Mutant protein related to Alzheimer disease, disease model and pharmaceutical composition

Abstract

The invention relates to a mutant protein related to Alzheimer's disease, a disease model and a pharmaceutical composition. The mutant protein is characterized in that phenylalanine at the 237th site of an amino acid sequence of PSEN1 protein is mutated into leucine (PSEN1p.F237L), and the mutant protein is associated with the Alzheimer's disease. The invention finds that the mutation affects the interaction of PSEN1 protein and calcium channel proteins (such as SERCA2 and RyR2), resulting in neuron calcium homeostasis imbalance, energy metabolism disorder and endoplasmic reticulum stress. The invention constructs a neuron animal disease model based on the mutation, simulates the pathological process of Alzheimer's disease, and is used for drug mechanism research and screening. Meanwhile, the invention provides a pharmaceutical composition aiming at a PSEN1 mutation related pathological process, and the pharmaceutical composition is used for improving calcium ion homeostasis, mitochondrial dysfunction and endoplasmic reticulum stress. The scheme provided by the invention can be used for Alzheimer disease pathogenic mechanism research, drug screening and treatment strategy development.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a mutant protein associated with Alzheimer's disease (AD), a disease model and a pharmaceutical composition. BACKGROUND

[0002] At present, the conventional drugs approved by FDA for improving cognitive symptoms of AD mainly include NMDA receptor antagonists and cholinesterase inhibitors. Memantine, as a non-competitive NMDA receptor antagonist, plays a neuroprotective role in moderate to severe AD patients by selectively blocking the over-activated NMDA receptor. Donepezil, as a typical cholinesterase inhibitor, improves cognitive function by inhibiting the degradation of acetylcholine, and is suitable for mild to severe AD patients.

[0003] In addition, there are also some candidate drugs in application or development targeting amyloid, tau protein, mitochondrial cascade, endoplasmic reticulum stress and other targets or mechanisms. However, the current AD drugs generally have limited efficacy and large side effects. Therefore, there is an urgent need in the art to develop new therapeutic means. SUMMARY

[0004] The mutant protein of the present application can cause AD phenotypes of neurons and mice: PSEN1p.F237L mutation causes various abnormal phenotypes such as neuronal atrophy, calcium homeostasis imbalance, energy metabolism disorder, endoplasmic reticulum stress; mice carrying the mutation show cognitive impairment, and Aβ deposition and Tau abnormal phosphorylation in the hippocampal brain region of mice.

[0005] The pharmaceutical composition of the present application can provide a new AD treatment strategy, improve the abnormal phenotypes of PSEN1p.F237L mutant neurons and mice, provide a new direction for AD treatment, and make up for the shortcomings of existing treatment programs.

[0006] The technical solutions of the present application are as follows: A mutant protein, which is PSEN1 protein mutated from phenylalanine at amino acid sequence position 237 to leucine (PSEN1p.F237L), is associated with AD.

[0007] A nucleic acid molecule encoding the above-mentioned mutant protein, in which the thymine at position c.709 is changed to cytosine.

[0008] The above-mentioned mutant protein, or the above-mentioned nucleic acid molecule has the following uses: (1) its use as a marker in the diagnosis of AD; (2) its use in the preparation of a drug for improving mitochondrial structure and dysfunction of neurons; (3) its use in improving PSEN1 p.F237L mutant neuron dendrite and synapse related protein expression; (4) its use in relieving PSEN1 p.F237L mutant neuron endoplasmic reticulum stress; (5) its use in preparing a drug for preventing and / or treating AD.

[0009] A disease model carrying the mutant protein described above, including neurons and transgenic mouse models.

[0010] The disease model described above is used in any of the following aspects: (1) its use in disease mechanism research; (2) its use in new drug screening, efficacy verification and safety evaluation; (3) its use in early diagnosis of diseases and screening and verification of biomarkers; (4) its use in the development and verification of new treatment strategies; (5) its use in the study of specific gene functions, signal pathway regulation and cell biology processes.

[0011] A pharmaceutical composition acting on the related pathways mediated by the mutant protein described above, including a SERCA2 agonist including CDN1163 and a RyR2 stabilizer including S107.

[0012] The pharmaceutical composition described above is used in any of the following aspects: (1) improving mitochondrial structure and dysfunction in neurons; (2) improving PSEN1 p.F237L mutant neuron dendrite and synapse related protein expression; (3) relieving PSEN1 p.F237L mutant neuron endoplasmic reticulum stress; (4) improving cognitive impairment in PSEN1 p.F237L mutant mice; (5) preventing and / or treating AD.

[0013] Preferably, the Alzheimer's disease is caused by PSEN1 p.F237L mutation, and the disease tissue or cell contains PSEN1 p.F237L mutant protein or its encoding gene.

[0014] The beneficial effects of the present application are as follows: The present application focuses on the neuron damage and cognitive impairment of mice caused by the specific factor PSEN1p.F237L mutation for the first time, reveals that the PSEN1p.F237L mutation affects the interaction of PSEN1 protein with calcium channel protein SERCA2 and RyR2, causes imbalance of neuronal calcium homeostasis and energy metabolism disorder, and provides a new direction for the selection of therapeutic targets, and breaks through the limitations of previous research on conventional pathogenic factors of AD.

[0015] The present application innovatively proposes a pharmaceutical composition, CDN1163 as a SERCA2 agonist, S107 as a RyR2 stabilizer, and the combination of the two can improve various abnormal phenotypes of PSEN1p.F237L mutant neurons. Compared with single drug treatment, CDN1163 combined with S107 can significantly improve the energy metabolism disorder of neurons, increase the oxygen consumption rate of neural stem cells, improve the complexity of neuron dendrites and synaptic plasticity, promote mitochondrial fusion, increase mitochondrial membrane potential, reduce the level of reactive oxygen species, improve endoplasmic reticulum stress, significantly improve the AD-related phenotype of PSEN1p.F237L mutant neurons, significantly reduce the pathological deposition in the hippocampal brain area of PSEN1p.F237L mutant mice, improve the ability of spatial learning and memory, and more comprehensively alleviate the problems of neuron atrophy, thereby providing a new combined drug treatment idea for AD treatment. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 : Induction differentiation and identification of neural stem cells and neurons, A: induction process of neural stem cells, hiPSCs cell suspension was uniformly distributed in a microwell plate on day 0, on day 5, a blastula was formed, on day 12, a neural tube-like rosette structure was formed, Scale Bar = 2 mm; B: immunofluorescence identification of the expression of neural stem cell marker proteins Nestin and SOX1, Scale Bar = 20 μm; C: qPCR detection of the transcription level of hiPSCs marker gene OCT-4 and neural stem cell marker gene PAX6; D: immunofluorescence identification of the expression of Tuj1 and MAP2 in mature neurons with PSEN1p.F237L mutation, Scale Bar = 25 μm. Indicates p<0.001.

