / or second protein markers to ensure that the peptide fragments are appropriately sized to optimize detection and quantification by mass spectrometry. The data acquisition template identifying polypeptide fragments detectable by the mass spectrometer for said first and / or second and / or third protein markers can be matched according to the predicted fragments to be analyzed.EXAMPLES

[0117] The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.Materials and MethodsBrain Tissue

[0118] Brain tissue was acquired under protocols with Institutional Review Board (IRB) approval at NYU Grossman School of Medicine and Rush University. Cases are a part of the Religious Orders Study (ROS) and Memory and Aging Project (MAP) cohorts (Bennett et al., “Religious Orders Study and Rush Memory and Aging Project,” J. Alzheimer s Dis. 64(sl): S 161— SI 89 (2018), which is hereby incorporated by reference in its entirety). Clinical assessment and neuropathology was performed at Rush University (Boyle et al., “Cerebral Amyloid Angiopathy and Cognitive Outcomes in Community-Based Older Persons,” Neurology 85: 1930-1936 (2015); Love et al., “Development, Appraisal, Validation and Implementation of a Consensus Protocol for the Assessment of Cerebral Amyloid Angiopathy in Post-Mortem Brain Tissue,” Am J. Neurodegener. Dis. 3(1): 19-32 (2014); Schneider et al., “Mixed Brain Pathologies Account for Most Dementia Cases in Community -Dwelling Older Persons,” Neurology 69(24):2197- 2204 (2007), each of which is hereby incorporated by reference in its entirety). Cases were stratified into Control, MCI, and advanced AD experimental groups using a combination of both clinical and neuropathological criteria. Cases were initially stratified by the clinical cognitive final consensus diagnosis that was generated by a neurologist with expertise in dementia by a review of all available cognitive data that was blinded to post-mortem data. Cases were excluded if they were designated to have a clinical diagnosis of AD and another cause of cognitive impairment. The following neuropathological inclusion criteria was then used to refine case selection in each group: neuropathological ABC score of A0-1 / B0-2 / C0-1 for Control, A2- 3 / B1-2 / C2-3 for MCI, and A3 / B3 / C3 for AD. Cases were prioritized to exclude those with high TDP-43 and Lewy body pathology. Formalin-fixed paraffin-embedded (FFPE) sections of small samples from the inferior temporal cortex were then obtained from Control (n = 62), MCI (n = 74), and AD cases (n = 56), which were then neuropathologically assessed to identify cases with sufficient CAA(+) vessels for localized proteomics analysis. Of these cases, n = 4 MCI and n = 6 AD had sufficient CAA(+) vessel area (2 mm2) for dissection in the inferior temporal cortex and were therefore selected for analysis. Control cases (n = 10) were included for comparison to CAA(-) vessels, and were age-matched to the other groups (p = 0.11, one-way ANOVA). Sample size was considered sufficient as in prior studies (Drummond et al., “Proteomic Differences in Amyloid Plaques in Rapidly Progressive and Sporadic Alzheimer’s Disease,” Acta Neuropathol. 133(6):933— 954 (2017); Drummond et al., “Phosphorylated Tau Interactome in the Human Alzheimer’s Disease Brain,” Brain 143(9):2803-2817 (2020); Hsieh et al., “Tau-Mediated Disruption of the Spliceosome Triggers Cryptic RNA Splicing andNeurodegeneration in Alzheimer’s Disease,” Cell Rep. 29(2):301-316.e310 (2019); Leitner et al., “Pilot Study Evaluating Everolimus Molecular Mechanisms in Tuberous Sclerosis Complex and Focal Cortical Dysplasia,” PLoS ONE 17(5):e0268597 (2022); Thierry et al., “The Influence of APOe4on the pTau Interactome in Sporadic Alzheimer's Disease,” Acta Neuropathol. 147(1 ):91 (2024), each of which is hereby incorporated by reference in its entirety). Case history is summarized in Table 3 below.Table 3. Case HistoryFurther details regarding case history is supplied in Supplemental Table 1 from Leitner et al., “Differences in the Cerebral Amyloid Angiopathy Proteome in Alzheimer's Disease and Mild Cognitive Impairment,” Acta Neuropathol.148(V) 9 (2024), doi: 10.1007 / s00401-024-02767-l, which is hereby incorporated by reference in its entirety.Screening Immunohistochemistry (IHC)

[0119] To identify cases with sufficient CAA(+) vessels for localized proteomics studies, one standard glass slide per case was sectioned from FFPE tissue by the NYU Center for Biospecimen Research and Development Core. Briefly, 8 pm sections were deparaffinized and rehydrated through a series of xylene and ethanol washes, followed by antigen retrieval with 88% formic acid, and 10 mM sodium citrate with 0.05% tween at pH 6. Sections were blockedin 10% normal horse serum for 1 hour, and incubated in a combination of 4G8 (1 : 1000, BioLegend #800711) and 6E10 (1 : 1000, BioLegend #803017) primary antibodies targeting Ap overnight at 4°C. After washing, sections were incubated in biotinylated mouse secondary antibody (1 : 1000, Vector Laboratories) for 1 hour, followed by avidin-biotin peroxidase (Vector Laboratories) for 1 hour. After washing, sections were incubated with diaminobenzidine (DAB) chromogen solution (ThermoFisher). After additional washes, sections were cover-slipped. A regional CAA score (0-5) and semi quantitative frequency of intravascular and perivascular score (+, ++, +++) was given for each slide to indicate relative level of pathology, excluding leptomeninges. Cases were included with intravascular and / or perivascular scores of +++ for MCI and AD cases, as well as microscopic confirmation of adequate CAA(+) vessels present.Laser Capture Microdissection (LCM)

[0120] FFPE tissue was cut into 8 pm sections onto LCM PET membrane slides (Drummond et al., “Isolation of Amyloid Plaques and Neurofibrillary Tangles from Archived Alzheimer’s Disease Tissue Using Laser-Capture Microdissection for Downstream Proteomics,” Methods Mol. Biol. 1723:319-334 (2018); Leitner et al., “Proteomics and Transcriptomics of the Hippocampus and Cortex in SUDEP and High-Risk SUDEP Patients,” Neurology 96(21):e2639- e2652 (2021); Leitner et al., “Proteomic Differences in Hippocampus and Cortex of Sudden Unexplained Death in Childhood,” Acta Neuropathol. 143(5):585-599 (2022); Pires et al., “Proteomic Differences in the Hippocampus and Cortex of Epilepsy Brain Tissue,” Brain Commun. 3(2):fcab021 (2021), each of which is hereby incorporated by reference in its entirety) by the NYU Experimental Pathology Core. CAA(+) (Ap positive) vessels and CAA(-) (Ap negative; non-CAA) vessels were visualized by IHC with 4G8 antibody followed by DAB chromogen counterstained with hematoxylin for all cases. Briefly, sections were deparaffinized and rehydrated through a series of xylenes and ethanol washes, followed by incubation in 0.3% H2O2 for 20 minutes, blocking in 10% normal goat serum for 1 hour, and incubated with 4G8 primary antibody (1 : 1000, BioLegend #800711) overnight at 4°C. After washing, sections were incubated in biotinylated mouse secondary antibody (1 : 1000, Vector Laboratories) for 1 hour, followed by avidin-biotin peroxidase (Vector Laboratories) for 1 hour. After washing, sections were incubated with DAB solution (ThermoFisher) for 10 minutes. Finally, sections were counterstained with hematoxylin (Sigma #MHS16) and air dried overnight in a loosely closed container.

