Protein expression profile screening and application of anxiety disorder of Alzheimer's disease

Abstract

The invention relates to the field of breeding technical equipment, and particularly provides protein expression profile screening and application of anxiety disorder of Alzheimer's disease. Specifically, for Alzheimer's disease anxiety disorder caused by amyloid beta protein accumulation, proteomics analysis finds that inhibition of aBLA-vCA1 pathway functions can effectively relieve social avoidance behaviors caused by amyloid beta protein accumulation; and the differential functional proteins of the anxiety disorder of the Alzheimer's disease, such as RPS6, UBQLN1, Eras2, Rpl30, SERBP1, CLU, APOE, C4B, C1QB, C1QA, C1QC, MAPK1, MAPK3, RHOG, PTK2B, ASPA, GAD1, GAD2, H3f3a, H4C8 and TSPYL4, are screened out. The different functional proteins can be used as biomarkers for detecting Alzheimer's disease anxiety disorder caused by accumulation of pulverulent beta protein, and can be used as potential therapeutic targets.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the screening and application of protein expression profiles for anxiety disorders in Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive dysfunction and memory loss. In addition to cognitive impairment, AD patients are often accompanied by psychiatric symptoms such as anxiety and depression, which seriously affect the quality of life of patients. Among them, social ability disorder is one of the common anxiety symptoms of AD patients, manifested as social withdrawal and avoidance of social activities, which has a serious impact on the social function and mental health of patients.

[0003] At present, the pathogenesis of AD is not fully understood, but the abnormal deposition of β-amyloid protein (Aβ) is considered to be one of its core pathological characteristics. Studies have shown that Aβ deposition not only causes neuronal damage and cognitive dysfunction, but may also cause psychiatric symptoms such as anxiety by affecting the function of neural circuits. However, the neural mechanism behind how Aβ deposition affects social ability remains unclear.

[0004] The basolateral amygdala (BLA) and ventral hippocampal CA1 region (vCA1) are key brain regions involved in emotion regulation, memory, and social behavior. The BLA-vCA1 pathway is thought to play an important role in fear memory, anxious behavior, and social interaction. However, the role of the BLA-vCA1 pathway in Aβ-related social impairment is still unclear.

[0005] Therefore, in-depth analysis of the protein network changes of the aBLA-vCA1 circuit in the context of Aβ pathology is necessary to clarify its causal relationship and specific mechanism. New biomarkers can be discovered for early diagnosis, disease monitoring, and efficacy evaluation of anxiety disorders in Alzheimer's disease. Summary of the invention

[0006] The present invention aims to disclose the protein expression profile screening and application of Alzheimer's disease anxiety disorder, so as to at least discover new biomarkers for detecting Alzheimer's disease anxiety disorder.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A biomarker for detecting anxiety disorder in Alzheimer's disease, wherein the anxiety disorder in Alzheimer's disease is caused by amyloid β protein, and the biomarker is selected from the following group: RPS6, UBQLN1, Eras2, Rpl30, SERBP1, CLU, APOE, C4B, C1QB, C1QA, C1QC, MAPK1, MAPK3, RHOG, PTK2B, ASPA, GAD1, GAD2, H3f3a, H4C8, and TSPYL4.

[0009] A method for screening biomarkers for detecting anxiety disorders in Alzheimer's disease comprises the following steps:

[0010] S1: Select experimental animals and divide them into model group and control group;

[0011] S2: Activate the basolateral amygdala-ventral hippocampal CA1 circuit through optogenetics, and evaluate the social behavior of model group animals and control group animals in combination with the three-chamber social test to identify social disorders;

[0012] S3: Obtain brain tissue samples from mice in the model group and control group;

[0013] S4: Labeling proteins using tandem mass tag technology and separating the proteome by HPLC;

[0014] S5: Compare the differences in protein expression between the model group and the control group by mass spectrometry analysis, and screen out proteins with significant differential expression;

[0015] S6: Combined with bioinformatics analysis tools, proteins associated with Alzheimer's disease-related anxiety disorders were identified as potential biomarkers.

