Application of collateral plexus overexpression in treatment of Alzheimer's disease

Abstract

The invention relates to the technical field of Alzheimer's disease treatment, in particular to application of collateral plexus overexpression in Alzheimer's disease treatment. The application comprises application of collateral plexus overexpression in treatment of Alzheimer's disease; the invention relates to application of venation plexus overexpression in reduction of beta-amyloid protein diffusion. According to the invention, the choice of a special target of the collateral plexus can become a new target for AD treatment, whether apoE synthesized by epithelial cells of the collateral plexus and secreted into cerebrospinal fluid participates in the disease process of AD or not is disclosed, the relation between the apoE of the collateral plexus and brain parenchyma A beta is disclosed, and a new thought can be opened for prevention and treatment of the Alzheimer's disease; according to the method, the influence of apoE4 overexpression and knock-down in the collateral cluster on brain parenchyma A beta is explored, and a new thought is possibly provided for reducing A beta diffusion so as to relieve AD (Alzheimer's disease).

Description

Technical Field

[0001] The present invention relates to the technical field of Alzheimer's disease treatment, and in particular to application of choroid plexus overexpression in the treatment of Alzheimer's disease. Background Art

[0002] The number of people with dementia is growing at an alarming rate worldwide, currently affecting more than 55 million people, and this number is expected to more than triple by 2050. Among the many types of dementia, Alzheimer's disease (AD) occupies a dominant position, accounting for approximately 50% of all autopsy and clinical cases. The pathological characteristics of AD are the deposition of amyloid plaques and neurofibrillary tangles (NFTs), which are composed of insoluble amyloid-β (Aβ) and hyperphosphorylated microtubule-associated protein tau, respectively [Tiwari S, Atluri V, Kaushik A, et al. Alzheimer's disease: pathogenesis, diagnostics, and therapeutics [J]. Int J Nanomedicine, 2019, 14: 5541-5554, Chen Y, Holtzman D M. New insights into innate immunity in Alzheimer's disease: from APOE protective variants to therapies [J]. Trends Immunol, 2024, 45 (10): 768-782]. The production and aggregation of Aβ are closely related to the onset of AD. Many studies support the view that the accumulation of Aβ in the brain is a key event in the pathogenesis of AD, while the rest of the disease process, including the formation of neurofibrillary tangles containing tau protein, is the result of an imbalance between Aβ production and clearance [Bading JR, Yamada S, Mackic JB, et al. Brain clearance of Alzheimer's amyloid-beta40 in the squirrel monkey: a SPECT study in a primate model of cerebral amyloid angiopathy [J]. J Drug Target, 2002, 10 (4): 359-68.]. Mutations in genes involved in Aβ biosynthesis, aggregation, and degradation can lead to an imbalance in Aβ production and clearance, which in turn leads to abnormal accumulation of Aβ in the brain. The accumulation of Aβ ultimately leads to the pathogenic cascade of AD [Hardy JA, Higgins GA. Alzheimer's disease: the amyloid cascade hypothesis [J]. Science, 1992, 256(5054): 184-5.].Aβ is mainly cleared through the following pathways: (1) phagocytosis by microglia and astrocytes. (2) Clearance through the blood-brain barrier (BBB). Aβ in the brain parenchyma crosses the BBB into the blood and reaches the periphery for clearance. In addition, Aβ can be cleared by entering the cervical lymph nodes through the perivascular pathway. Some AD patients may have increased Aβ production in the central nervous system due to inherited mutations in the APP gene (i.e., Swedish mutation) or presenilin 1 or 2 genes [Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics [J]. Science, 2002, 297 (5580): 353-6]. However, most non-hereditary or late-onset AD patients do not have increased Aβ production in the central nervous system or APP overexpression. The reason for Aβ accumulation in these patients may be due to the clearance disorder of Aβ in the central nervous system, i.e., defects in the Aβ transport and efflux mechanism in the BBB or incorrect degradation in the central nervous system.