[0121] Blood vessels with a lumen and vessel wall in gray matter were microdissected at a consistent area per case of 2 mm2into mass spectrometry (MS) grade water (Thermo Scientific)from n = 10 Control [CAA(-) vessels], n = 4 MCI [CAA(+) vessels and CAA(-) vessels collected separately], and n = 6 AD [CAA(+) vessels and CAA(-) vessels collected separately] cases. Both larger vessels and capillaries were included. Vessels in the leptomeninges and white matter were excluded. Microdissected samples were centrifuged for 2 min at 14,000 g and stored at - 80°C. LCM was performed at 10x magnification with a Leica LMD6500 microscope equipped with a UV laser. Schematic overview depicts workflow, partially generated in Biorender (FIG. 1).Label-Free Quantitative Mass Spectrometry (LFQ-MS)

[0122] Tissue samples were solubilized and digested using the modified SPEED sample prep workflow (Doellinger et al., “Sample Preparation by Easy Extraction and Digestion (SPEED) — A Universal, Rapid, and Detergent-Free Protocol for Proteomics Based on Acid Extraction,” Mol. Cell Proteom. 19(l):209-222 (2020), which is hereby incorporated by reference in its entirety) by the NYU Proteomics Laboratory. Proteins were extracted in 10 pl of formic acid (FA) for 5 minutes at 73 °C. FA was then neutralized with 90 pl of 2 M TRIS containing 10 mM TCEP and 20 mM chloroacetamide (CAA), and samples were incubated at 90°C for 30 minutes. For enzymatic digestion, samples were diluted 6* (v:v) with water containing 0.2 pg of sequencing-grade trypsin (Promega) and digested at 37°C overnight. Digestion was halted with acidification to 2% of trifluoroacetic acid (TFA). Peptides were loaded on Evosep Pure C18 tips and separated on Evosep One LC system using 88 min ACN gradient (SPD15 method) on the analytical column packed with Dr Maisch C18 AQ 1.9 pm C18 beads (150 pm ID, 15 cm length, #EV-1106). Peptides were identified and quantified in data-independent (DIA) acquisition mode on a QExactive HF-X mass spectrometer (ThermoFisher). High-resolution full MS spectra were acquired with a resolution of 120,000, an AGC target of 3e6, a maximum ion injection time of 60 ms, and a scan range of 350-1650 m / z. Following each full MS scan, 22 data-independent HCD MS / MS scans were acquired at the resolution of 30,000, AGC target of 3e6, and stepped NCE of 22.5, 25, and 27.5.

[0123] MS raw data were analyzed using Spectronaut software in directDIA (library -free) pipeline against the Homo sapiens UniProt reference database concatenated with a list of common lab contaminants. Database searches were performed in the integrated search engine Pulsar. Independent quantification of Ap was manually curated and included in search results, as in previous studies (Drummond et al., “Proteomic Differences in Amyloid Plaques in Rapidly Progressive and Sporadic Alzheimer’s Disease,” Acta NeuropathoL 133(6):933— 954 (2017); Seyfried et al., “A Multi-Network Approach Identifies Protein-Specific Co-Expression inAsymptomatic and Symptomatic Alzheimer’s Disease,” Cell Syst. 4(l):60-72.e64 (2017), each of which is hereby incorporated by reference in its entirety). Ap intensity was determined by integrating the area under the curve for peptide LVFFAEDVGSNK (Ap peptide, SEQ ID NO: 1). This peptide corresponds to amino acids 17-28 of Ap and does not discriminate from cleaved or full length sequences, but this peptide does show strong enrichment and correlation to Ap pathology (Drummond et al., “Proteomic Differences in Amyloid Plaques in Rapidly Progressive and Sporadic Alzheimer’s Disease,” Acta NeuropathoL 133(6):933-954 (2017); Handa et al., “Proteomics-Based Investigation of Cerebrovascular Molecular Mechanisms in Cerebral Amyloid Angiopathy by the FFPE-LMD-PCT-SWATH Method,” Fluids Barriers CNS 19(1): 56 (2022); Leitner et al., “Similar Brain Proteomic Signatures in Alzheimer’s Disease and Epilepsy,” Acta NeuropathoL 147(1):27 (2024); Seyfried et al., “A Multi-Network Approach Identifies Protein-Specific Co-Expression in Asymptomatic and Symptomatic Alzheimer’s Disease,” Cell Syst. 4(1): 60-72. e64 (2017), each of which is hereby incorporated by reference in its entirety). Data were log transformed and normalized using median intensity across all samples. Subsequent data analysis was performed in Perseus (Tyanova et al., “The Perseus Computational Platform for Comprehensive Analysis of (Prote)omics Data,” Nat. Methods 13(9):731-740 (2016), which is hereby incorporated by reference in its entirety), the R environment, or GraphPad Prism. Raw data is available on the MassIVE server under accession MSV000094400.

[0124] The protein expression matrix (n = 2188) was filtered to contain only proteins that were human, non-lab contaminant, and quantified in at least 50% of samples from at least one of the five groups (n = 2026). For principal component analysis (PCA), missing values were imputed from the normal distribution with a width of 0.3 and a downshift of 1.8 (relative to measured protein intensity distribution) in Perseus (Tyanova et al., “The Perseus Computational Platform for Comprehensive Analysis of (Prote)omics Data,” Nat. Methods 13(9):731-740 (2016), which is hereby incorporated by reference in its entirety). Multiple linear regression was performed to determine whether sample type or disease group contributed to differences in PCA1, in GraphPad Prism. Paired t tests were performed in Perseus v. 1.6.2.3 (Tyanova et al., “The Perseus Computational Platform for Comprehensive Analysis of (Prote)omics Data,” Nat. Methods 13(9):731-740 (2016), which is hereby incorporated by reference in its entirety) to detect significant changes in protein expression between CAA(+) and CAA(-) vessel samples in MCI and in AD. Unpaired t tests were performed in Perseus to detect significant changes in protein expression between CAA(-) vessel samples from Control cases compared to MCI or AD CAA(-) vessel samples. Significance was considered at < 0.05 and fold-change > 1.5. Cell-type annotations were derived from a previous vascular single cell RNAseq study (Winkler et al., “A Single-Cell Atlas of the Normal and Malformed Human Brain Vasculature,” Science 375(6584):eabi7377 (2022), which is hereby incorporated by reference in its entirety), which included an association of 13 cell-type annotations. Twelve cell-type annotations were detected in the current dataset, red blood cell proteins were not detected in the current study and duplicate gene IDs were excluded from annotations (303 annotations overlap with detected proteins). Cell-type enrichment analysis was performed among significantly altered proteins with a Fisher’s exact test in GraphPad Prism and the R environment. A comparison of proteins by disease group were evaluated by Venn diagram generated from InteractiVenn (Heberle et al., “InteractiVenn: A Web-Based Tool for the Analysis of Sets through Venn Diagrams,” BMC Bioinform. 16(1): 169 (2015), which is hereby incorporated by reference in its entirety). Correlation analyses were performed by Pearson correlation in GraphPad Prism. Unsupervised hierarchical clustering and heatmaps were evaluated in the R environment with ComplexHeatmap . Individual protein plots depict significance for pairwise comparisons performed.Gene Ontology (GO) Term Enrichment Analysis

[0125] GO cell component (GOCC) term over-representation analysis was performed in R v4.3.0 using the packages enrichplot vl.20.0, clusterProfiler 4.8.1, with the genome wide annotation for human, org.Hs.eg.db v3.17.0. GO over-representation analysis was performed on gene subsets, with increased and decreased proteins separated, in CAA(+) vessels in MCI or AD and for proteins altered in CAA(-) vessels of MCI vs Control and AD vs Control. Prior to analyses, gene IDs were mapped to Entrez IDs with the ‘bitr’ function of clusterProfiler . GO terms were assessed for enrichment via one-way Fisher’s exact test, and multiple comparisons correction was performed with Benjamini -Hochberg to obtain an adjusted p value. GO terms were filtered to include those at an adjusted p < 0.05. The full list of proteins detected across all samples was used as the background list (2026 proteins) as this is a highly targeted dataset. ‘Lollypop’ plots of the top 10 up and down annotations were generated in R with the packages ggplot2 v3.4.2 and ggpubr v0.6.0. Simplify function at 0.7 semantic similarity was used to reduce redundant terms in figures.Comparison with Previous Studies