[0016] Furthermore, in step S1, the model group of experimental animals includes 5×FAD transgenic mice, and the control group includes wild-type C57BL / 6 mice.

[0017] Furthermore, the optogenetic technique used in step S2 activates or inhibits the neural circuit by injecting the AAV5-CaMKIIa-eNpHR3.0-eYFP viral vector into the basolateral amygdala region.

[0018] Furthermore, the brain tissue samples obtained in step S3 are from the basolateral amygdala and ventral hippocampal CA1 region, which are used for proteomic analysis.

[0019] Furthermore, in step S4, proteins are labeled by tandem mass tags for comparison of differential expression between the model group and the control group.

[0020] Furthermore, the bioinformatics analysis tools used in step S6 include the STRING protein interaction network, which is used to analyze the interactions between differentially expressed proteins.

[0021] A potential therapeutic target for anxiety disorder in Alzheimer's disease, the potential therapeutic target being selected from the following group: RPS6, UBQLN1, Eras2, Rpl30, SERBP1, CLU, APOE, C4B, C1QB, C1QA, C1QC, MAPK1, MAPK3, RHOG, PTK2B, ASPA, GAD1, GAD2, H3f3a, H4C8, and TSPYL4.

[0022] The use of an inhibitor of aBLA-vCA1 circuit function in the preparation of a treatment for anxiety disorders caused by Alzheimer's disease, wherein the anxiety disorders caused by Alzheimer's disease are caused by amyloid β protein.

[0023] The present invention has at least one of the following beneficial effects:

[0024] 1. The present invention, through proteomic analysis, found that inhibiting the function of the aBLA-vCA1 pathway can effectively alleviate the social avoidance behavior caused by the accumulation of amyloid β protein, and the preparation of aBLA-vCA1 pathway function inhibitors can be used as therapeutic drugs for the treatment of anxiety disorders in Alzheimer's disease caused by amyloid β protein pathology.

[0025] 2. The screened differentially functional proteins, such as RPS6, UBQLN1, Eras2, Rpl30, SERBP1, CLU, APOE, C4B, C1QB, C1QA, C1QC, MAPK1, MAPK3, RHOG, PTK2B, ASPA, GAD1, GAD2, H3f3a, H4C8, TSPYL4, etc., can be used as biomarkers for detecting anxiety disorders in Alzheimer's disease caused by amyloid β protein, and provide potential targets for the development of new therapeutic drugs.

[0026] 3. It provides valuable reference for the research on the treatment of anxiety disorders in Alzheimer's disease. Through in-depth research on these differentially functional proteins, we can better understand the molecular mechanism of the disease, provide valuable reference for research in related fields, and have extremely high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0028] Figure 1 The experimental results of the social ability of 5×FAD mice;

[0029] Figure 2 The experimental results show the relationship between aBLA-vCA1 circuit inactivation and social deficits in 5×FAD mice;

[0030] Figure 3 The results of aBLA proteomics detection in 5×FAD mice and wild-type (WT) mice;

[0031] Figure 4 The results of proteomic analysis of 5×FAD mouse pBLA and WT mouse pBLA;

[0032] Figure 5 The results of proteomic analysis of WT mouse aBLA and WT mouse pBLA;

[0033] Figure 6 These are the proteomic detection results of aBLA and pBLA of 5×FAD mice. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0035] Glossary: ​​Alzheimer's disease (AD), amyloid-βpeptide (Aβ), basolateral amygdala (BLA), hippocampus (HP), ventral hippocampal CA1 region (vCA1).

[0036] The research process involved in the present invention includes the following contents:

[0037] 1. Experimental Methods

[0038] 1. Select experimental animals and divide them into model group and control group.

[0039] Specifically, adult male C57BL / 6 mice (6 months old) and 5×FAD mice (6 months old) were purchased from GENEAND Pence (Yangzhou, Jiangsu, China). The animals were grouped (5-4 / cage) and raised at a stable temperature (23-25°C) with a 12 h light / dark cycle (lights on at 6:00 pm and off at 6:00 am). Food and water were freely available. (Animal husbandry and experiments were conducted in accordance with ethical regulations regarding animal experiments and research.)