[0003] Currently, monoclonal antibody therapies targeting beta-amyloid protein (Aβ), such as Lecanemab and Donanemab, are already on the market or are about to enter the market. These drugs can selectively bind to and clear Aβ fibrils in the brain, showing potential in delaying cognitive decline and reducing Aβ deposition. Nevertheless, they may also trigger a series of adverse reactions, such as binding to the Fc receptors of microglia, activating these cells and causing them to release proinflammatory cytokines, which may lead to problems such as cell edema or small blood vessel rupture and bleeding. L, Johannesson M, Nygren P, et al. Lecanemab, Aducanumab, and Gantenerumab-Binding Profiles to Different Forms of Amyloid-Beta Might Explain Efficacy and Side Effects in Clinical Trials for Alzheimer's Disease[J]. Neurotherapeutics, 2023, 20(1): 195-206, Rofo F, Buijs J, Falk R, et al. Novel multivalent design of a monoclonal antibody improves binding strength to soluble aggregates of amyloid beta[J]. Transl Neurodegener, 2021, 10(1): 38]. In the field of tau protein treatment, although there is currently a lack of clear pathogenesis and molecular mechanisms and no effective treatment methods, some studies have begun to explore monoclonal antibody therapy targeting tau protein and methods of reducing tau gene expression through antisense oligonucleotides (ASOs). However, these treatments may reduce the level of normal tau protein, thus bringing potential side effects [Jabbari E, Duff K E. Tau-targeting antibody therapies: too late, wrong epitope or wrong target? [J]. Nat Med, 2021, 27(8): 1341-1342., Congdon EE, Sigurdsson E M. Tau-targeting therapies for Alzheimer disease [J]. Nat Rev Neurol, 2018, 14(7): 399-415, Wang Y, Mandelkow E. Tau inphysiology and pathology [J]. Nat Rev Neurosci, 2016, 17(1): 5-21]. In addition to antibody treatment strategies that directly target Aβ or tau protein, genetic factors can also be used to reduce Aβ deposition or tau fiber tangles from upstream key pathways.The most important genetic risk factor for Alzheimer's disease (AD) is apolipoprotein E (APOE) [Serrano-Pozo A, Das S, Hyman BT. APOE and Alzheimer's disease: advances ingenetics, pathophysiology, and therapeutic approaches [J]. Lancet Neurol, 2021, 20(1): 68-80.]. The APOE gene is located on chromosome 19 and has three polymorphic alleles: E2, E3, and E4, of which APOE4 is the strongest genetic risk factor for late-onset AD. Various intervention attempts targeting APOE aim to reduce the factors that drive the disease process from the source, which has great practical significance for the treatment of AD. This approach may open up new avenues for AD treatment and may help delay or prevent the progression of the disease by reducing the key drivers of disease development.

[0004] APOE is expressed in astrocytes, microglia, neurons, and choroid plexus, and multiple studies have shown that APOE can have different effects on the pathological process of Alzheimer's disease (AD) in a cell- and subtype-specific manner. In the Aβ mouse model, knocking out APOE in astrocytes can significantly reduce the deposition of Aβ plaques and inhibit the activation of microglia surrounding the plaques [Mahan TE, Wang C, Bao X, et al. Selective reduction of astrocyte apoE3 and apoE4 strongly reduces Aβ accumulation and plaque-related pathology in a mouse model of amyloidosis [J]. Mol Neurodegener, 2022, 17 (1): 13]. Another study found that when APOE3 was overexpressed in an APOE systemic knockout mouse model, the cognitive function of the mice was improved, the number of microglia around the plaques increased, and the deposition of Aβ plaques and dystrophic neurites were significantly reduced.

[0005] (Dystrophic neurites, DNs) coverage area; while overexpression of APOE4 in the same background will lead to the disappearance of protective function due to damage to lipid metabolism [Liu CC, Wang N, Chen Y, et al. Cell-autonomous effects of APOE4 in restricting microglial response in brainhomeostasis and Alzheimer's disease[J]. Nat Immunol, 2023, 24(11): 1854-1866]. In mouse models of tauopathy, knocking out APOE4, but not APOE3, in astrocytes [Wang C, Xiong M, Gratuze M, et al. Selective removal of astrocytic APOE4 strongly protects against tau-mediated neurodegeneration and decreases synaptic phagocytosis by microglia[J]. Neuron, 2021, 109(10): 1657-1674.e7.] or neurons [Koutsodendris N, Blumenfeld J, Agrawal A, et al. Neuronal APOE4 removal protects against tau-mediated gliosis, neurodegeneration and myelin deficits[J]. Nat Aging, 2023, 3(3): 275-296] can significantly attenuate neuroinflammation and slow down the progression of tau pathology. However, another study showed that knocking out APOE in microglia did not effectively reduce tau pathology and related neurodegenerative lesions [Yin Z, Rosenzweig N, Kleemann KL, et al. APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFβ-mediated checkpoints [J]. Nat Immunol, 2023, 24 (11): 1839-1853]. These research results reveal the specific effects of APOE and its subtypes from different cell sources on the pathological process of AD.