[0017] Figure 2: PSEN1 p.F237L mutant neurons present related phenotypes, A: representative images of control and PSEN1 p.F237L mutant neurons, Scale Bar = 10 μm; B: Sholl Analysis analysis of neuron dendritic complexity; C: Western-Blot detection of MAP2 and SNAP25 expression; D: quantification analysis of MAP2 and SNAP25 expression; E: Western-Blot detection of t-Tau and p-Tau expression in neurons; F: quantification analysis of t-Tau and p-Tau expression. p < 0.05, p < 0.01, p < 0.001.

[0018] Figure 3 : Genotype identification of PSEN1 p.F237L mutant mice, A: agarose gel electrophoresis results of mouse tail identification, the target band size is 472 bp; B: Sanger sequencing results of PSEN1 gene of wild type, PSEN1 p.F237L heterozygous and homozygous mutant mice, wild type mice are single peak "T", heterozygous mutant mice are "T" and "C" double peak, wild type mice are single peak "C".

[0019] Figure 4 : Hippocampal neuron dendritic complexity of PSEN1 p.F237L mutant mice is reduced, A: representative images of mouse hippocampal neurons, Scale Bar = 20 μm; B: quantification analysis of mouse hippocampal neuron dendritic complexity; C: Western-Blot detection of MAP2 and SNAP25 expression in mouse hippocampal neurons; D: quantification analysis of MAP2 and SNAP25 expression. p < 0.05, p < 0.001.

[0020] Figure 5 : Hippocampal brain regions of PSEN1 p.F237L mutant mice present Aβ42 pathological deposition and Tau abnormal phosphorylation, A: immunohistochemical detection of Aβ42 pathological deposition in mouse hippocampal brain regions; B: quantification analysis of the number of Aβ deposition plaques; C: Western-Blot detection of t-Tau and p-Tau expression in mouse hippocampal brain regions; D: quantification analysis of t-Tau and p-Tau expression; Scale Bar = 100 μM. p < 0.05, p < 0.01, p < 0.001.

[0021] Figure 6: PSEN1 p.F237L mutant neurons exhibit endoplasmic reticulum calcium store depletion and endoplasmic reticulum stress, A-B: Pseudocolor images of endoplasmic reticulum (A, Scale Bar = 2 μm) and cytosol (B, Scale Bar = 10 μm) calcium ion concentration in neurons, color from red to blue represents calcium ion concentration from high to low, Pre indicates resting state before TG treatment, Peak indicates peak value after TG treatment, Plateau indicates stable plateau after TG treatment; C: Changes of endoplasmic reticulum and cytosol calcium ion concentration before and after TG treatment; D: Standardized curve of endoplasmic reticulum and cytosol calcium ion concentration changes; E: Quantitative analysis of endoplasmic reticulum calcium ion concentration at resting state, peak value and plateau, δCa2+ indicates the amplitude of endoplasmic reticulum calcium ion decrease induced by TG; F: Quantitative analysis of cytosol calcium ion concentration at resting state, peak value and plateau, δCa2+ indicates the amplitude of cytosol calcium ion concentration increase induced by TG. p < 0.05, p < 0.01, p < 0.001, ns indicates no significant difference (p ≥ 0.05).

[0022] Figure 7 : PSEN1 p.F237L mutant neurons exhibit endoplasmic reticulum calcium store depletion and endoplasmic reticulum stress, A-B: Pseudocolor images of endoplasmic reticulum (A, Scale Bar = 2 μm) and cytosol (B, Scale Bar = 10 μm) calcium ion concentration in neurons, color from red to blue represents calcium ion concentration from high to low, Pre indicates resting state before TG treatment, Peak indicates peak value after TG treatment, Plateau indicates stable plateau after TG treatment; C: Changes of endoplasmic reticulum and cytosol calcium ion concentration before and after TG treatment; D: Standardized curve of endoplasmic reticulum and cytosol calcium ion concentration changes; E: Quantitative analysis of endoplasmic reticulum calcium ion concentration at resting state, peak value and plateau, δCa2+ indicates the amplitude of endoplasmic reticulum calcium ion decrease induced by TG; F: Quantitative analysis of cytosol calcium ion concentration at resting state, peak value and plateau, δCa2+ indicates the amplitude of cytosol calcium ion concentration increase induced by TG. p < 0.001.

[0023] Figure 8: PSEN1p.F237L mutation alters its interaction with SERCA2 and RyR2 and affects their associated phosphorylation, A: IP-MS analysis of differentially interacting proteins between wild type and PSEN1p.F237L mutant PSEN1, including SERCA2 and RyR2, which are related to endoplasmic reticulum calcium homeostasis; B: Analysis of the ratio of peptide peak area of SERCA2 and RyR2 in IP and Input samples in control and mutant neurons; C: Co-IP detection of the binding ability of PSEN1 and its PSEN1p.F237L mutant to SERCA2; D: Co-IP detection of the binding ability of PSEN1 and its PSEN1p.F237L mutant to RyR2; E: Western-Blot detection of the protein expression of SERCA2, PLN, p-PLN-Ser16, RyR2 and p-RyR2-Ser2814 in neurons; F: Quantitative analysis of the expression of SERCA2, PLN, p-PLN-Ser16, p / t-PLN, RyR2, p-RyR2-Ser2814 and p / t-RyR2. p < 0.05, p < 0.01, p < 0.001.