[0040] Six-month-old mice were subjected to stereotaxic surgery to establish the animal model. Mice were anesthetized with sodium pentobarbital (1%, 30 mg / kg) and fixed in a stereotaxic injector (RWD 68046&68055, China). After the scalp was disinfected and balanced, a small hole was gently made. Under a stereotaxic instrument (World Precision Instruments, USA), the virus was injected into the aBLA (AP: -1.34 mm, ML: ±3.4 mm, DV: -4.8 mm) and vCA1 (AP: -3.28 mm, ML: ±3.3 mm, DV: -4.6 mm) using a 10 μL gastight mixing syringe (KF019, China). The injection rate was 50–100 nL / min (for virus injection) or 30 nL / min (for CTB injection). After each injection, the needle was placed for 5 minutes to avoid virus transmission and inhalation. Lincomycin lidocaine gel was evenly applied to the skull, and then the skin was sutured with absorbable sutures. The mice were placed on a heated blanket and waited for awakening. Finally, the injection site was confirmed by fluorescence expression. Mice with incorrect viral expression were excluded.

[0041] Then, cholera toxin subunit B (CTB) was used to retrogradely track the social control ability of the aBLA-vCA1 circuit. Cholera toxin subunit B (recombinant) was obtained from BrainVTA (CTB-555). CTB (100nl) was stereotaxically delivered to vCA1 (AP: -3.28mm, ML: ±3.3mm, DV: -4.6mm). After 7 days, the mice were anesthetized and perfused, the brains were removed, and the expression of CTB in the aBLA was detected under a confocal microscope (LSM780, Zeiss, Germany).

[0042] 2. Use optogenetics to activate the basolateral amygdala-ventral hippocampal CA1 circuit, and combine it with a three-chamber social test to evaluate the social behavior of the model group and the control group animals to identify social disorders.

[0043] AAV5-CaMKIIa-eNpHR3.0-eYFP and AAV5-CaMKIIa-eYFP were purchased from Brain Case Co., Ltd., Shenzhen, China. Optogenetic manipulations were performed in freely moving mice by injecting viruses (150–200 nL, >1012 vg / mL) at a rate of 0.1 μL min-1 into the aBLA (AP: -1.34 mm, mL: ±3.4 mm, DV: -4.8 mm). Virus injections were balanced across the left and right aBLA. Approximately 4 weeks later, an optical fiber (core = 200 μm; numerical aperture = 0.37) was implanted into the vCA1 (AP: -3.28 mm, ML: ±3.3 mm, DV: -4.1 mm). Mice were given a week to recover before behavioral testing.

[0044] Subsequently, WT mice, 5×FAD mice treated with acipimox (OLB), and 5×FAD mice were subjected to proteomic experiments using tandem mass tags (TMT). Each sample was digested into peptides using mass spectrometry trypsin (Promega, V5072), then labeled with different TMTs (ThermoFisher 90406), mixed together, and separated into 15 fractions by high performance liquid chromatography (HPLC) for subsequent experiments. The dried fractions were dissolved in 0.1% formic acid (FA) and captured with a silica capillary column filled with C18 resin (Varian, Lexington, MA, USA) for subsequent Q Exactive (ThermoScientific, NJ, USA) mass spectrometer analysis, with the following parameters: 400-1800 m / z, 70000 resolution; MS / MS scan (100-1800 m / z). MS / MS data were searched against the Uniport human database using Proteome Discoverer 2.1 software (Thermo Scientific).