[0006] As one of the important sources of APOE in the brain, the APOE expressed in the choroid plexus may directly affect the brain parenchyma in a subtype-specific manner. This may directly enter the brain parenchyma through the lymphatic system, or directly or indirectly regulate the neurophysiological function of nerve cells by regulating the secretion of choroid plexus growth factor. In addition, APOE in the choroid plexus may also interfere with the clearance rate of interstitial fluid in the brain parenchyma and change the metabolism of the central nervous system by affecting the blood-cerebrospinal fluid barrier and perivascular macrophages, thereby participating in the occurrence and development of AD pathology.

[0007] In view of this, the present invention intends to use an animal model, with the first purpose of exploring the effect of apoE derived from the choroid plexus on apoE in the brain parenchyma, and the mechanism of Aβ clearance of the choroid plexus apoE in the brain parenchyma; the second purpose of the present invention is to explore the specific mechanism of action of choroid plexus apoE in the progression of AD disease, and to clarify whether choroid plexus apoE can serve as a specific target for Aβ clearance in the brain parenchyma; the third purpose of the present invention is that this study will provide a theoretical and practical basis for Aβ clearance in the brain parenchyma, and choroid plexus targeted gene therapy may serve as a potential therapeutic target for Aβ clearance. Summary of the invention

[0008] The purpose of the present invention is to provide an application of choroid plexus overexpression in the treatment of Alzheimer's disease, and to provide a new idea for clearing Aβ and treating AD.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The present invention provides application of choroid plexus overexpression in treating Alzheimer's disease.

[0011] Furthermore, the application method is: injecting choroid plexus-specific AAV virus in a stereotaxic manner to promote the choroid plexus to synthesize and secrete apoE into the cerebrospinal fluid, thereby participating in the treatment of Alzheimer's disease.

[0012] The present invention provides application of choroid plexus overexpression in reducing the diffusion of beta-amyloid protein.

[0013] The present invention has at least the following beneficial effects:

[0014] The present invention provides the selection of the choroid plexus as a special target that can become a new target for the treatment of AD, whether apoE synthesized by choroid plexus epithelial cells and secreted into the cerebrospinal fluid is involved in the disease progression of AD, and reveals the connection between choroid plexus apoE and brain parenchyma Aβ, which may open up a new idea for the prevention and treatment of Alzheimer's disease; the present invention explores the effects of overexpression and knockdown of apoE4 in the choroid plexus on brain parenchyma Aβ, which may provide a new idea for reducing the spread of Aβ and thus alleviating AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0016] Figure 1 Schematic diagram of virus testing to verify whether the produced AAV is specifically expressed in the choroid plexus;

[0017] Figure 2 This is a comparison of amyloid plaque Aβ in the APOE2, APOE3, APOE4 and control groups of 5xFAD / APOE-KO (5AEKO) mice by immunofluorescence;

[0018] Figure 3 Schematic diagram of the comparison of X34, Aβ, and LAMP1 in the APOE2, APOE3, APOE4 and control groups of mice. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0020] The main purpose of the present invention is to 1. reveal the effect of apoE from the choroid plexus on apoE in the brain parenchyma, and the mechanism of Aβ clearance of choroid plexus apoE in the brain parenchyma. 2. clarify the specific mechanism of action of choroid plexus apoE in the AD disease process, and clarify whether choroid plexus apoE can serve as a specific target for Aβ clearance in the brain parenchyma. 3. provide theoretical and practical basis for Aβ clearance in the brain parenchyma, and choroid plexus targeted gene therapy may serve as a potential therapeutic target for Aβ clearance. The specific technical scheme is as follows:

[0021] 1. Choroid Plexus-specific AAV Production

[0022] We selected choroid plexus-specific 2 / 5 serotype AAV viruses, which carry different subtypes of APOE2 / APOE3 / APOE4. The AAV viruses used in the present invention were purchased from Wuhan Shumi Brain Science Technology Co., Ltd.

[0023] 2. Establishment of AD mouse model with overexpression of apoE2 / 3 / 4 in the choroid plexus and control model

[0024] The laboratory introduced 5×FAD mice and ApoE-KO (apolipoprotein E knockout) mice, Aβ plaque mouse models that simulate Alzheimer's disease pathology, from David Holtzman's laboratory. 5xFAD / APOE-KO (5AEKO) double-gene transgenic mice were obtained by hybridizing 5×FAD mice and ApoE-KO mice. The 1.2-month-old 5xFAD / apoE-KO mice were divided into 4 groups, and the AAV viruses overexpressing apoE2, apoE3, and apoE4 in the choroid plexus and their control viruses were injected into the bilateral lateral ventricles of the mice by stereotaxic injection. After the model was successfully established, the mice were raised in the same environment with unlimited feed and water. Brains were removed and analyzed at 4.5 months of age.