[0024] Figure 9 : PSEN1p.F237L mutant neurons exhibit insufficient mitochondrial energy metabolism, A: Western-Blot detection of MFN2 and Fis1 content in control and mutant neurons; B: Quantitative analysis of MFN2 and Fis1 protein expression; C: Representative images of MMP fluorescence in control and mutant neurons; D: Quantitative analysis of MMP levels; E: Representative images of mitochondrial ROS fluorescence in control and mutant neurons; F: Quantitative analysis of mitochondrial ROS content; G: Oxygen consumption rate curves of control and mutant neural stem cells; H: Quantitative analysis of basal respiration, maximum respiration and oxygen consumption rate for ATP synthesis of neural stem cells. Scale Bar = 5 μm. p < 0.05, p < 0.01, p < 0.001.

[0025] Figure 10 : Endoplasmic reticulum stress-related protein expression in neurons, A: Western-Blot detection of the expression levels of endoplasmic reticulum stress-related proteins BIP, PERK, ATF4, ATF6, IRE1, p-IRE1 in normal control and PSEN1p.F237L mutant neurons; B: Quantitative analysis of the expression levels of endoplasmic reticulum stress-related proteins. p < 0.05, ns p ≥ 0.05.

[0026] Figure 11 Schematic representation of the target modulation of SERCA2 and RyR2 by calcium homeostasis modulators.

[0027] Figure 12 Calcium homeostasis modulators improve endoplasmic reticulum calcium homeostasis in PSEN1 p.F237L mutant neurons, A: Changes in endoplasmic reticulum calcium concentration and its normalization curve before and after TG treatment; B: Quantitative analysis of endoplasmic reticulum calcium concentration at resting state; C: Quantitative analysis of the amplitude of endoplasmic reticulum calcium decrease induced by TG; D: Changes in cytosolic calcium concentration and its normalization curve before and after TG treatment; E: Quantitative analysis of cytosolic calcium concentration at resting state; F: Quantitative analysis of the amplitude of cytosolic calcium increase induced by TG. p < 0.05, p < 0.001, ### p < 0.001 (mutant neurons vs. normal control neurons), ns not significant (p ≥ 0.05).

[0028] Figure 13 Calcium homeostasis modulators restore mitochondrial calcium homeostasis in PSEN1 p.F237L mutant neurons, A: Changes in endoplasmic reticulum calcium concentration and its normalization curve before and after ARN11391 treatment; B: Quantitative analysis of endoplasmic reticulum calcium concentration at resting state; C: Quantitative analysis of the amplitude of endoplasmic reticulum calcium decrease induced by ARN11391; D: Changes in mitochondrial calcium concentration and its normalization curve before and after ARN11391 treatment; E: Quantitative analysis of mitochondrial calcium concentration at resting state; F: Quantitative analysis of the amplitude of mitochondrial calcium increase induced by ARN11391. p < 0.01, p < 0.001, ### p < 0.001 (mutant neurons vs. normal control neurons).

[0029] Figure 14 Calcium homeostasis modulators improve mitochondrial fusion-fission balance in PSEN1 p.F237L mutant neurons, A, C, E: Western-Blot detection of MFN2 and Fis1 expression after CDN1163 (A), S107 (C), CDN1163 and S107 (E) intervention in neurons; B, D, F: Quantitative analysis of MFN2 and Fis1 protein expression. p < 0.05, p < 0.01, p < 0.001, ns not significant (p ≥ 0.05).

[0030] Figure 15Calcium homeostasis modulators enhanced mitochondrial function in PSEN1 p.F237L mutant neurons, A: Representative images of MMP fluorescence; B: Quantitative analysis of MMP fluorescence intensity; C: Representative images of mitochondrial ROS fluorescence; D: Quantitative analysis of mitochondrial ROS level fluorescence intensity; E: Seahorse assay plot of mitochondrial oxygen consumption rate in neural stem cells; F: Quantitative analysis of basal respiration, maximal respiration and ATP production related oxygen consumption rate in neural stem cells. Scale Bar = 5 pm. p < 0.05, p < 0.01, p < 0.001, ## p < 0.01, ### p < 0.001 (mutant neurons vs. normal control neurons).

[0031] Figure 16 Calcium homeostasis modulators alleviated endoplasmic reticulum stress in PSEN1 p.F237L mutant neurons, A, C, E: Western-Blot analysis of endoplasmic reticulum stress protein BIP expression after CDN1163 (A), S107 (C), CDN1163 and S107 (E) intervention; B, D, F: Quantitative analysis of BIP expression. p < 0.05, p < 0.01, p < 0.001, ns, no significant difference (p ≥ 0.05).

[0032] Figure 17 Calcium homeostasis modulators increased dendritic complexity in PSEN1 p.F237L mutant neurons, A: Morphological performance of control and PSEN1 p.F237L mutant neurons after CDN1163, S107 intervention alone or in combination; B: Sholl Analysis quantitative results on neuronal dendritic complexity; C, E, G: Western-Blot analysis of MAP2 and SNAP25 expression in neurons after CDN1163 (C), S107 (E), CDN1163 and S107 (G) intervention; D, F, H: Quantitative analysis of MAP2 and SNAP25 expression. p < 0.05, p < 0.01, p < 0.001, ns, no significant difference (p ≥ 0.05).

[0033] Figure 18: 10 μM CDN1163 combined with 10 μM S107 intervention reduced Tau abnormal phosphorylation in PSEN1 p.F237L mutant neurons, A, C, E: Western-Blot detected t-Tau and p-Tau protein expression in neurons after CDN1163 (A), S107 (C) and CDN1163 + S107 (E) intervention; B, D, F: Quantitative analysis of t-Tau and p-Tau protein expression. p < 0.05, p < 0.01, p < 0.001, ns means no significant difference (p ≥ 0.05).

[0034] Figure 19 : CDN1163 combined with S107 intervention increased the dendritic complexity of PSEN1 p.F237L mutant mouse hippocampal neurons, A: Golgi staining showed the results of Sholl Analysis of mouse hippocampal neurons, Scale Bar = 50 μm; B: Quantitative analysis of the number of dendritic branches and total length of neurons, C: Golgi staining showed the morphology of dendritic spines of mouse hippocampal neurons, Scale Bar = 10 μm; D: Quantitative analysis of the density of mushroom-shaped dendritic spines. E: Western-Blot detected the expression of MAP2 and SNAP25 in mouse hippocampal brain regions; F: Quantitative analysis of MAP2 and SNAP25 protein expression. p < 0.05, p < 0.01, p < 0.001, ns means no significant difference (p ≥ 0.05).