[0045] The three-chamber sociability test is used to identify anxiety disorders in WT mice and 5×FAD mice. The three-chamber sociability test is derived from the inherent tendency of mice to engage in social interactions and investigate unfamiliar stimuli. The test apparatus was constructed using custom rectangular plexiglass resin (60 cm long × 40 cm wide × 30 cm high). The dimensions of each chamber are 20 cm by 40 cm. Custom-designed resin cups (8 cm in diameter) are inverted and symmetrically placed in the two conical parts of the chamber, allowing adolescent mice (3-4 weeks) to move while restricting them to move in designated areas. In the initial stage, the experimental mouse was placed in the center of the chamber and allowed 5 minutes for unrestricted exploration (habituation). Over the next 5 minutes, the test mouse was confined to the center of the test chamber for 30 seconds, while an adolescent mouse (3-4 weeks) was placed in an inverted resin cup in the designated social chamber. An empty resin cup was placed on the side designated as the non-social chamber. Subsequently, the test mouse was placed in the exploration chamber for 5 minutes. Each test mouse participated in a two-day optogenetic stimulation social interaction test, such as Figure 2 (g) As shown. The positions of adolescent mice and the light stimulation sequences of test mice were randomly assigned in a balanced manner. Social behavior was characterized by the exploration of the test mouse within a 2 cm radius, especially by sniffing or orienting its nose or head, around the inverted resin cup containing the adolescent mouse. Checking the empty inverted resin cup on the opposite side was considered as non-social behavior. The complete experimental process was recorded by using tracking software and a camera system. After each experiment, the bottom of the three chambers and the resin cups were thoroughly cleaned with 70% ethanol.

[0046] 3. Obtain brain tissue samples from mice in the model group and control group.

[0047] 90 minutes after the sociability test, mice were killed with a lethal dose of sodium pentobarbital and the brains were subjected to immunofluorescence staining. The mice were intracardially perfused with saline and 4% paraformaldehyde (PFA), and the brains were removed and postfixed in 4% PFA overnight. Afterwards, they were transferred to a 25-30% sucrose solution in PBS for 3 hours and cut into 30 μm thick coronal sections. The sections were washed with PBS-T (PBS containing 0.1% Triton x-100) and permeabilized with PBS containing 0.5% Triton x-100 for 20-30 minutes. QuickBlock TM Blocking buffer (P0260, Beyotime) was used for blocking. Polyclonal rabbit c-Fos antibody (1:200; catalog number sc-52, Santa Cruz) or monoclonal mouse 6E10 antibody (1:300; catalog number 803001, BioLegend) was then used for immunostaining in QuickBlock TMThe sections were incubated in primary antibody dilution buffer (P0262) for 20 hours. Afterwards, the sections were washed with PBS-T (three times, 5 minutes each time) and then incubated with secondary antibodies (1:500, catalog number A-21206 or A-21202 Invitrogen) for 1 hour at 37°C. Finally, the sections were washed three times and counterstained with DAPI.

[0048] 4. Compare the differences in protein expression between the model group and the control group mice through mass spectrometry analysis, and screen out proteins with significant differential expression.

[0049] 5. Combined with bioinformatics analysis tools, identify proteins associated with Alzheimer's disease-related anxiety disorders as potential biomarkers.

[0050] Among them, the specific methods adopted in parts 4 and 5 are standard process operations and will not be described in detail in this application.

[0051] 2. Experimental results:

[0052] Compared with the WT group, immunofluorescence staining using 6E10 antibody showed strong fluorescence signals in the aBLA region of 7-month-old 5×FAD mice (Fig. Figure 1 Quantitative analysis showed that the 6E10+ signal density in the aBLA of 5×FAD mice was higher than that of the control group (e.g. Figure 1 This finding suggests that the aBLA is susceptible to Aβ pathology.

[0053] Behavioral tests were then performed to assess sociability in the presence of Aβ. In the three-chamber sociability test, no differences were observed in the time spent by 5×FAD and WT mice in the right and left areas of the testing chamber during the habituation trial, indicating that the mice had no place preference, as Figure 1 (c). Five minutes after habituation, an intruder was introduced into the chamber and placed under an inverted cup (social zone). WT mice spent more time in the social zone than in the nonsocial zone (an empty cup), whereas 7-month-old 5×FAD mice spent less time exploring the social zone ( Figure 1 d), decreased social / non-social ratio (e.g. Figure 1 These data suggest that the sociability of 5×FAD mice is impaired in the presence of Aβ accumulation in the aBLA.