[0025] 3. Mouse Brain Tissue Extraction

[0026] (1) Infusion

[0027] 1) Anesthesia: 5% chloral hydrate was injected into the mouse at a ratio of 100 ul / g by intraperitoneal injection. After the mouse's respiratory rate stabilized, the limbs were fixed, the chest cavity was cut open to expose the heart, and a needle that had been rinsed with 0.5 M EDTA in advance was pointed at the apex of the heart to draw apical blood. The blood was centrifuged at 5,500 g for 10 minutes at 4°C, and the supernatant was obtained as a plasma sample, which was stored at -80°C for later use.

[0028] 2) Use a peristaltic pump to perfuse pre-chilled phosphate buffered saline (PBS) at a flow rate of 11 ml / min for 3 minutes until the liver turns white, then stop perfusion.

[0029] 3) Anesthetize the mice by intraperitoneal injection of 5% chloral hydrate at a ratio of 100 ul / g. After the respiratory rate of the mice stabilized, the limbs were fixed, the chest cavity was cut open to expose the heart, and the apical blood was drawn by aligning a needle that had been rinsed with 0.5 M EDTA in advance to the apex. The blood was centrifuged at 5,500 g for 10 minutes at 4°C. The supernatant was obtained as a plasma sample and stored at -80°C for later use.

[0030] 4) Use a peristaltic pump to perfuse pre-chilled phosphate buffered saline (PBS) at a flow rate of 11 ml / min for 3 minutes until the liver turns white, then stop perfusion.

[0031] (2) Tissue fixation and dehydration

[0032] 1) Carefully remove the mouse brain with forceps and place it on an ice plate. The left half of the brain is placed in pre-cooled 4% paraformaldehyde (PFA) at 4°C overnight. The other half is peeled off according to the location of different brain regions, such as the anterior and posterior cortex, hippocampus, cerebellum, thalamus, etc., and immediately frozen at -80°C for later use.

[0033] 2) The left brain was treated with 4% PFA at 4°C overnight and then transferred to 25% sucrose overnight. The next day, the brain was transferred to 30% sucrose until it was completely precipitated. The slices were cut using a Leica slicer and placed in a pre-prepared freezing solution at -20°C for later use.

[0034] Please refer to Figure 1-Figure 2 .

[0035] Figure 1 To verify whether the produced AAV is specifically expressed in the choroid plexus, we conducted a virus test. The mice were injected at 1.5 months of age and samples were taken at 4 months of age. It can be seen that the constructed virus can be expressed very efficiently in the choroid plexus tissue. And continue to spread to the hippocampus. In previous studies, in order to verify the specific expression of APOE in the choroid plexus and whether APOE in the choroid plexus can spread to adjacent brain tissues with CSF circulation, we selected AAV2 / 5 serotype APOE4 virus and its control EGFP. The mice were injected at 1.5 months of age and samples were taken at 4 months of age. Immunofluorescence staining showed that the constructed virus can be expressed very efficiently in the choroid plexus tissue. And continue to spread to the hippocampus.

[0036] Figure 2 To compare the amyloid plaques Aβ in the APOE2, APOE3, APOE4 and control groups of 5xFAD / APOE-KO (5AEKO) mice by immunofluorescence, the plaques formed by amyloid deposition are one of the most important pathological manifestations of AD. We selected two brain sections of the same unprecedented dimension for each mouse for X-34, Aβ, and LAMP1 staining. X-34 stains the core dense amyloid plaques, EGFP is green fluorescence, Aβ stains the loose amyloid plaques, and LAMP1 stains the dystrophic neurites. In 5AEKO mice, the choroid plexus overexpresses E2, E3, and E4. We collected brain slices for immunofluorescence staining and found that compared with the control group, the X-34, Aβ, and LAMP indicators of the thalamus in the APOE4 group increased significantly, the amyloid plaque deposition increased, and the nerve damage was more severe, indicating that APOE4 aggravated the pathology of Alzheimer's disease; the X-34, Aβ, and LAMP indicators of the thalamus in the APOE2 group decreased, the amyloid plaque deposition decreased, and the nerve damage was alleviated, indicating that the pathology of Alzheimer's disease was alleviated, and the APOE2 gene has a protective effect on Alzheimer's disease. It can be seen that different subtypes of choroid plexus APOE have different effects on the deposition of amyloid plaques and nerve damage pathology in Alzheimer's disease, verifying the feasibility of the choroid plexus as a target for the treatment of Alzheimer's disease.