[0035] Figure 20 : CDN1163 combined with S107 intervention reduced pathological deposits in the hippocampal brain regions of PSEN1 p.F237L mutant mice, A: Immunohistochemical detection of Aβ42 plaque deposition in the hippocampal brain regions of mice; B: Quantitative analysis of Aβ42 plaque in the hippocampal brain regions of mice. C: Western-Blot detected the expression of t-Tau and p-Tau in the hippocampal brain regions of mice; D: Quantitative analysis of t-Tau and p-Tau expression; Scale Bar = 100 μM. p < 0.05, p < 0.01, p < 0.001, ns means no significant difference (p ≥ 0.05).

[0036] Figure 21Figure 6. CDN1163 and S107 combined intervention improves the spatial learning and memory ability of PSEN1p.F237L mutant mice. A: Change curve of escape latency of mice in the training stage of 1-5 days; B: Representative trajectory of mice swimming from the contralateral quadrant to the hidden platform on the training day 5; C: Quantitative analysis results of escape latency on the training day 5; D: Heat map of swimming trajectory of mice in the spatial exploration stage on the day 6; E: Quantitative analysis of the number of times of mice crossing the platform area; F: Quantitative analysis of the time of mice staying in the target quadrant. p < 0.05, p < 0.01, p < 0.001, and ns represents no significant difference (p ≥ 0.05). DETAILED DESCRIPTION

[0037] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. The specific conditions not indicated in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by purchase.

[0038] A pharmaceutical composition acting on the PSEN1p.F237L-mediated related pathway, comprising CDN1163 and S107.

[0039] The above-mentioned pharmaceutical composition is used in any of the following aspects: (1) improving the structure and dysfunction of neuronal mitochondria; (2) improving the expression of PSEN1p.F237L mutant neuron dendrite and synapse-related proteins; (3) relieving the endoplasmic reticulum stress of PSEN1p.F237L mutant neurons; (4) preventing and / or treating AD.

[0040] For the activation strategy of SERCA2, the existing drugs include Pimobendan, Istaroxime and CDN1163, etc. Among them, Pimobendan and Istaroxime have lower targeting in the nervous system, while CDN1163 as a typical allosteric activator can pass through the blood-brain barrier, directly enhance the function of SERCA2 calcium pump, promote the endoplasmic reticulum calcium recovery, and effectively improve the calcium disorder and related dysfunction in neurons, myocardial cells and other cells.

[0041] Current drugs targeting RyR2 include JTV519, Dantrolene and S107. Among them, JTV519 and Dantrolene have limited blood-brain barrier penetration, while S107 reduces abnormal calcium leakage by enhancing the stability of RyR2-Calstabin2 binding. Its advantages are that it can retain normal calcium release function, has fewer side effects and can cross the blood-brain barrier, and it has shown protective effects on endoplasmic reticulum calcium homeostasis in heart failure and cardiogenic dementia models.

[0042] This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University, and the written informed consent of the subjects was obtained.

[0043] Example 1: PSEN1p.F237L mutant neurons and mouse disease models exhibit AD-related phenotypes I. Construction of PSEN1p.F237L mutant neuron model Patient-derived and family control (patient's sister) skin fibroblasts were induced into human induced pluripotent stem cells (hiPSCs) using reprogramming technology. Then, normal and PSEN1p.F237L mutant hiPSCs were induced to differentiate into neural stem cells (NSCs) and neurons. Figure 1 A). Immunofluorescence staining results showed that both strains of neural stem cells expressed neural stem cell marker proteins Nestin and SRY-Box Transcription Factor 1 (SOX1) Figure 1 B). qPCR detection results further showed that as the differentiation process progressed, the transcription levels of hiPSCs marker gene OCT-4 in both strains of neural stem cells decreased significantly, and the transcription levels of neural stem cell marker gene PAX6 increased significantly Figure 1 C), indicating that hiPSCs had been induced to differentiate into neural stem cells. On this basis, Neurobasal medium (purchased from Thermo Fisher) was used to further induce the two strains of neural stem cells to differentiate into neurons. Immunofluorescence identification results showed that neurons expressed neuron marker proteins Tuj1 and MAP2 after 30 days of differentiation Figure 1 D), indicating that the neuron cell model was successfully constructed.

[0044] II. PSEN1p.F237L mutant neurons exhibit AD-related phenotypes The pCDH-CMV-MCS-EF1a-EGFP plasmid (purchased from GenScript) was transfected into differentiated neurons using the calcium phosphate transfection method, so that the neurons expressed green fluorescent protein. The morphology of neurons was imaged using a laser confocal microscope Figure 2A). Neuronal dendrites were traced using the Simple Neurite Tracer plugin, and the complexity of neuronal dendrites was quantified using Sholl Analysis. The results showed that, compared with the control group, the dendritic complexity of PSEN1p.F237L mutant neurons was reduced ( Figure 2 B). Western blotting results further showed that the expression of microtubule-associated protein MAP2 and synaptic-associated protein SNAP25 was significantly decreased in PSEN1p.F237L mutant neurons. Figure 2 C, D). Furthermore, compared to controls, mutant neurons showed significantly higher expression of both t-Tau and p-Tau (C, D). Figure 2 E, F). The above results suggest that PSEN1p.F237L mutant neurons exhibit AD-related pathological phenotypes, characterized by decreased neuronal dendritic complexity, downregulated expression of dendritic and synaptic-related proteins, and increased expression of t-Tau and p-Tau.

[0045] III. Construction of PSEN1p.F237L mutant mouse model To investigate the mechanism by which the PSEN1p.F237L mutation leads to Alzheimer's disease (AD) in vivo and to explore potential intervention strategies, this invention also constructed a transgenic mouse model carrying this mutation. Genomic DNA was extracted from F1 generation mice, and the PSEN1 gene was amplified to obtain a target band of 472 bp. Figure 3 A), Sanger sequencing results showed that the present invention successfully constructed a mouse model of PSEN1p.F237L mutation ( Figure 3 B).