[0126] Proteins that were significantly enriched or depleted in amyloid plaques in comparison to neighboring non-plaque tissue were sourced from published datasets that identified proteins significantly enriched / depleted in amyloid plaques in early onset AD andDown syndrome (Drummond et al., “Proteomic Differences in Amyloid Plaques in Rapidly Progressive and Sporadic Alzheimer’s Disease,” Acta NeuropathoL 133(6):933-954 (2017), which is hereby incorporated by reference in its entirety), and proteins significantly enriched / depl eted in amyloid plaques in sporadic AD and preclinical AD (Xiong et al., “Quantitative Proteomics Reveals Distinct Composition of Amyloid Plaques in Alzheimer’s Disease,” Alzheimers Dement. 15(3):429-440 (2019), which is hereby incorporated by reference in its entirety). Proteins were considered significantly enriched or depleted in plaques at < 0.05 (t test) and fold change > 1.5 between plaque and non-plaque tissue for (Drummond et al., “Proteomic Differences in Amyloid Plaques in Rapidly Progressive and Sporadic Alzheimer’s Disease,” Acta NeuropathoL 133(6):933-954 (2017), which is hereby incorporated by reference in its entirety) and a ratio > 1.5 between plaque and non-plaque tissue for (Xiong et al., “Quantitative Proteomics Reveals Distinct Composition of Amyloid Plaques in Alzheimer’s Disease,” Alzheimers Dement. 15(3):429-440 (2019), which is hereby incorporated by reference in its entirety). Remaining proteins that were detected, but not significant between groups were designated “present”. For this comparative analysis, proteins were designated as significantly enriched or depleted in CAA(+) vessels from proteins that were either significantly different between CAA(+) and CAA(-) vessels or exclusive identification in the majority of either CAA(+) or CAA(-) vessels within a disease group (z.e., > 3 samples for MCI or > 4 samples for AD) and 0 samples in the comparative blood vessel group. Remaining proteins that were detected, but not significant between groups were designated “present”. Proteins that were enriched in at least one CAA(+) vessel analysis and at least one plaque analysis were considered to be “CAA and plaque enriched” proteins. Proteins that were enriched in at least one CAA(+) vessel analysis and present or depleted in at least one plaque analysis and vice versa were considered to be “CAA enriched” or “plaque enriched” proteins, respectively. Proteins enriched only in one type of pathology and not detected in the other type of pathology were considered potentially enriched when found in more than half of the pathology groups (i.e., both CAA groups and 3 of 4 plaque groups). Proteins that were inconsistently enriched or depleted within CAA(+) vessel experimental groups or within plaque experimental groups were excluded from the analysis.Validation IHC

[0127] Follow up histology was performed on COL6A2, SEMA3G, and SMOC1 in inferior temporal cortex sections from progressive stages of disease (Control, n = 12; Preclinical AD, n = 10; MCI, n = 12; and AD, n = 12). Cases were age-matched between groups (p = 0.35, one-way ANOVA), and included all cases evaluated by proteomics as well as additional cases to increase the power of the analysis. Additional AD and MCI cases were selected from those with the highest CAA and plaque scores by IHC screening studies. All Control cases that had a combined plaque and CAA score of > 3 in IHC screening studies (indicating presence of moderate amyloid pathology in the absence of cognitive impairment) were included in the preclinical AD group. Additional Control cases were selected from cases that had a score of 0 for both plaques and CAA in IHC screening studies. Where possible, groups were balanced for sex, age, and APOE genotype, detailed in Supplemental Table 1 from Leitner et al., “Differences in the Cerebral Amyloid Angiopathy Proteome in Alzheimer's Disease and Mild Cognitive Impairment,” Acta NeuropatholA^( y.9 (2024), doi: 10.1007 / s00401-024-02767-l, which is hereby incorporated by reference in its entirety. Briefly, FFPE sections (8 pm) were deparaffinized and rehydrated in a series of xylenes and ethanol dilutions. After washing, sections were incubated in 88% formic acid for 7 minutes for COL6A2 and SEMA3G, and 99% formic acid for 7 minutes for SMOC1. Heat-induced antigen retrieval was performed with 10 mM sodium citrate, 0.05% triton-x 100 pH 6 for COL6A2 and SEMA3G; and 10 mM sodium citrate, 0.05% Tween-20 pH 6 for SMOC1. Blocking with 10% normal goat serum or normal donkey serum was followed by primary antibodies for COL6A2 (1 : 100, Novus Biologicals #H00001292-M01), Ap40 (1 : 100, (Miller et al., “High-Affinity Rabbit Monoclonal Antibodies Specific for Amyloid Peptides Amyloid-P40 and Amyloid-P42,” J. Alzheimer s Dis. 23(2):293- 305 (2011), which is hereby incorporated by reference in its entirety)), SEMA3G (1 : 100, Sigma HPA001761), SMOC1 (1 : 100, Abeam ab200219), and 4G8 (1 : 1000, BioLegend #800701) overnight at 4°C. Sections were incubated with goat anti-mouse 647 and goat anti-rabbit 488 secondary antibodies (1 :500, Jackson Immuno Research Laboratories), donkey anti-rabbit 647 and donkey anti -mouse 488 (1 : 1000, ThermoFisher), counterstained with DAPI (Sigma D9542) or Hoescht 33342 (1 : 1000, Sigma B2261), and coverslipped. Whole slide images were acquired using a Leica Aperio Versa 8 Scanner or an Olympus VS200 Slide Scanner at 10x magnification, with empty channel 568 used to capture autofluorescence for subtraction. For COL6A2 and SEMA3G, representative 40* images were obtained with the Leica Aperio Versa 8 Scanner, and for SMOC1, representative 60* images were captured on a Nikon C2 Confocal microscope.IHC Quantification

[0128] Slide scan images for COL6A2, SEMA3G, and SMOC1 were imported into QuPath (v0.5.0) and gray matter manually annotated to exclude tissue folds, tears, and staining artifacts.A blood vessel classifier was trained using the autofluorescence channel (568) and applied to gray matter annotations in all images. Each image was subsequently checked to ensure classifier accuracy. Images were exported as .tif tiles with corresponding masks for blood vessels and total gray matter. Autofluorescence was subtracted from Ap and COL6A2, SEMA3G, or SM0C1 channels in ImageJ2 (v2.14.0 / 1.54f). To determine COL6A2, SEMA3G, or SM0C1 positive staining in CAA(+) and CAA(-) vessels, blood vessel masks were applied to each image and areas of Ap-positive or Ap-negative staining subtracted, respectively. COL6A2, SEMA3G, or SM0C1 signal was then thresholded, and positive area and integrated density measured. For analysis, COL6A2, SEMA3G, or SM0C1 positive area in CAA(-) and CAA(+) vessels was normalized to total blood vessel area for each section. Integrated density was normalized to either CAA(-) or CAA(+) area to give average gray value per pixel. Results were visualized in GraphPad Prism (vl0.0.3) and analyzed using Kruskal-Wallis test with Dunn’s post hoc test for the same pairwise comparisons performed in the proteomics analyses.Example 1 - Proteomic Analysis of Cerebral Amyloid Angiopathy

[0129] From LFQ-MS, 2,026 proteins were detected in microdissected CAA(+) and CAA(-) blood vessels from age-matched Control (n = 10), MCI (n = 4), and AD (n = 6) cases (FIG. 1). PCA showed significant segregation of CAA(+) vessel samples from CAA(-) vessel samples in PCA2 (p < 0.0001), as well as some segregation in PCA1 (p = 0.027, FIG. 2). There was no segregation by disease group among all samples on the PCA nor was this seen by multiple linear regression (Table 4 below).Table 4. Multiple Linear Regression of PCA for Group and Tissue Type

[0130] Markers of interest were reviewed to characterize dissected samples, including the Ap peptide and vessel markers. The Ap peptide LVFFAEDVGSNK (SEQ ID NO: 1) corresponds to amino acids 17-28 of Ap, does not discriminate from cleaved or full-length sequences, but this peptide does show strong enrichment and correlation to Ap pathology (Drummond et al., “Proteomic Differences in Amyloid Plaques in Rapidly Progressive and Sporadic Alzheimer’s Disease,” Acta Neuropathol. 133(6):933-954 (2017); Handa et al., “Proteomics-Based Investigation of Cerebrovascular Molecular Mechanisms in Cerebral Amyloid Angiopathy by the FFPE-LMD-PCT-SWATH Method,” Fluids Barriers CNS 19(1):56 (2022); Leitner et al., “Similar Brain Proteomic Signatures in Alzheimer’s Disease and Epilepsy,” Acta Neuropathol. 147(1):27 (2024); and Seyfried et al., “A Multi-Network Approach Identifies Protein-Specific Co-Expression in Asymptomatic and Symptomatic Alzheimer’s Disease,” Cell Syst. 4(l):60- 72.e64 (2017), each of which is hereby incorporated by reference in its entirety). The Ap peptide was significantly enriched in CAA(+) vessels in comparison to CAA(-) vessels in both MCI and AD (MCI: p = 7.80 x 1Q-3, 24.5-fold; AD: p = 2.07 x 10^, 47.8-fold; FIG. 3A). The endothelial cell marker PECAM1 (CD31) (Lertkiatmongkol et al., “Endothelial Functions of Platelet / Endothelial Cell Adhesion Molecule- 1 (CD31),” Curr. Opin. Hematol. 23(3):253-259 (2016), which is hereby incorporated by reference in its entirety) was detected in all samples and was present at a similar level in all groups (FIG. 3B). Specific blood vessel markers associated with capillaries or larger vessels (Trimm and Red-Horse, “Vascular Endothelial Cell Development and Diversity,” Nat. Rev. Cardiol. 20(3): 197-210 (2023), which is hereby incorporated by reference in its entirety) were detected (PTGDS, BSG, SLC3A2, SLC7A5, HSPA1 A, PRSS23, TFRC). Among the detected markers, 4 were differentially abundant in AD CAA(+) vessels (PTGDS, BSG, SLC3A2, SLC7A5; FIGS. 3C-3F) and the same was observed in MCI except with no change in SLC3 A2. Three of these proteins are associated with capillaries (BSG, SLC3A2, SLC7A5) and one with veins (PTGDS) (Trimm and Red-Horse, “Vascular Endothelial Cell Development and Diversity,” Nat. Rev. Cardiol. 20(3): 197-210 (2023), which is hereby incorporated by reference in its entirety). In the CAA(-) vessels, these proteins were not altered in either disease group when compared to Control cases. These differences may indicate fewer CAA(+) capillaries in the microdissected CAA(+) vessels and a mix of vessel types in the CAA(-) vessels, consistent with what was observed when dissectionwas performed and with pathogenesis typically involving larger vessels (Charidimou et al., “Emerging Concepts in Sporadic Cerebral Amyloid Angiopathy,” Brain 140: 1829-1850 (2017), which is hereby incorporated by reference in its entirety).Example 2 - CAA(+) vs CAA(-) Vessel Comparisons