[0054] However, there was no significant difference in travel distance between the WT group and the 5×FAD group (e.g. Figure 1 (f) ), indicating that their motor ability is not affected by Aβ deposition. This reinforces the view that the observed changes in social behavior are specifically related to social anxiety rather than general movement disorders.

[0055] Using retrograde tracing combined with c-Fos staining, it was shown that excessive activation of the aBLA-vCA1 circuit by Aβ impaired social ability. Specifically, a retrograde tracer (CTB, cholera toxin subunit B) was stereotaxically injected into the vCA1 region. One week later, c-Fos staining was performed 90 minutes after the social ability test. The results showed that there was a significant CTB signal in the aBLA region (e.g. Figure 2 (a)), indicating a physical connection between the aBLA and vCA1. Notably, 5×AD mice exhibited a higher number of c-Fos+ and CTB+ neurons in the aBLA compared with WT mice (e.g. Figure 2 (b) ), indicating that the aBLA-vCA1 circuit is overactivated in 5xFAD mice.

[0056] To investigate the role of the aBLA-vCA1 pathway in Aβ-induced impairment of sociability, we introduced adenovirus AAV5-CaMKIIa-eNpHR3.0-eYFP into the aBLA region (referred to as NpHR-5×FAD mice, e.g. Figure 2 As shown in (c) in Figure 5), AAV5 CaMKIIa eYFP (referred to as eYFP-5×FAD mice) was administered as a control group. After 4 weeks of viral expression, expression in aBLA neurons was confirmed by green fluorescence (eYFP) signals in aBLA, as shown in Figure 5. Figure 2 (d). Optical fibers were then implanted bilaterally above the vCA1 region of NpHR-5×FAD and eYFP-5×FAD mice. Subsequently, the mice were given a one-week recovery period after the surgical procedure. Behaviorally, in the three-chamber sociability test, no difference was observed in the time spent in the social zone between NpHR-5×FAD and eYFP-5×FAD mice during the start-up trial. Compared with eYFP-5×FAD mice, NpHR-5×FAD mice spent more time in the social zone during the light period, as shown in Figure 5. Figure 2 In addition, NpHR-5×FAD mice showed a higher social / nonsocial ratio during the light period, indicating that they spent more time exploring the social area during light stimulation, as shown in (e, f). Figure 2 In contrast, the social / nonsocial ratio of eYFP-5×FAD mice did not change significantly between the on and off periods (Fig. Figure 2 In addition, when comparing the motor activities during the two periods, it was found that there was no difference in the distance traveled by the two groups during the lights-on period compared with the lights-off period, e.g. Figure 2 (i) in .

[0057] Proteomic analysis shows a comparison between WT mice and 5×FAD mice. Figure 3As shown in (a), 767 differentially expressed proteins were detected in the comparison of 5×FAD-aBLA / WT-aBLA, including up-regulated proteins such as Apoe, Ctsd, and Rps13, and down-regulated proteins such as Pacsin1, Basp1, and C2cd21. Figure 4 As shown in (a), 574 differentially expressed proteins were detected in the comparison of 5×FAD-pBLA / WT-pBLA, including up-regulated proteins such as Apoe, Clu, Hexb, Grn, Ctsd, etc., and down-regulated proteins such as Glmx5, Tppp, Gap43, Sprin1, etc. Figure 5 As shown in (a), 125 differentially expressed proteins were detected in the comparison between WT-aBLA and WT-pBLA, including up-regulated proteins such as Cnp, Penk, Ina, Mog, etc., and down-regulated proteins such as Ncam2, Crym, Grpin1, etc. Figure 6 As shown in (a), 42 differential proteins were detected in the comparison of 5×FAD-aBLA / 5×FAD-pBLA, the upregulated proteins included Ache, Fth1, Apfs2, etc., and the downregulated proteins included Palmd, H4c1, etc.

[0058] In addition, GO enrichment analysis showed that proteins related to cytoplasmic protein translation, intracellular and extracellular protein transport, regulation of postsynaptic neurotransmitter receptor activity, central nervous system myelination, DNA template transcription and initiation, nucleosome assembly, negative regulation of megakaryocyte differentiation, and DNA replication with / without nucleosome assembly were upregulated (e.g. Figure 3 (b) in Figure 4 (b) in Figure 5 (b) in Figure 6 (b) in the figure.