[0046] IV. The PSEN1p.F237L mutant mouse model exhibits AD-related phenotypes. In this study, pCDH-CMV-MCS-EF1a-EGFP plasmid was transfected into mature primary mouse hippocampal neurons. Neuronal morphology was imaged using laser confocal microscopy, and dendritic complexity was assessed using Sholl analysis. The results showed that, compared to wild-type mice, the dendritic complexity of hippocampal neurons in PSEN1p.F237L mutant mice was significantly reduced. Figure 4 A, B); Western blotting results further showed that the levels of MAP2 and SNAP25 proteins in the hippocampal neurons of mutant mice were both low (A, B); Figure 4 C, D). Immunohistochemical analysis in this study revealed an increase in the number of Aβ42 deposits in the hippocampus of PSEN1p.F237L mutant mice compared to the control group. Figure 5 A, B); Meanwhile, Western blotting results showed that, compared with the control group, the expression of t-Tau and p-Tau in the hippocampus of mutant mice was significantly increased (A, B); Figure 5 C, D).

[0047] Example 2: PSEN1p.F237L mutation induces calcium disturbance via SERCA2 and RyR2, which leads to mitochondrial dysfunction and endoplasmic reticulum stress, resulting in AD I. PSEN1p.F237L mutation reduces endoplasmic reticulum and mitochondrial calcium ion concentration and increases cytosolic calcium ion concentration in neurons Calcium ion is an important second messenger in cells, which is involved in neuronal signal transduction, synaptic plasticity, neurotransmitter release, etc. The endoplasmic reticulum is a key organelle for calcium ion storage and regulation, and the endoplasmic reticulum transports calcium ions through its membrane calcium channel transporters, such as sarcoplasmic / endoplasmic reticulum calcium-ATPase (SERCA), ryanodine receptor (RyR), and inositol 1,4,5-trisphosphate receptor (IP3R), to achieve calcium ion storage and release.

[0048] In this embodiment, specific Ca 2+ ATPase inhibitor TG was used to treat neurons, and the changes in endoplasmic reticulum and cytosolic calcium ion concentrations before and after TG treatment were compared to evaluate the specific situation of endoplasmic reticulum calcium disturbance in mutant neurons. The results showed that before TG treatment, the endoplasmic reticulum calcium ion concentration in PSEN1p.F237L mutant neurons was significantly lower than that in the control group ( Figure 6 A, C, E), while the cytosolic calcium ion concentration was significantly higher than that in the control group ( Figure 6 B, C, F), suggesting that in the resting state, PSEN1p.F237L mutant neurons have endoplasmic reticulum calcium disturbance, characterized by decreased endoplasmic reticulum calcium ion concentration and increased cytosolic calcium ion concentration. After 10 μM TG treatment, whether in the peak phase of calcium ion release or in the subsequent plateau phase, the endoplasmic reticulum calcium ion concentration in mutant neurons was always lower than that in the control group ( Figure 6 A, C, E), while the cytosolic calcium ion concentration was consistently higher than that in the control group ( Figure 6 B, C, F), and the amplitude of endoplasmic reticulum calcium ion decrease ( Figure 6 E) and cytosolic calcium ion increase ( Figure 6 F) caused by TG were significantly reduced compared with the control group, further suggesting that the endoplasmic reticulum calcium reserve in mutant neurons was significantly reduced. The above research results support that PSEN1p.F237L mutation induces endoplasmic reticulum calcium disturbance, which is characterized by decreased endoplasmic reticulum calcium reserve and increased cytosolic calcium concentration.

[0049] In this study, neurons were transfected with the ER-GCaMP6-210 plasmid (purchased from Addgene) and the 4COX8-GCaMP6s plasmid (purchased from Addgene) to detect calcium ion concentrations in the endoplasmic reticulum (ER) and mitochondria. Neurons were treated with ARN11391, and changes in ER and mitochondrial calcium ion concentrations before and after treatment were compared to investigate whether a decrease in ER calcium reserves subsequently affects mitochondrial calcium homeostasis. Results showed that before ARN11391 treatment, the ER-GCaMP6-210 plasmid (purchased from Addgene) in PSEN1p.F237L mutant neurons (…) Figure 7 The concentrations of A, C, and E, as well as mitochondrial calcium ions, were significantly lower than those in the control group. Figure 7 (B, C, F) This indicates that under resting conditions, calcium imbalance exists in both the endoplasmic reticulum and mitochondria of mutant neurons, manifested as insufficient calcium reserves. After treatment with ARN11391, at both the peak and plateau phases, the calcium ion concentrations in the endoplasmic reticulum and mitochondria of mutant neurons remained consistently lower than those in the control group. Figure 7 AF). Furthermore, in mutant neurons, the decrease in endoplasmic reticulum calcium ions induced by ARN11391 was smaller ( Figure 7 E), the increase in mitochondrial calcium ions is also limited ( Figure 7 The F) indicates a significant reduction in calcium transport between the endoplasmic reticulum and mitochondria. In summary, PSEN1p.F237L mutant neurons exhibit endoplasmic reticulum-mitochondrial calcium disorder, specifically manifested as insufficient endoplasmic reticulum calcium reserves, reduced calcium transport between the endoplasmic reticulum and mitochondria, and decreased mitochondrial calcium ion concentration.

[0050] The above results suggest that the PSEN1p.F237L mutation causes an imbalance in neuronal endoplasmic reticulum-mitochondrial calcium homeostasis, specifically manifested as a decrease in endoplasmic reticulum and mitochondrial calcium ion concentrations and an increase in cytoplasmic calcium ion concentrations.