[0131] Proteins differentially abundant in CAA(+) vessels when compared to neighboring CAA(-) vessels in each disease group were identified by paired t tests at / ? < 0.05 and with fold change > 1.5. There were 257 proteins differentially abundant in MCI, and 289 proteins in AD (FIG. 4A and FIG. 4B).

[0132] Cell-type annotations (Winkler et al., “A Single-Cell Atlas of the Normal and Malformed Human Brain Vasculature,” Science 375(6584):eabi7377 (2022), which are hereby incorporated by reference in its entirety) were evaluated to determine cell-type enrichment of altered proteins. The majority of proteins were “Undefined” as they are expressed by multiple cell types or it is unknown, and thus would require other detailed follow up cell-type characterization analysis. From annotated proteins in MCI CAA(+) vessels, there was enrichment for astrocyte (p = 2.71 x 102) and oligodendrocyte (p = 5.70 x 103) proteins. The majority of the astrocyte proteins (75%; 3 / 4) and oligodendrocyte proteins (85%; 11 / 13) were decreased. In AD CAA(+) vessels, there was enrichment for perivascular fibroblast (p = 1.26 x 102) and oligodendrocyte (p = 5.80 x 103) proteins. The majority of perivascular fibroblast proteins (60%; 3 / 5) were decreased and half of the annotated oligodendrocyte proteins (50%; 7 / 14) were decreased.

[0133] Top significant GOCC terms (FDR adjusted p value < 0.05) were determined for the differentially abundant proteins (FIG. 4C and FIG. 4D; see also Supplemental Tables 4-5 from Leitner et al., “Differences in the Cerebral Amyloid Angiopathy Proteome in Alzheimer's Disease and Mild Cognitive Impairment,” Acta Neuropathol.148(1 ): 9 (2024), doi:10.1007 / s00401-024-02767-l, which is hereby incorporated by reference in its entirety). The most significant GOCC term associated with increased proteins for both MCI and AD was collagen-containing extracellular matrix (MCI: adj . / ? = 4.15 x 10l 4, 26 proteins; AD: adj. p = 1.51 x 106, 29 proteins). The most significant GOCC term associated with decreased proteins for both MCI and AD was ribonucleoprotein complex (MCI: adj . p = 1.1244 proteins; AD: adj. / ? = 3.33 x 10l 9, 53 proteins).

[0134] Collagen protein subtypes were notably increased in CAA(+) vessels in both MCI and AD. In MCI, increased collagen proteins included 8 proteins from the collagen I, IV, VI, and XVIII families, and for AD 2 proteins from the IV and XVIII families (FIGS. 12A-12M; seealso Supplemental Table 3 from Leitner et al., “Differences in the Cerebral Amyloid Angiopathy Proteome in Alzheimer's Disease and Mild Cognitive Impairment,” Acta NeuropatholA48(V) 9 (2024), doi: 10.1007 / s00401-024-02767-l, which is hereby incorporated by reference in its entirety). In addition to the altered collagen proteins associated with the GOCC collagen- containing extracellular matrix term shared by both MCI and AD, this GOCC term also included increased laminin proteins (LAMA5, LAMC1), APOE, CLU, SMOC1, TGFBI, HTRA1, EFEMP1, NPNT, SPON1, and EMILIN1.

[0135] Ribosome proteins and ribonucleoproteins were notably decreased in both MCI and AD CAA(+) vessels. Of the proteins associated with the GOCC ribonucleoprotein complex term, 22 proteins were shared between both disease groups (unique proteins: MCI 22, AD 31). Key BBB proteins that had robust expression in CAA(-) vessels were significantly decreased in CAA(+) vessels (FIGS. 13A-13C), including OCLN (MCI: 2.7-fold decrease, / ? = 2.03 x | Q2; AD: not detected), TJP1 (also known as zona occludens-1; ZO-1; MCI: 2.7-fold decrease, / ? = 4.45xI O3; AD: 1.7-fold decrease, p= 1.76 x I O3), and SLC2A1 (also known as GLUT1; MCI: 2.2-fold, / ? = 0.059; AD: 1.7-fold, / ? = 0.030).

[0136] Protein changes in CAA(+) vessels were similar in MCI and AD. There were 86 proteins significantly altered in both MCI and AD, of which 84 / 86 were changed in the same fold-change direction (FIG. 5A). The remaining two proteins (DNA2 and WIPF3) were significantly decreased in CAA(+) vessels in MCI and increased in AD. While there were seemingly many proteins uniquely altered in only one group (FIG. 5 A), there was a strong positive correlation (p < 0.0001, R2= 0.62) between protein changes in MCI and AD (FIG. 5B) suggesting that differences between groups were likely due to the power of our study rather than biologic differences. There were 87% (400 / 460) of proteins altered in either MCI or AD that changed in the same fold-change direction (FIG. 5B). The 84 shared significant CAA(+) vessel proteins were evaluated by unsupervised hierarchical clustering, which indicated clustering by tissue type (FIG. 5C). Of those proteins altered in both disease groups, there was particular representation of decreased ribosome proteins, as was also seen most significantly by GOCC enrichment.Example 3 - Non-CAA Vessel Comparisons

[0137] Significant protein changes were also observed in CAA(-) vessels in MCI and AD. To identify protein differences in CAA(-) vessels from MCI or AD cases, CAA(-) vessels in both disease groups were compared to Control CAA(-) vessels by unpaired t tests at / ? < 0.05 andwith fold change > 1.5. There were 61 proteins differentially abundant in MCI, and 112 proteins in AD (FIG. 6A and FIG. 6B).

[0138] After cell-type annotation analysis, the majority of proteins were “Undefined” as they are expressed by multiple cell types or it is unknown. In MCI CAA(-) vessels, there was enrichment for perivascular fibroblast (p = 4.12 x 102) and oligodendrocyte (p = 1.90 x 103) proteins. Both of the perivascular fibroblast proteins (100%; 2 / 2) were increased and the majority of the oligodendrocyte proteins (67%; 4 / 6) were decreased. In AD CAA(-) vessels, there was enrichment for neuron (p = 4.30 x 103) proteins, with the majority decreased (75%; 3 / 4).

[0139] Top significant GOCC terms (FDR adj . p < 0.05) were determined for the differentially abundant proteins (FIG. 6C and FIG. 6D; see also Supplemental Tables 6-7 from Leitner et al., “Differences in the Cerebral Amyloid Angiopathy Proteome in Alzheimer's Disease and Mild Cognitive Impairment,” Acta Neuropathol.148(1 ): 9 (2024), doi:10.1007 / s00401-024-02767-l, which is hereby incorporated by reference in its entirety). The most significant GOCC terms associated with increased proteins for MCI included three terms: extracellular matrix, external encapsulating structure, and collagen-containing extracellular matrix (adj. p = 5.57 x 104, 12 proteins for all three terms). For AD, the top significant GOCC term was collagen trimer (adj. p = 4.65 x I O3, 5 proteins). Decreased proteins were not significantly associated with GOCC terms (see Supplemental Tables 6-7 from Leitner et al., “Differences in the Cerebral Amyloid Angiopathy Proteome in Alzheimer's Disease and Mild Cognitive Impairment,” Acta Neuropathol.148(1):9 (2024), doi: 10.1007 / s00401-024- 02767-1, which is hereby incorporated by reference in its entirety).