[0059] Visualized protein interaction networks and protein annotations established by PPI Figure 4 (c) and (d) in Figure 5 (c) and (d) in Figure 6As shown in (c) and (d) in . The proteins with different functions in neuronal cells and synapses include NEDD8 (Neural precursor cell expressed developmentally downregulated protein 8), APP (Gamma-secretase C-terminal fragment 50), SHANK2 (SH3 and multiple ankyrin repeat domains protein 2), SHISA6 (Protein shisa-6), DLGAP1 (Diskslarge-associated protein 1), GRIN2B (Glutamate receptorionotropic NMDA2B), GRIN1 (Glutamate receptorionotropic NMDA 1), SHANK1 (SH3 and multiple ankyrinrepeat domains protein 1), GRIA3 (Glutamatereceptor 3, glutamate receptors), SYNGAP1 (Ras / Rap GTPase-activating protein SynGAP, Ras / Rap GTPase activating protein), CAMK2A (Calcium / calmodulin-dependent protein kinase type II subunit alpha, calcium / calmodulin-dependent protein kinase II subunit alpha), OLFM1 (Noelin), ASS1 (Argininosuccinate synthase, argininosuccinate synthase). The main functional areas involved are: ubiquitin-like proteins in neural precursor cells, interactions between adjacent cells to promote synapse formation, regulation of the structure and function of dendritic spines and synapses, prevention of synaptic inhibition, postsynaptic scaffolds in neuronal cells, regulation of glutamate receptor ligand-gated calcium channels in the central nervous system, postsynaptic signaling involved in synaptic plasticity, neurotransmitter release and long-term potentiation. By exploring the AD anxiety protein expression profile, it is shown that the occurrence of AD anxiety may involve pathological processes caused by changes in the synaptic function of neuronal cells.

[0060] The differentially functional proteins screened out are:

[0061] RPS6 (40S ribosomal protein S6, 40S ribosomal protein): may play an important role in controlling cell growth and proliferation by selectively translating specific categories of mRNA. It belongs to the eukaryotic ribosomal protein es6 family.

[0062] UBQLN1 (Ubiquilin-1, ubiquitin-1) plays an important role in regulating different protein degradation mechanisms and pathways, including the ubiquitin-proteasome system (UPS), autophagy, and the endoplasmic reticulum-associated protein degradation (ERAD) pathway. It mediates the proteasomal targeting of misfolded or accumulated proteins for degradation by binding (through the UBA domain) to their polyubiquitin chains and by interacting with subunits of the proteasome (through the ubiquitin-like domain).

[0063] Eras2 (Glutamyl-TRNA Synthetase 2, possible glutamate-tRNA ligase): catalyzes the connection between glutamate and tRNA (Glu) in mitochondria through a two-step reaction: glutamate is first activated by ATP to form Glu-AMP, and then transferred to the receptor end of tRNA (Glu).

[0064] Rpl30 (Ribosomal protein L30, large ribosomal subunit protein eL30): It is a huge ribonucleoprotein complex in the ribosome, responsible for the synthesis of intracellular proteins

[0065] SERBP1 (Serpine1 mRNA-binding protein 1): SERBP1 is the first example of an RNA-binding protein acting as a central regulator of tumor metabolism and an indirect regulator of epigenetic regulation in glioblastoma.

[0066] CLU (Clusterin alpha chain): functions as an extracellular chaperone to prevent aggregation of non-native proteins. Prevents stress-induced aggregation of plasma proteins. Inhibits in vitro formation of amyloid fibrils from APP, APOC2, B2M, CALCA, CSN3, SNCA, and aggregation-prone LYZ variants. Does not require ATP. Maintains partially unfolded proteins in a state suitable for subsequent refolding by other chaperones (e.g., HSPA8 / HSC70).