[0051] II. The PSEN1p.F237L mutation induces neuronal endoplasmic reticulum-mitochondrial calcium homeostasis imbalance through SERCA2 and RyR2. This study used IP-MS to reveal the impact of this mutation on PSEN1 interacting proteins, and to identify the specific molecular targets of the endoplasmic reticulum-mitochondrial calcium homeostasis imbalance caused by the PSEN1p.F237L mutation. The results showed that the PSEN1p.F237L mutation altered the PSEN1 interacting protein profile, including proteins related to endoplasmic reticulum calcium homeostasis such as SERCA2 and RyR2. Figure 8 A). By analyzing the ratio of the peak areas of SERCA2 and RyR2 peptides in the IP sample to the peak areas in the Input sample, it was found that the PSEN1p.F237L mutation reduced the binding of SERCA2 and increased the binding of RyR2. Figure 8B), and then this experiment verified through immunoprecipitation that the PSEN1p.F237L mutation weakens the binding of PSEN1 to SERCA2, while enhancing the interaction between PSEN1 and RyR2. Figure 8 C, D).

[0052] SERCA2 and RyR2 are located in the endoplasmic reticulum membrane of neurons and are responsible for calcium uptake and release, respectively. The calcium pump function of SERCA2 is usually reflected by the phosphorylation of its regulator PLN. The dephosphorylation state of PLN indicates that the calcium pump function is inhibited, while its phosphorylation state indicates that the calcium pump function is activated. The function of RyR2 is regulated by its own phosphorylation. Phosphorylation activates the opening of calcium channels, thereby promoting calcium release from the endoplasmic reticulum.

[0053] This study used Western blotting to detect changes in the expression and related phosphorylation of SERCA2 and RyR2, assessing whether the altered binding pathways of PSEN1p.F237L mutation to SERCA2 and RyR2 affect their function. Results showed that, compared to the control group, PSEN1p.F237L mutant neurons exhibited decreased phosphorylation of PLN, a regulator of SERCA2, and a significantly reduced p / t-PLN ratio. Figure 8 E, F), suggesting that the calcium pump function of SERCA2 is inhibited. Simultaneously, compared to the control group, RyR2 expression was downregulated in mutant neurons, while RyR2 phosphorylation was significantly increased, and the p / t-RyR2 ratio was increased ( Figure 8 E, F), indicating that the RyR2 calcium release channel is activated.

[0054] III. Calcium homeostasis imbalance caused by PSEN1p.F237L mutation impairs mitochondrial energy metabolism. This study further examined mitochondrial function in mutant neurons to investigate whether endoplasmic reticulum-mitochondrial calcium disorder induced by PSEN1p.F237L mutation participates in pathogenesis by affecting mitochondrial energy metabolism. Western blot results showed that, compared with the control group, the expression level of mitochondrial fusion protein MFN2 was decreased and the expression of mitochondrial fission protein Fis1 was significantly upregulated in mutant neurons. Figure 9 A, B), indicating a dynamic homeostasis disorder in the mitochondria of the mutant neurons; MMP detection using Rh800 dye revealed that the MMP levels in the mutant neurons were significantly lower than those in the control group (A, B). Figure 9 C, D); Mito-SOX dye was used to detect mitochondrial ROS, showing that the ROS in the mitochondria of mutant neurons was significantly increased (C, D). Figure 9 E, F); In addition, energy metabolism analysis showed that the basal oxygen consumption, maximum respiratory capacity, and ATP synthesis-related oxygen consumption of mutant neural stem cells were significantly lower than those of the control group (E, F); Figure 9G, H). The above results suggest that the imbalance of ER-mitochondria calcium homeostasis caused by PSEN1p.F237L mutation damages the energy metabolism of mitochondria.

[0055] Four, mitochondrial energy metabolism disorder of PSEN1p.F237L mutant neurons leads to endoplasmic reticulum stress Mitochondrial energy disorder can trigger endoplasmic reticulum stress, and the two interact to form a vicious cycle, playing a core role in the pathogenesis of neurodegenerative diseases such as AD. Endoplasmic reticulum stress is mainly regulated by three pathways: Protein kinase R-like endoplasmic reticulum kinase (PERK) pathway: This pathway can inhibit the synthesis of new proteins to reduce the burden on the endoplasmic reticulum, and it can also induce the expression of activating transcription factor 6 (ATF4, ATF4) and participate in physiological processes such as amino acid metabolism and redox balance regulation; Activating transcription factor 6 (ATF6) pathway: When endoplasmic reticulum stress occurs, the ATF6 pathway is activated, and its main function is to enhance the protein folding capacity of the endoplasmic reticulum and improve the efficiency of degrading misfolded proteins, thereby maintaining the homeostasis of the endoplasmic reticulum; Inositol-requiring enzyme 1 (IRE1) pathway: When the endoplasmic reticulum is stressed, IRE1 is phosphorylated into phosphorylated IRE1 (p-IRE1), which regulates the transcription of related genes to handle unfolded proteins and restore endoplasmic reticulum homeostasis.

[0056] In this implementation, Western-Blot technology was used to detect the main markers of endoplasmic reticulum stress BIP and the key proteins of the three pathways PERK, ATF4, ATF6, IRE1 and p-IRE1 in normal control and PSEN1p.F237L mutant neurons. The results showed that in PSEN1p.F237L mutant neurons, BIP, PERK, ATF4, ATF6 and p / t-IRE1 were up-regulated Figure 10 A, B). It is suggested that the mitochondrial energy metabolism disorder caused by PSEN1p.F237L mutation leads to endoplasmic reticulum stress.

[0057] Example 3: Drug combination CDN1163 combined with S107 intervention improves the AD phenotype of PSEN1p.F237L mutant neurons This implementation employs the following three strategies to intervene in neurons: (1) applying SERCA2 calcium pump activator CDN1163 alone ( Figure 11 A); (2) Application of RyR2 calcium channel stabilizer S107 alone ( Figure 11 B); (3) Combined application of CDN1163 and S107 ( Figure 11 C). Then, the effects of different interventions on calcium homeostasis, mitochondrial function, endoplasmic reticulum stress, and AD-related phenotypes in PSEN1p.F237L mutant neurons were compared.