[0140] Among the most significantly increased proteins, multiple collagen protein subtypes were increased in both MCI and AD CAA(-) vessels. In MCI, increased collagen proteins included 4 proteins from the VI and XII families, and for AD 5 proteins from I, VI, and XII families (FIGS. 12A-12M; see also Supplemental Table 3 from Leitner et al., “Differences in the Cerebral Amyloid Angiopathy Proteome in Alzheimer's Disease and Mild Cognitive Impairment,” Acta Neuropathol A^(V).9 (2024), doi: 10.1007 / s00401-024-02767-l, which is hereby incorporated by reference in its entirety).

[0141] Of the differentially abundant proteins, 22 were shared by both MCI and AD and all changed in the same fold-change direction (FIG. 7A). Among significant proteins in at least one disease group (151 proteins), there was a mild positive correlation (p < 0.0001, R2= 0.18) with 85% (129 / 151) changing in the same fold-change direction (FIG. 7B). The 22 shared significant CAA(-) vessel proteins were evaluated by unsupervised hierarchical clustering, which indicatedclustering of Control from MCI and AD CAA(-) vessel samples (FIG. 7C). Shared proteins included several increased collagens (COL6A1, COL6A2, COL6A3, COL12A1). As COL6A2 was previously associated with CAA severity (Handa et al., “Proteomics-Based Investigation of Cerebrovascular Molecular Mechanisms in Cerebral Amyloid Angiopathy by the FFPE-LMD- PCT-SWATH Method,” Fluids Barriers CNS 19( 1 ): 56 (2022), which is hereby incorporated by reference in its entirety), COL6A2 was selected for further validation using immunohistochemistry in the current study. Increased intensity of COL6A2 was observed in CAA(+) vessels of preclinical AD, MCI, and AD cases and not in CAA(-) vessels (FIG. 8). Among those proteins significant in only one disease group, MCI included increased ribosome proteins (RPL28, RPL35A, RPS10), and AD included decreased 14-3-3 family proteins (YWHAB, YWHAE, YWHAG, YWHAH, YWHAZ; as well as YWHAQ in both AD and MCI).Example 4 - Comparison of CAA and Plaque Proteomic Studies

[0142] Amyloid-associated proteins were evaluated further to identify CAA(+) vessel and amyloid-plaque-enriched proteins. The CAA proteomic results in this study were compared to two datasets that identified proteins significantly enriched or depleted in amyloid plaques relative to neighboring non-plaque tissue from sporadic AD, preclinical AD, early onset AD, and Down syndrome cases (Drummond et al., “The Amyloid Plaque Proteome in Early Onset Alzheimer’s Disease and Down Syndrome,” Acta NeuropathoL Commun. 10(l):53 (2022); Xiong and Ge, “Quantitative Proteomics Reveals Distinct Composition of Amyloid Plaques in Alzheimer’s Disease,” Alzheimers Dement. 15(3):429-440 (2019), each of which is hereby incorporated by reference in its entirety). This comparison showed that for proteins identified in both CAA and plaque datasets, 39 / 235 CAA enriched proteins were also enriched in amyloid plaques. Notably, this included 9 proteins that were significantly enriched in both CAA and plaques in all experimental groups examined in all 3 datasets (APP, APOE, SMOC1, SPON1, GPNMB, C4A, HTRA1, OLFML3, NRXN1), indicating a particularly robust enrichment of these proteins in both CAA and plaques. The enrichment of one of these proteins, SMOC1, was evaluated in CAA using immunohistochemistry and confirmed significant enrichment of SMOC1 in CAA(+) vessels in preclinical AD, MCI, and advanced AD (FIG. 9). There were also 106 proteins that were potentially only enriched in CAA and 90 proteins that were potentially only enriched in plaques (FIGS. 10A-B, see also Supplemental Table 8 from Leitner et al., “Differences in the Cerebral Amyloid Angiopathy Proteome in Alzheimer's Disease and Mild Cognitive Impairment,” Acta NeuropatholA48( ) 9 (2024), doi: 10.1007 / s00401-024-02767-l, which is hereby incorporated by reference in its entirety). Of these, SEMA3G emerged as apromising potential CAA specific marker, as it was detected in CAA(+) vessels from both MCI and AD, was not detected or at low levels in CAA(-) vessels (detected in one control), correlated to Ap peptide levels (p = 0.0016; R2= 0.69), and was not detected in the previous plaque proteomic studies. SEMA3G was evaluated by immunohistochemistry and showed enrichment in CAA(+) vessels in preclinical AD, trending in MCI, and enrichment in advanced AD (FIG. 11), with variation in CAA levels. Immunohistochemical SEMA3G levels correlated with CAA pathology levels (p < 0.0001; R2= 0.90). Other proteins reported herein to be enriched in either CAA or plaques could indicate proteins that selectively accumulate in one type of pathology. Comparison of functional associations showed that proteins enriched in both CAA and plaques, proteins enriched in CAA, and proteins enriched in plaques largely belonged to the same protein networks, particularly collagen-containing extracellular matrix proteins (FIG. 10B) that suggest proteins enriched in CAA and plaques are largely similar.Discussion of Examples 1-4

[0143] This is the first study to evaluate both the CAA(+) and neighboring CAA(-) vessel proteomes separately, as well as first to evaluate the vessel proteomes in MCI. 257 proteins were identified that were differentially abundant in CAA(+) vessels in MCI and 289 proteins that were differentially abundant in CAA(+) vessels in AD, most significantly associated with increased collagen-containing extracellular matrix and decreased ribonucleoprotein complex and BBB proteins. The CAA(+) vessel proteome was found to be similar in MCI and AD cases. Interestingly, differences were also observed in CAA(-) vessels in both MCI and AD in comparison to Control cases, suggesting changes in blood vessel integrity in the absence of CAA. Finally, a comparison between the CAA and amyloid-plaque proteomes confirmed that many of the same amyloid-associated proteins were enriched in both CAA and plaques.

[0144] In CAA(+) vessels, many collagen proteins were increased in both MCI and AD. Collagens are a component of the basement membrane, which undergoes thickening with aging and in AD (Thomsen et al., “The Vascular Basement Membrane in the Healthy and Pathological Brain,” J. Cereb. Blood Flow Metab. 37(10):3300-3317 (2017), which is hereby incorporated by reference in its entirety). Similar to previous histologic studies (Fisher et al., “Pathological Changes within the Cerebral Vasculature in Alzheimer’s Disease: New Perspectives,” Brain Pathol. 32:el3061 (2022), which is hereby incorporated by reference in its entirety), increased collagen IV proteins, as well as collagen I, VI, and XVIII family proteins were identified for MCI and XVIII family proteins in AD. These findings extend a previous CAA proteomics study that reported elevated COL6A2 in CAA cases (Handa et al., “Proteomics-Based Investigation ofCerebrovascular Molecular Mechanisms in Cerebral Amyloid Angiopathy by the FFPE-LMD- PCT-SWATH Method,” Fluids Barriers CNS 19(1):56 (2022), which is hereby incorporated by reference in its entirety), as in Examples 1-4. Further, basement membrane protein changes have been observed in previous studies that in addition to increased collagen IV included fibronectin, agrin, and perlecan (also known as HSPG2) (Howe et al., “The Role of Basement Membranes in Cerebral Amyloid Angiopathy,” Front Physiol. 25: 11 :601320, which is hereby incorporated by reference in its entirety). Similarly, increased collagen IV subtype proteins were identified in CAA(+) vessels for COL4A1, COL4A2 in MCI and decreased COL4A3 in AD; with trends seen across disease groups. By contrast, changes in fibronectin or agrin were not observed in the pairwise comparisons evaluated. Increased collagens in vessels with Ap deposition suggest vascular matrix reorganization, which may contribute to vascular dysfunction in both MCI and AD.