[0067] APOE (Apolipoprotein E): A protein that binds to lipid particles and transports lipids between organs via lipoproteins in plasma and interstitial fluid.

[0068] C4B (Complement C4-B alpha chain): A non-enzymatic component of the C3 and C5 convertases and therefore essential for the propagation of the classical complement pathway. Covalently binds immunoglobulins and immune complexes, enhances the solubilization of immune aggregates, and clears ICs via erythrocyte CR1. The C4A isoform is responsible for efficient binding to immune aggregates or protein antigens to form amide bonds, while the C4B isoform catalyzes the transacylation of the thioester carbonyl to form ester bonds with carbohydrate antigens.

[0069] C1QB (Complement C1q subcomponent subunit B): C1q combines with proenzymes C1r and C1s to produce C1, the first component of the serum complement system. The collagen-like region of C1q interacts with the Ca(2+)-dependent C1r(2)C1s(2) proenzyme complex, and efficient activation of C1 occurs on the interaction of the globular head of C1q with the Fc region of IgG or IgM antibodies present in the immune complex.

[0070] C1QA (Complement C1q subcomponent subunit A): C1q combines with proenzymes C1r and C1s to produce C1, the first component of the serum complement system. The collagen-like region of C1q interacts with the Ca(2+)-dependent C1r(2)C1s(2) proenzyme complex, and the efficient activation of C1 occurs on the interaction of the globular head of C1q with the Fc region of IgG or IgM antibodies present in the immune complex.

[0071] C1QC (Complement C1q subcomponent subunit C): C1q combines with proenzymes C1r and C1s to produce C1, the first component of the serum complement system. The collagen-like region of C1q interacts with the Ca(2+)-dependent C1r(2)C1s(2) proenzyme complex, and efficient activation of C1 occurs on the interaction of the globular head of C1q with the Fc region of IgG or IgM antibodies present in the immune complex.

[0072] MAPK1 (Mitogen-activated protein kinase 1, serine / threonine kinase 1): is an important component of the MAP kinase signal transduction pathway. MAPK1 / ERK2 and MAPK3 / ERK1 are two MAPKs that play an important role in the MAPK / ERK cascade. They are also involved in the signal cascade initiated by activated KIT and KITLG / SCF. Depending on the different cellular environments, the MAPK / ERK cascade mediates different biological functions such as cell growth, adhesion, survival and differentiation by regulating transcription, translation, and cytoskeletal rearrangement.

[0073] MAPK3 (Mitogen-activated protein kinase 3, serine / threonine kinase 3): is an important component of the MAP kinase signal transduction pathway. MAPK1 / ERK2 and MAPK3 / ERK1 are two MAPKs that play an important role in the MAPK / ERK cascade. They are also involved in the signal cascade initiated by activated KIT and KITLG / SCF. Depending on the different cellular environments, the MAPK / ERK cascade mediates different biological functions such as cell growth, adhesion, survival and differentiation by regulating transcription, translation, and cytoskeleton rearrangement.

[0074] RHOG (Rho-related GTP-binding protein RhoG): Required for the formation of membrane ruffles during macropinocytosis. Plays a role in cell migration and is required for the formation of cup-like structures during transendothelial migration of leukocytes. In the case of enteric Salmonella infection, it is activated by SopB and ARHGEF26 / SGEF, inducing cytoskeletal rearrangements and facilitating bacterial entry. Belongs to the small GTPase superfamily.

[0075] PTK2B (Protein-tyrosine kinase 2-beta): A non-receptor protein tyrosine kinase that regulates reorganization of the actin cytoskeleton, cell polarization, cell migration, adhesion, spreading, and bone remodeling. It plays a role in regulating humoral immune responses and is required for normal levels of splenic marginal B cells and normal migration of splenic B cells. It is required for normal macrophage polarization and migration to sites of inflammation.

[0076] ASPA (Aspartoacylase): catalyzes the deacetylation of N-acetylaspartate (NAA) to produce acetic acid and L-aspartate. NAA is present in high concentrations in brain tissue, and its hydrolysis plays an important role in maintaining the integrity of brain white matter. In other tissues, it acts as a scavenger of NAA in body fluids.