[0058] I. Treatment with CDN1163 combined with S107 for 4 weeks improves calcium homeostasis in PSEN1p.F237L mutant neurons This study observed the regulatory effects of different intervention methods on endoplasmic reticulum (ER) calcium ion concentration in mutant neurons. Neurons treated with TG (transferase in calcium) with CDN1163, S107, or a combination of both were treated, and the changes in ER and cytoplasmic calcium ion concentrations were recorded before and after TG treatment. The results showed that CDN1163, S107, and combined interventions all increased the resting ER calcium ion concentration in mutant neurons to varying degrees. Figure 12 The decrease in endoplasmic reticulum calcium ions induced by TG also showed varying degrees of increase, with the combined intervention showing the most significant effect. Figure 12 A, C). Furthermore, all three interventions were able to reduce the cytoplasmic calcium ion concentration in the resting state of mutant neurons to varying degrees. Figure 12 D, E), the increase in cytoplasmic calcium ions induced by TG also showed varying degrees of increase ( Figure 12 The combined intervention (D, F) showed the most significant effect. These results suggest that all three intervention methods can improve endoplasmic reticulum calcium homeostasis in mutant neurons to varying degrees, manifested as increased endoplasmic reticulum calcium reserves and decreased cytoplasmic calcium ion concentration. The combined intervention of CDN1163 and S107 showed the best effect.

[0059] Next, this study measured the calcium ion concentrations in the endoplasmic reticulum and mitochondria of neurons to further evaluate the effects of CDN1163 and S107 interventions, alone or in combination, on calcium transport capacity and mitochondrial calcium homeostasis in MAMs. The results showed that all three interventions could increase the endoplasmic reticulum calcium ion concentration in mutant neurons at rest to varying degrees. Figure 13 A, B), increases the rate of calcium ion decrease in the endoplasmic reticulum ( Figure 13 A, C); at the same time, the three intervention methods also increased the mitochondrial calcium ion concentration in the resting state of mutant neurons to varying degrees. Figure 13 D, E), and increase the rise in mitochondrial calcium ions (D, E), and increase the magnitude of mitochondrial calcium ion rise ( Figure 13D, F), in which the effect of combined intervention was the most significant. The above results showed that CDN1163, S107 and their combined intervention could promote calcium transport of MAMs and increase mitochondrial calcium ion concentration to different degrees, and the combined intervention improved the effect best, further suggesting that targeted regulation of SERCA2 and RyR2 could restore endoplasmic reticulum-mitochondrial calcium homeostasis.

[0060] II. CDN1163 combined with S107 treatment for 4 weeks improves mitochondrial function of PSEN1p.F237L mutant neurons To explore whether the restoration of endoplasmic reticulum-mitochondrial calcium homeostasis can improve the mitochondrial function of mutant neurons, the present embodiment further evaluates the improvement effect of the above interventions on the mitochondrial function of mutant neurons. First, Western-Blot was used to detect the influence of CDN1163, S107 and combined intervention on the mitochondrial fusion-fission related proteins in PSEN1p.F237L mutant neurons. The results showed that CDN1163 intervention did not significantly change the expression of fusion protein MFN2 and fission protein Fis1 in mutant neurons ( Figure 14 A, B); S107 intervention did not affect the expression of MFN2 in mutant neurons, but down-regulated the expression of Fis1 ( Figure 14 C, D); and CDN1163 combined with S107 intervention significantly up-regulated the expression of MFN2 in mutant neurons, and the expression of Fis1 was also significantly reduced ( Figure 14 E, F). The above results suggest that S107 intervention alone and combined intervention can improve the mitochondrial fusion-fission balance of mutant neurons to different degrees, and the combined intervention has the best improvement effect.

[0061] To further evaluate the improvement effect of calcium homeostasis regulators on the mitochondrial function of mutant neurons, the MMP, ROS of neurons and the oxygen consumption rate of neural stem cells were detected. The results showed that CDN1163 and S107 alone or in combination could improve the MMP of mutant neurons to different degrees, and the combined intervention had the best effect ( Figure 15 A, B). The three interventions also reduced mitochondrial ROS in mutant neurons to different degrees, and the combined intervention had the most obvious effect on reducing ROS ( Figure 15 C, D). The three intervention methods could improve the oxygen consumption rate of mutant neural stem cells to different degrees, including basal oxygen consumption rate, maximum respiration and oxygen consumption rate for ATP production, and the combined intervention had the best improvement effect ( Figure 15 E, F).

[0062] III. CDN1163 combined with S107 treatment for 4 weeks relieves endoplasmic reticulum stress of PSEN1p.F237L mutant neurons This study examined the effects of three interventions on endoplasmic reticulum stress in PSEN1p.F237L mutant neurons. Results showed that CDN1163 ( Figure 16 A, B) or S107 ( Figure 16 C and D) Individual interventions had almost no effect on the expression of the endoplasmic reticulum stress protein BIP, while combined intervention with CDN1163 and S107 significantly downregulated BIP expression. Figure 16 E, F), further suggesting that combined intervention can significantly alleviate endoplasmic reticulum stress in PSEN1p.F237L mutant neurons.

[0063] IV. Treatment with CDN1163 combined with S107 for 4 weeks improves the phenotype of PSEN1p.F237L mutant neurons. In this study, neurons were cultured to maturity in media containing CDN1163 and S107, and CDN1163 and S107, respectively, and then transfected with pCDH-CMV-MCS-EF1a-EGFP plasmid. Neuronal morphology was imaged using laser confocal microscopy, and dendritic complexity was quantitatively analyzed using Sholl Analysis. The results showed that CDN1163, S107, and combined interventions increased dendritic complexity in PSEN1p.F237L mutant neurons to varying degrees, with the combined intervention showing the most significant improvement. Figure 17 A, B).

[0064] In further research, this study examined the effects of the aforementioned interventions on the expression of dendrites and synapse-related proteins in mutant neurons. Western blotting results showed that CDN1163 intervention upregulated the expression levels of MAP2 and SNAP25 in mutant neurons. Figure 17 C, D); S107 intervention can increase MAP2 expression, but has no significant effect on SNAP25 ( Figure 17 E, F); After combined intervention, the expression of MAP2 and SNAP25 in mutant neurons was significantly upregulated (E, F); Figure 17 G, H).

[0065] The results suggest that the three intervention methods can increase the dendritic complexity of PSEN1p.F237L mutant neurons to varying degrees and upregulate the expression of MAP2 and SNAP25, with the combined intervention showing the most significant effect.