[0145] Proteins enriched in CAA and plaques were largely similar, both at the individual protein level and at the protein network level. Proteins enriched in both CAA and plaques included many present in the matrisome protein cluster previously reported to strongly correlate with amyloid pathology, tau pathology and cognitive status (z.e., APP, APOE, SMOC1, SPON1, GPNMB, HTRA1, OLFML3, PTN, SPOCK2, NTN1, MD) (Johnson et al., “Large-Scale Deep Multi-Layer Analysis of Alzheimer’s Disease Brain Reveals Strong Proteomic Disease-Related Changes not Observed at the RNA Level,” Nat. Neurosci. 25(2):213-225 (2022); Levites et al., “Ap Amyloid Scaffolds the Accumulation of Matrisome and Additional Proteins in Alzheimer’s Disease,” bioRxiv 7:2023.11.29.568318 (2023), each of which is hereby incorporated by reference in its entirety). The results presented herein provide new evidence that many of these matrisome module proteins are highly enriched in CAA in human AD brain tissue and further support the hypothesis that accumulation of extracellular matrix proteins is a key feature of both CAA and plaque pathology. While many proteins had apparent enrichment in either CAA or plaques, these results should be interpreted cautiously as they are reliant on low-powered datasets. While some preliminary findings are provided about proteins that may be enriched in either CAA or plaques, knowledge in this area will continue to evolve as future higher-powered studies examining CAA or plaque-enriched proteins are published. One protein that did emerge as different in plaques and CAA was semaphorin 3G (SEMA3G), which is a class 3 secreted semaphorin (Taniguchi et al., “Identification and Characterization of a Novel Member of Murine Semaphorin Family,” Genes Cells 10(8):785-792 (2005), which is hereby incorporated by reference in its entirety). The results presented herein showed enrichment of SEMA3G in CAA(+) vessels, correlation to regional CAA levels, which was not detected in other CAAproteomic studies with the approaches used (Handa et al., “Proteomics-Based Investigation of Cerebrovascular Molecular Mechanisms in Cerebral Amyloid Angiopathy by the FFPE-LMD- PCT-SWATH Method,” Fluids Barriers CNS 19(1):56 (2022); Hondius et al., “Proteomics Analysis Identifies New Markers Associated with Capillary Cerebral Amyloid Angiopathy in Alzheimer’s Disease,” Acta. Neuropathol. Commun. 6(1):46 (2018); Inoue et al., “Sushi Repeat- Containing Protein 1 : A Novel Disease-Associated Molecule in Cerebral Amyloid Angiopathy,” Acta. NeuropathoL 134(4):605— 617 (2017); Manousopoulou et al., “Systems Proteomic Analysis Reveals that Clusterin and Tissue Inhibitor of Metalloproteinases 3 Increase in Leptomeningeal Arteries Affected by Cerebral Amyloid Angiopathy,” Neuropathol Appl Neurobiol. 43(6):492- 504 (2017); Ojo et al., “Molecular Pathobiology of the Cerebrovasculature in Aging and in Alzheimers Disease Cases with Cerebral Amyloid Angiopathy,” Front. Aging Neurosci.13:658605 (2021); Wojtas et al., “Proteomic Changes in the Human Cerebrovasculature in Alzheimer’s Disease and Related Tauopathies Linked to Peripheral Biomarkers in Plasma and Cerebrospinal Fluid,” Alzheimers Dement. 20(6):4043-4065(2024); Zellner et al., “Proteomic Profiling in Cerebral Amyloid Angiopathy Reveals an Overlap with CADASIL Highlighting Accumulation of HTRA1 and its Substrates,” Acta Neuropathol. Commun. 10:6 (2022), each of which is hereby incorporated by reference in its entirety), not detected in plaque tissue (Drummond et al., “The Amyloid Plaque Proteome in Early Onset Alzheimer’s Disease and Down Syndrome,” Acta Neuropathol. Commun. 10(l):53 (2022); Drummond et al., “Proteomic Differences in Amyloid Plaques in Rapidly Progressive and Sporadic Alzheimer’s Disease,” Acta Neuropathol. 133(6):933-954 (2017), each of which is hereby incorporated by reference in its entirety), and not detected in other AD proteomic studies (Askenazi et al., “Compilation of Reported Protein Changes in the Brain in Alzheimer’s Disease,” Nat. Commun. 14(1):4466 (2023), which is hereby incorporated by reference in its entirety). Further, a recent study indicated that SEMA3G secretion in the brain modulates synaptic function in an animal model (Tan et al., “Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2 / PlexinA4,” Neuron 101(5):920-937.e913 (2019), which is hereby incorporated by reference in its entirety). SEMA3G is detectable in human CSF (Tan et al., “Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2 / PlexinA4,” Neuron 101(5):920-937.e913 (2019); Vervuurt et al., “Cerebrospinal Fluid Shotgun Proteomics Identifies Distinct Proteomic Patterns in Cerebral Amyloid Angiopathy Rodent Models and Human Patients,” Acta Neuropathol. Commun. 12( 1 ): 6 (2024); Wojtas et al., “Proteomic Changes in the Human Cerebrovasculature in Alzheimer’s Disease and Related Tauopathies Linked to Peripheral Biomarkers in Plasma and CerebrospinalFluid,” Alzheimers Dement. 20(6):4043-4065(2024), each of which is hereby incorporated by reference in its entirety), making it a potentially useful diagnostic biomarker of CAA. Because there are currently no commercially available biomarkers for CAA, this may facilitate CAA diagnosis and evaluation of patients at risk for ARIA when administered currently available immunotherapies.

[0146] Many proteins were significantly decreased in CAA(+) vessels, most significantly ribonucleoprotein complex and ribosome proteins in both MCI and AD, which are associated with protein translation. Previous mouse models show decreased protein synthesis occurs early with aging (Skariah and Todd PK, “Translational Control in Aging and Neurodegeneration,” Wiley Interdiscip. Rev. RNA 12(4):el628 (2021), which is hereby incorporated by reference in its entirety), and AD mouse model studies indicate decreased ribosomal proteins correlate to increased phosphorylated tau (pTau) levels (Evans et al., “Decreased Synthesis of Ribosomal Proteins in Tauopathy Revealed by Non-Canonical Amino Acid Labelling,” EMBO J.38:el01174 (2019), which is hereby incorporated by reference in its entirety). Further, ribosome protein expression is linked to the cell stress response (Kavanagh et al., “Tau Interactome and RNA Binding Proteins in Neurodegenerative Diseases,” Mol. Neurodegener. 17( 1 ): 66 (2022); Pfister, “Emerging Role of the Nucleolar Stress Response in Autophagy,” Front. Cell. Neurosci. 30: 13:156 (2019), each of which is hereby incorporated by reference in its entirety). Previous CAA proteomics studies have not identified ribonucleoprotein complex protein differences with the approaches used ((Handa et al., “Proteomics-Based Investigation of Cerebrovascular Molecular Mechanisms in Cerebral Amyloid Angiopathy by the FFPE-LMD-PCT-SWATH Method,” Fluids Barriers CNS 19(1):56 (2022); Hondius et al., “Proteomics Analysis Identifies New Markers Associated with Capillary Cerebral Amyloid Angiopathy in Alzheimer’s Disease,” Acta. NeuropathoL Commun. 6(1):46 (2018); Inoue et al., “Sushi Repeat-Containing Protein 1 : A Novel Disease-Associated Molecule in Cerebral Amyloid Angiopathy,” Acta. NeuropathoL 134(4):605-617 (2017); Wojtas et al., “Proteomic Changes in the Human Cerebrovasculature in Alzheimer’s Disease and Related Tauopathies Linked to Peripheral Biomarkers in Plasma and Cerebrospinal Fluid,” Alzheimers Dement. 20(6):4043-4065(2024); Zellner et al., “Proteomic Profiling in Cerebral Amyloid Angiopathy Reveals an Overlap with CADASIL Highlighting Accumulation of HTRA1 and its Substrates,” Acta NeuropathoL Commun. 10:6 (2022), each of which is hereby incorporated by reference in its entirety), although some protein differences are seen with other pairwise comparisons when evaluating aging and low CAA to age-matched Control cases. Other non-CAA studies have linked the ribonucleoprotein HNRNPA1 to APP splicing, with a suggestion that increased HNRNPA1 levels may be protective (Low et. al.,“Heterogeneous Nuclear Ribonucleoproteins: Implications in Neurological Diseases,” Mol. Neurobiol. 58(2):631-646 (2021), which is hereby incorporated by reference in its entirety). HNRNPA1 was decreased in MCI CAA(+) vessels and a similar trend was observed in AD CAA(+) vessels, as well as CAA(-) vessels, thus HNRNPA1 may be associated with development and / or progression of vessel pathology.