[0077] GAD1 (Glutamate decarboxylase 1): catalyzes the production of GABA GAD2 (Glutamate decarboxylase 2): catalyzes the production of GABA H3f3a (H3.3 histoneA): a variant histone H3 that replaces conventional H3 in large nucleosomes of active genes. It constitutes the major form of histone H3 in non-dividing cells and is incorporated into chromatin independently of DNA synthesis. It is deposited at sites of nucleosome displacement throughout transcribed genes, suggesting that it represents an epigenetic imprint of transcriptionally active chromatin. Nucleosomes wrap and compact DNA into chromatin, restricting DNA access to cellular machinery that requires DNA as a template.

[0078] H4C8 (H4 clustered histone 8): core component of nucleosomes. Nucleosomes package and compact DNA into chromatin, restricting DNA access to cellular machinery that requires DNA as a template. Histones therefore play a central role in transcriptional regulation, DNA repair, DNA replication, and chromosome stability. DNA accessibility is regulated by a complex series of histone post-translational modifications (also known as the histone code) and nucleosome remodeling.

[0079] TSPYL4 (Testis-specific Y-encoded-like protein 4, testis-specific Y protein): encoded-like protein 4, TSPY-like protein 4.

[0080] 5×FAD mice have a large number of changes in protein networks compared to WT mice. Interpreting protein expression profiles may be an important means of intervening in anxiety caused by Aβ accumulation.

[0081] By characterizing the unique protein spectrum destroyed by Aβ accumulation in the aBLA of 5×FAD mice, it is a feasible solution for detecting anxiety disorders in Alzheimer's disease (AD) caused by Aβ, providing valuable reference for research in related fields, with extremely high application value, and will help in the screening of preclinical candidate drugs and monitoring of disease progression.

[0082] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A biomarker for detecting anxiety disorders in Alzheimer's disease, characterized in that: The biomarkers include one or more of the following proteins: RPS6, UBQLN1, Eras2, Rpl30, SERBP1, CLU, APOE, C4B, C1QB, C1QA, C1QC, MAPK1, MAPK3, RHOG, PTK2B, ASPA, GAD1, GAD2, H3f3a, H4C8, and TSPYL4.

2. A method for screening biomarkers for detecting anxiety disorders in Alzheimer's disease, characterized in that: The following steps are involved: S1: Establishment of experimental animal model of anxiety disorder in Alzheimer's disease; S3: Obtain brain tissue samples from mice in the model group and control group; S4: Labeling proteins using tandem mass tag technology and separating the proteome by HPLC; S5: Compare the differences in protein expression between the model group and the control group by mass spectrometry analysis, and screen out proteins with significant differential expression; S6: Combined with bioinformatics analysis tools, proteins associated with Alzheimer's disease-related anxiety disorders were identified as potential biomarkers.

3. The screening method according to claim 2, characterized in that The model group of experimental animals in step S1 includes 5×FAD transgenic mice, and the control group includes wild-type C57BL / 6 mice.

4. The screening method according to claim 2, characterized in that The biomarkers described in step S6 include RPS6, UBQLN1, Eras2, Rpl30, SERBP1, CLU, APOE, C4B, C1QB, C1QA, C1QC, MAPK1, MAPK3, RHOG, PTK2B, ASPA, GAD1, GAD2, H3f3a, H4C8, and TSPYL4.

5. Use of a reagent for detecting RPS6, UBQLN1, Eras2, Rpl30, SERBP1, CLU, APOE, C4B, C1QB, C1QA, C1QC, MAPK1, MAPK3, RHOG, PTK2B, ASPA, GAD1, GAD2, H3f3a, H4C8, and TSPYL4 in the preparation of a product for diagnosing anxiety disorders in Alzheimer's disease.

6. Application of aBLA-vCA1 circuit function inhibitors in the preparation of drugs for the treatment of anxiety disorders in Alzheimer's disease.

7. The use according to claim 6, characterized in that: Amyloid-β deposition overactivates the aBLA-vCA1 circuit.

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