[0066] Subsequently, this study further evaluated the intervention effect of the aforementioned calcium homeostasis regulators on abnormal Tau phosphorylation in mutant neurons. Western blotting results showed that CDN1163 intervention could reduce the expression levels of total Tau and p-Tau in mutant neurons. Figure 18 A, B); S107 intervention also shows a similar trend (Figure 18 C, D); while the combined intervention showed the most significant trend of decreasing total Tau and p-Tau (E, F). These results indicated that CDN1163, S107 and their combined intervention could reduce Tau abnormal phosphorylation to different extents, and the combined intervention showed the most significant improvement effect. Figure 18

[0067] In summary, the combined intervention of CDN1163 and S107 could improve the AD-related phenotypes of PSEN1p.F237L mutant neurons, including increasing the complexity of neuronal dendrites and reducing Tau abnormal phosphorylation. The combined intervention of CDN1163 and S107 showed the best synergistic improvement effect, which was superior to the effect of CDN1163 or S107 alone. Especially considering that endoplasmic reticulum stress is one of the main causes of neuronal death, CDN1163 (A, B) or S107 (C, D) alone almost had no effect on the expression of endoplasmic reticulum stress protein BIP, while the combined intervention of CDN1163 and S107 could significantly down-regulate the expression of BIP (E, F), indicating that only the combined intervention of CDN1163 and S107 could alleviate the endoplasmic reticulum stress of PSEN1p.F237L mutant neurons and thus improve the AD phenotype of PSEN1p.F237L mutant neurons. Figure 16 Figure 16 Figure 16

[0068] Example 4: Combined intervention of CDN1163 and S107 improves the AD phenotype of PSEN1p.F237L mutant mice To evaluate the effect of the restoration of endoplasmic reticulum-mitochondrial calcium homeostasis on the complexity of hippocampal neuron dendrites, Golgi staining was performed in this embodiment, and the results showed that the combined intervention of CDN1163 and S107 increased the dendritic branching and total length of hippocampal neurons of PSEN1p.F237L mutant mice (A, B), as well as the density of mushroom-shaped dendritic spines (C, D); and the combined intervention also significantly up-regulated the expression of MAP2 and SNAP25 of hippocampal neurons of PSEN1p.F237L mutant mice (E, F), which supported the improvement effect of calcium homeostasis regulators on the AD phenotype of PSEN1p.F237L mutant mice at the level of neurons. Figure 19 Figure 19 Figure 19

[0069] In this embodiment, immunohistochemical method was used to observe the deposition of Aβ42 in the hippocampal brain region of mice, and the results showed that the combined intervention of CDN1163 and S107 significantly reduced the deposition of Aβ (A, B). Western-Blot results showed that the combined intervention of CDN1163 and S107 significantly reduced the content of t-Tau and pTau in the brain tissue of mutant mice (C, D). Figure 20 Figure 20 ​​​​​​​​C, D). The above results suggest that CDN1163 combined with S107 significantly reduces pathological deposition in the hippocampus of PSEN1p.F237L mutant mice.

[0070] Further, the present embodiment evaluates the spatial learning and memory ability of mice by water maze experiment. The results show that, compared with wild type, PSEN1p.F237L mutant mice exhibit longer escape latency in training stage Figure 21 A-C), less crossing of platform in spatial exploration stage Figure 21 D, E), and shorter time of staying in target quadrant Figure 21 D, F). After CDN1163 combined with S107 intervention, the escape latency of mutant mice is significantly shortened Figure 21 A-C), the number of crossing of platform is significantly increased Figure 21 D, E), and the time of staying in target quadrant is also significantly prolonged Figure 21 D, F), suggesting that CDN1163 combined with S107 intervention has an improving effect on the impaired spatial learning and memory ability of PSEN1p.F237L mutant mice.

[0071] Although the present application has been described in detail with general principles and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art.

Claims

1. A mutant protein, characterized in that, The PSEN1 protein has a mutation at position 237 where phenylalanine is replaced by leucine (PSEN1p.F237L), which is associated with Alzheimer's disease.

2. A nucleic acid molecule, characterized in that, It encodes the mutant protein as described in claim 1.

3. The use of the mutant protein of claim 1, or the nucleic acid molecule of claim 2, in any of the following aspects: (1) Its use as a biomarker in the diagnosis of Alzheimer's disease; (2) Its use in the preparation of drugs that improve neuronal mitochondrial structure and function disorders; (3) Its use in improving the expression of dendrites and synaptic-related proteins in PSEN1p.F237L mutant neurons; (4) Its use in alleviating endoplasmic reticulum stress in PSEN1p.F237L mutant neurons; (5) Its use in the preparation of medicines for the prevention and / or treatment of Alzheimer's disease.

4. An Alzheimer's disease model carrying the mutant protein of claim 1, characterized in that, This includes neuronal and transgenic mouse models.

5. The use of the disease model according to claim 4 in any of the following aspects: (1) Its applications in disease mechanism research; (2) Its uses in new drug screening, efficacy verification and safety evaluation; (3) Its use in early disease diagnosis and biomarker screening and validation; (4) Its use in the development and validation of novel treatment strategies; (5) Its use in studying specific gene functions, signaling pathway regulation and cell biological processes.

6. A pharmaceutical composition, characterized in that, It acts on the relevant pathway mediated by the mutant protein of claim 1, including a SERCA2 agonist and a RyR2 stabilizer, wherein the SERCA2 agonist includes CDN1163 and the RyR2 stabilizer includes S107.

7. Use of the pharmaceutical composition of claim 6 in any of the following aspects: (1) Improve neuronal mitochondrial structure and functional disorders; (2) Improves the expression of dendrites and synaptic-related proteins in PSEN1p.F237L mutant neurons; (3) Alleviating endoplasmic reticulum stress in PSEN1p.F237L mutant neurons; (4) Improves cognitive impairment in PSEN1p.F237L mutant mice; (5) Prevention and / or treatment of Alzheimer's disease.

8. The use as described in claim 7, characterized in that, The Alzheimer's disease is caused by the PSEN1p.F237L mutation, and the diseased tissues or cells contain the PSEN1p.F237L mutant protein or its encoding gene.

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