[0147] Of particular interest among decreased proteins in CAA(+) vessels were altered proteins important for BBB integrity, including tight junction proteins, SLC2A1 (GLUT1), and basement membrane proteins. OCLN and TJP1 were decreased in CAA(+) vessels of both MCI and AD cases, which was not different or not detected in other human CAA proteomics studies with the approaches used (Handa et al., “Proteomics-Based Investigation of Cerebrovascular Molecular Mechanisms in Cerebral Amyloid Angiopathy by the FFPE-LMD-PCT-SWATH Method,” Fluids Barriers CNS 19(1):56 (2022); Hondius et al., “Proteomics Analysis Identifies New Markers Associated with Capillary Cerebral Amyloid Angiopathy in Alzheimer’s Disease,” Acta. NeuropathoL Commun. 6(1):46 (2018); Wojtas et al., “Proteomic Changes in the Human Cerebrovasculature in Alzheimer’s Disease and Related Tauopathies Linked to Peripheral Biomarkers in Plasma and Cerebrospinal Fluid,” Alzheimers Dement. 20(6):4043-4065(2024); Zellner et al., “Proteomic Profiling in Cerebral Amyloid Angiopathy Reveals an Overlap with CADASIL Highlighting Accumulation of HTRA1 and its Substrates,” Acta NeuropathoL Commun. 10:6 (2022), each of which is hereby incorporated by reference in its entirety). In previous non-proteomic CAA studies, TJP1 was decreased histologically in frontal cortex, TJP1 and OCLN were decreased in leptomeninges as measured biochemically, and OCLN and TJP1 were decreased in occipital lobe histologically in CAA cases with capillary involvement (Carrano et al., “Amyloid Beta Induces Oxidative Stress-Mediated Blood-Brain Barrier Changes in Capillary Amyloid Angiopathy,” Antioxid Redox Signal 15(5): 1167-1178 (2011); Cheng et al., “Occludin Deficiency with BACE1 Elevation in Cerebral Amyloid Angiopathy,” Neurology 82(19): 1707-1715 (2014); and Freeze et al., “Blood-Brain Barrier Dysfunction in Small Vessel Disease Related Intracerebral Hemorrhage,” Front. Neurol. 12:9:926 (2018), which is hereby incorporated by reference in its entirety). In AD, with cases having a range of CAA severity, changes to BBB proteins included decreased OCLN in several cortical regions as well as correlations to CAA severity particularly in the inferior temporal cortex as measured by ELISA in brain homogenate relative to CD31 levels (Yamazaki et al., “Selective Loss of Cortical Endothelial Tight Junction Proteins During Alzheimer’s Disease Progression,” Brain142(4): 1077-1092 (2019), which is hereby incorporated by reference in its entirety). Changes in TJP1 and OCLN have also been reported in AD animal models (Freeze et al., “Blood-BrainBarrier Dysfunction in Small Vessel Disease Related Intracerebral Hemorrhage,” Front. Neurol. 12:9:926 (2018); Situ et al., “Transcriptomic Profile of Blood-Brain Barrier Remodeling in Cerebral Amyloid Angiopathy,” Front. Cell Neurosci. 22: 16:931247, each of which is hereby incorporated by reference in its entirety). SLC2A1 (GLUT1) is expressed by endothelial cells (Simpson et al., “Supply and Demand in Cerebral Energy Metabolism: The Role of Nutrient Transporters,” J. Cereb. Blood Flow Metab. 27(11): 1766-1791 (2007), which is hereby incorporated by reference in its entirety), and it is enriched in vessels as measured by proteomics but not different by CAA status in mixed A0+ / - vessels (Handa et al., “Proteomics-Based Investigation of Cerebrovascular Molecular Mechanisms in Cerebral Amyloid Angiopathy by the FFPE-LMD-PCT-SWATH Method,” Fluids Barriers CNS 19(1):56 (2022), which is hereby incorporated by reference in its entirety). When SLC2A1 is decreased it is associated with vascular dysfunction (Fisher et al., “Pathological Changes within the Cerebral Vasculature in Alzheimer’s Disease: New Perspectives,” Brain Pathol. 32:el3061 (2022), which is hereby incorporated by reference in its entirety), and it is decreased as measured by other approaches in AD as well as indirectly in MCI on brain imaging (Fisher et al., “Pathological Changes within the Cerebral Vasculature in Alzheimer’s Disease: New Perspectives,” Brain Pathol. 32:el3061 (2022); Zenaro et al., “The Blood-Brain Barrier in Alzheimer’s Disease,” Neurobiol. Dis. 107:41-56 (2017), each of which is hereby incorporated by reference in its entirety). Similarly in the current study, SLC2A1 was detected in all samples, with a decrease in AD and trending decrease in MCI CAA(+) vessels. In addition to the collagen basement membrane proteins, metalloproteinases (MMPs) and their inhibitors (TIMPs) (Dewing et al., “The Diverse Roles of TIMP-3: Insights into Degenerative Diseases of the Senescent Retina and Brain,” Cells 9(1):39 (2019), which is hereby incorporated by reference in its entirety) also contribute to BBB integrity. Previous histologic studies in occipital lobe showed an altered ratio of MMP9 and TIMP3 in leptomeningeal vessels of CAA cases with intracerebral hemorrhage (Jakel et al., “Disturbed Balance in the Expression of MMP9 and TIMP3 in Cerebral Amyloid Angiopathy- Related Intracerebral Haemorrhage,” Acta NeuropathoL Commun. 8(1):99 (2020), which is hereby incorporated by reference in its entirety). MMP proteins were not detected in the current study, but TIMP3 was detected in all CAA(+) vessel samples as well as one Control CAA(-) vessel sample indicating that the inhibitor of MMP9 is elevated in CAA(+) samples. The results presented herein support deficits in BBB integrity and vascular function in both MCI and AD, which are relevant to the underlying disease mechanisms and provide implications for therapeutics like parenchymal drug delivery and ARIA-related risk associated with recently available immunotherapies (Salloway et al., “Amyloid-Related Imaging Abnormalities in 2Phase 3 Studies Evaluating Aducanumab in Patients with early Alzheimer Disease,” JAMA Neurol. 79(1): 13-21 (2022); Stem et al., “Impaired Intracortical Inhibition Demonstrated in vivo in People with Dravet Syndrome,” Neurology 88(17): 1659-1665 (2017), each of which is hereby incorporated by reference in its entirety).

[0148] In neighboring CAA(-) vessels, there were protein changes identified in both MCI and AD that were associated with increased collagens and the extracellular matrix. In MCI, increased collagen proteins included 4 proteins from the VI and XII families, and for AD 5 proteins from I, VI, and XII families. Additionally, proteins altered only in AD CAA(-) vessels included decreased 14-3-3 family proteins, with similar trends seen in MCI CAA(-) vessels as well as in CAA(+) vessels of both MCI and AD. From previous CAA proteomics studies, this protein family was not altered (Handa et al., “Proteomics-Based Investigation of Cerebrovascular Molecular Mechanisms in Cerebral Amyloid Angiopathy by the FFPE-LMD-PCT-SWATH Method,” Fluids Barriers CNS 19(1):56 (2022); Hondius et al., “Proteomics Analysis Identifies New Markers Associated with Capillary Cerebral Amyloid Angiopathy in Alzheimer’s Disease,” Acta. NeuropathoL Commun. 6(1):46 (2018); Zellner et al., “Proteomic Profiling in Cerebral Amyloid Angiopathy Reveals an Overlap with CADASIL Highlighting Accumulation of HTRA1 and its Substrates,” Acta NeuropathoL Commun. 10:6 (2022), each of which is hereby incorporated by reference in its entirety), and was decreased in brain tissue from some AD proteomic studies and increased or present in plaque proteomic studies (Askenazi et al., “Compilation of Reported Protein Changes in the Brain in Alzheimer’s Disease,” Nat. Commun. 14(1):4466 (2023), which is hereby incorporated by reference in its entirety). The 14-3-3 proteins are phospho-binding proteins that have many cellular regulatory functions, and in AD colocalize with neurofibrillary tangles, interact with AD pathology associated proteins, and are decreased in AD choroid plexus when compared to Control cases (Foote and Zhou, “14-3-3 Proteins in Neurological Disorders,” Int. J. Biochem. Mol. Biol. 3(2): 152-164 (2012); Leitner et al., “Localized Proteomic Differences in the Choroid Plexus of Alzheimer’s Disease and Epilepsy Patients,” Front. Neurol. 14: 14: 1221775 (2023); Pennington et al., “The Dynamic and Stress-Adaptive Signaling Hub of 14-3-3: Emerging Mechanisms of Regulation and Context- Dependent Protein-Protein Interactions,” Oncogene 37(42):5587-5604 (2018), each of which is hereby incorporated by reference in its entirety). Proteomic differences in CAA(-) vessels from those cases with CAA is expected, as the mechanisms of CAA previously described indicate Ap deposition occurs in a spiral-like patchy pattern (Charidimou et al., “Emerging Concepts in Sporadic Cerebral Amyloid Angiopathy,” Brain 140: 1829-1850 (2017), which is hereby incorporated by reference in its entirety) and thus it would be expected that neighboring cellswithout Ap deposition are impacted. Further, it is proposed that there is a bidirectional relationship between vascular dysfunction and Ap clearance (Fisher et al., “Pathological Changes within the Cerebral Vasculature in Alzheimer’s Disease: New Perspectives,” Brain Pathol. 32:el3061 (2022), which is hereby incorporated by reference in its entirety). To expand on this point, the demonstrated results in CAA(-) vessels may include a mix of protein changes as a result of both the effects of reactive changes from neighboring Ap accumulation as well as protein changes that may occur before Ap deposition as a consequence of various genetic and / or environmental factors (diet, exercise, stress) (Masurkar et al., “Factors Affecting Resilience and Prevention of Alzheimer’s Disease and Related Dementias,” Ann Neurol. 96(4):633-649 (2024), which is hereby incorporated by reference in its entirety). To better understand these protein changes, it will be of interest in future studies to evaluate protein differences that correspond to disease duration, CAA pathology severity, differences across brain regions (i.e., temporal lobe vs. the more CAA vulnerable occipital lobe), and the contribution of other co-occurring vascular pathologies to protein changes, i.e., hypertension, infarction, atherosclerosis.