[0037] We further conducted experimental verification of the effects of overexpression and knockdown of apoE in the choroid plexus:

[0038] We injected 1.2-month-old 5xFAD / APOE-KO (5AEKO) mice with choroid plexus-specific AAV viruses of APOE2, APOE3, and APOE4 by stereotaxic means, and harvested their brains for immunofluorescence analysis at 4.5 months of age.

[0039] A. Fixation of brain tissue: The left cerebral hemisphere of the mouse was immersed in 4% paraformaldehyde solution overnight to fix the tissue.

[0040] B. Dehydration: The fixed brain tissue samples were immersed in 30% sucrose solution for at least 24 hours for dehydration.

[0041] C. Sectioning: Dehydrated brain tissue was cut into 50 μm thick serial coronal sections using a Leica sliding microtome.

[0042] D. Washing: Wash the slices three times in PBS, 5 minutes each time.

[0043] E. Permeabilization: After washing, the sections were incubated in 0.25% Triton X-100 in PBS at room temperature on a shaker for 20 minutes.

[0044] F. Then wash again in PBS 3 times, 5 minutes each time.

[0045] G. Perform X-34 staining and incubate the prepared X-34 staining solution (40% ETOH, 60% PBS, 1:500 10NNaOH, 1:5,000X-34) at room temperature for 20 minutes.

[0046] H. Wash three times with 40% ETOH + 60% PBS, 2 minutes each time.

[0047] I. Wash 3 times with PBS for 5 minutes each time.

[0048] J. Blocking: Block the sections in 5% bovine serum albumin for 1 hour at room temperature.

[0049] K. Primary antibody incubation: Incubate with primary antibodies, including Aβ and LAMP1, overnight at 4°C.

[0050] L. Secondary antibody incubation: On the second day, after washing 3 times in PBS, the sections were incubated with the corresponding fluorescent labeled secondary antibody at room temperature for 1 hour, and then washed 3 times in PBS, 5 minutes each time, and then sealed.

[0051] M. Imaging and Analysis: All fluorescent staining in this section was imaged using an Olympus laser spinning disk confocal microscope.

[0052] All results are reported as mean ± standard error (SEM), and all statistical analyses were performed in Prism 9 (GraphPad) software. The significant difference between the two groups was determined by the ROUT method, and the outliers in each group were identified by Q = 0.2%. After removing the outliers, the one-way ANOVA multiple comparison method was used to determine the significant differences between more than two groups. The comparison between the two groups was performed using an unpaired t-test of two independent samples. A p value less than 0.05 (p < 0.05) was considered to be a statistically significant difference. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0053] For details, please refer to Figure 2 and Figure 3 , Immunofluorescence staining of X34, Aβ, and LAMP1 of APOE2, APOE3, and APOE4 in 5AEKO female mice, 20×, scale bar = 500 μm. 5AEKO female mice were injected with APOE2, APOE3, APOE4, and empty control viruses. In mice injected with APOE4 virus, amyloid plaque deposition in the thalamus increased significantly, and neurological damage was more severe. In mice injected with APOE2 virus, amyloid plaque deposition in the thalamus decreased, and neurological damage was alleviated. A brain slice from a different location was selected for each mouse, and the same location was selected for each group. The final results were averaged, and the one-way ANOVA multiple comparison method was used to determine the significant differences between more than two groups. The comparison between the two groups was performed using the unpaired t-test of two independent samples. *P<0.05, **P<0.01, ***P<0.001.

[0054] Combining the above, we can know that:

[0055] (1) The present invention provides the selection of the choroid plexus as a special target, which can become a new target for the treatment of AD. To date, it is still unclear whether apoE synthesized by choroid plexus epithelial cells and secreted into the cerebrospinal fluid is involved in the disease progression of AD and its mechanism of action. The present invention reveals the connection between choroid plexus apoE and brain parenchyma Aβ, which may open up a new idea for the prevention and treatment of Alzheimer's disease. (2) Exploring the effects of overexpression and knockdown of apoE4 in the choroid plexus on brain parenchyma Aβ may provide new ideas for reducing the spread of Aβ and thus alleviating AD.

[0056] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. Application of choroid plexus overexpression in the treatment of Alzheimer's disease.

2. The use according to claim 1, characterized in that: The application method is: injecting choroid plexus-specific AAV virus in a stereotaxic manner to promote the choroid plexus to synthesize and secrete apoE into the cerebrospinal fluid, thereby participating in the treatment of Alzheimer's disease.

3. Application of choroid plexus overexpression in reducing the spread of β-amyloid protein.

Images