[0149] There were several limitations to our study, including small sample size that was hindered by the low levels of CAA in the inferior temporal cortex. Our technique is less sensitive in detecting membrane proteins, insoluble proteins, and low abundance proteins. Heterogeneous clinical variables warrant further evaluation in future studies with larger samples, including age, disease duration, APOE genotype, neuropathology across the severity spectrum, cognitive scores, medications, co-occurring vascular pathologies.

[0150] In summary, protein changes in CAA(+) and neighboring CAA(-) vessels were identified in both MCI and AD that were associated with vascular matrix reorganization, protein translation deficits, and BBB breakdown. These changes can contribute to vascular dysfunction, and therefore inform diagnostic testing at earlier disease timepoints as well as therapeutic intervention prior to what is currently feasible.

[0151] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED:

1. An analytical method for diagnosing a patient having cognitive impairment with Cerebral Amyloid Angiopathy (CAA) comprising the steps of: a) performing an assay on at least one biological sample from said patient to obtain quantifying data for said one or more than one first protein marker and optionally one or more than one second protein marker; b) generating a result of said assay, said result comprising (i) at least the presence or absence of an increase in the level of said one or more than one first protein marker in said sample based on a comparison of said quantifying data for said one or more than one first protein marker to a control or standard range for said one or more than one first protein marker, and optionally (ii) at least the presence or absence of a decrease in the level of said one or more than one second protein marker in said sample based on a comparison of said quantifying data for said one or more than one second protein marker to a control or standard range for said one or more than one second protein marker; and c) assigning the patient as having or not having CAA based on the result of said generating, wherein an increase in the level of said one or more than one first protein marker in the sample from the patient relative to the control or standard range indicates that the patient has CAA and / or wherein a decrease in the level of said one or more than one second protein marker in the sample from the patient relative to the control or standard range indicates that the patient has CAA.

2. An analytical method for detecting presence and quantity of a protein marker in a patient sample, comprising the steps of: providing at least one sample from a patient having cognitive impairment; and performing a quantitative assay on the at least one sample using either a mass spectrometer or a biological reagent that reacts specifically with one or more than one protein marker in said sample, thereby generating quantifying data for said one or more than one protein marker; and wherein the protein marker is one or more than one first protein marker identified in Table 1 as being elevated in individuals having Cerebral Amyloid Angiopathy (CAA), the protein marker is one or more than one second protein marker identified in Table 2 as being decreased in individuals having Cerebral Amyloid Angiopathy (CAA), or both.

3. The method according to claim 1 or 2, wherein the assay is carried out by mass spectrometry.

4. The method of claim 3, wherein the mass spectrometer is equipped with a chromatographic system.

5. The method according to claim 3, wherein the mass spectrometer is a quadrupole or triple quadrupole mass spectrometer.

6. The method according to claim 1 or 2, wherein the assay is carried by immunoassay.

7. The method according to claim 6, wherein the immunoassay is an ELISA, a label- free immunoassay, or electrochemical luminescence assay.

8. The method of claim 1 or 2, wherein the sample is a whole blood sample, a subfraction of whole blood, a blood serum sample, a blood plasma sample.

9. The method of claim 1 or 2, wherein the sample is a CSF sample.

10. The method according to any one of claims 1 to 9, wherein the first protein marker is SEMA3G.

11. The method according to any one of claims 1 to 10, wherein the first protein marker further comprises a protein marker associated with collagen-containing extracellular matrix.

12. The method according to claim 11, wherein the protein marker associated with collagen-containing extracellular matrix is selected from COL6A1, COL6A2, COL6A3, COL4A1, COL4A2, COL4A3, COL1A1, COL1A2, COL12A1, COL18A1, and any combination thereof.

13. The method according to any one of claims 1 to 10, wherein the first protein marker further comprises a protein marker selected from APP, APOE, SMOC1, SPON1, GPNMB, C4A, HTRA1, OLFML3, NRXN1, and combinations thereof.

14. The method according to any one of claims 1 to 10, wherein the second protein marker is associated with ribonucleoprotein complex.

15. The method according to any one of claims 1 to 10, wherein the second protein marker is DNA2 or WIPF3, or a combination thereof.

16. The method according to any one of claims 1 to 10, wherein the second protein marker is associated with disruption of the blood brain barrier, and is selected from OCLN, TJP1, SLC2A1 (GLUT1), and combinations thereof.

17. The method according to any one of claims 1 to 10, wherein the first protein marker is any one or more of hFGF, hPLGF, hAng-2, hHB-EGF, hVEGF, and hHGF.

18. The method according to any one of claims 1 to 10 further comprising: detecting and / or quantifying amyloid in the patient sample using a reagent that binds specifically to amyloid.

19. The method according to claim 18, wherein one or more of Ap40, Ap42, total -tau, pTau-181, or pTau-217 is detected and quantified.

20. The method according to any one of claims 1 to 19 further comprising: imaging the brain of the patient from which the sample was obtained and identifying amyloid-related imaging abnormalities in the brain image.

21. A method for diagnosing and treating a patient having cognitive impairment with Cerebral Amyloid Angiopathy (CAA) comprising the steps of: performing said method according to any one of claims 1 to 20 to diagnose the patient as having CAA; and administering one or more therapeutic agents to the patient under conditions effective to reduce the risk of first-time or recurrent lobar intracerebral hemorrhages or cerebral microbleeds, manage hypertension, manage CAA-related inflammation, slow vascular deposition of AP and / or remove insoluble AP from vasculature.

22. The method according to claim 21, wherein the therapeutic agent is selected from the group of angiotensin-converting enzyme inhibitors, alpha-blockers, beta-blockers, calcium channel blockers, central agonists, angiotensin II receptor blockers, and diuretics.

23. The method according to claim 21, wherein the therapeutic agent is a statin.

24. The method according to claim 21, wherein the therapeutic agent is selected from insulin and insulin derivatives, insulin sensitizers, insulin release secretagogues, DPP-4inhibitors, SGLT2 inhibitors, GLP-1 Receptor Agonists, alpha-glucosidase inhibitors, amylin analogs, and non-sulfonylureas.

25. The method according to claim 21, wherein the therapeutic agent is an antiinflammatory steroid.

26. The method according to claim 21, wherein the therapeutic agent is an anti- amyloid-p.

27. The method according to claim 21 or 26, wherein said performing includes identifying quantitative data for one or more than one third protein marker associated with neuroinflammation or neuronal damage; and said administering further comprises adjusting dosage or dosing scheduling of the therapeutic agent, administering a different therapeutic agent, or discontinuing administration of the therapeutic agent.

28. The method according to claim 27, wherein the third protein marker is any one or more of NfL, GFAP, IFN-y, IL-1, IL-4, IL-5-IL-6, IL-8, IL-10, IL-12p70, IL-22, and TNFa.

29. The method according to any one of claims 1 to 28, further comprising determining the patient ApoE s4 status.

30. A kit for carrying out the methods of any one of claims 1 to 29.

31. The kit according to claim 30 comprising: an antibody reagent, or antigen-binding fragment thereof, that binds specifically to one of the first protein markers; and / or an antibody reagent, or antigen-binding fragment thereof, that binds specifically to one of the second protein markers.

32. The kit according to claim 31 further comprising an antibody reagent, or binding fragment thereof, that binds specifically to one of the third protein markers.

33. The kit according to claim 31 further comprising: protein extraction reagents for extraction of proteins from patient samples; and / or an enzyme capable of cleaving the first and / or second protein markers; and / ora data acquisition template identifying polypeptide fragments detectable by the mass spectrometer for said first and / or second and / or third protein markers.

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