Screening and application of mitochondrial marker for anxiety disorder of Alzheimer's disease

Abstract

The invention provides screening and application of mitochondrial markers for anxiety disorder of Alzheimer's disease, and belongs to the technical field of medical treatment. The mitochondrial protein marker related to the Alzheimer's disease anxiety disorder, provided by the invention, can be used as a mitochondrial biomarker for detecting the Alzheimer's disease anxiety disorder caused by amyloid beta protein, and provides a potential target for developing a new therapeutic drug.

Description

Technical Field

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

[0002] Alzheimer's disease (AD), a common progressive dementia in the elderly, has core pathological features including memory and cognitive decline, as well as neuroinflammatory plaques formed by β-amyloid deposition in the brain and abnormal phosphorylation of tau protein. It is a neurodegenerative disease that seriously endangers the health of the elderly. Among them, anxiety and depression are the most common emotional disorders in AD. Epidemiological data show that about 50% of AD patients experience anxiety and depression symptoms.

[0003] In recent years, changes in mitochondrial function have been shown to play an important role in the pathogenesis and development of AD, including impaired mitochondrial autophagy, mitochondrial DNA damage, and mitochondrial dysfunction. Mitochondrial stability is significantly impaired in AD, and autolysosomal acidification in AD mouse models induces autophagic Aβ accumulation in neurons and produces senile plaques. Therefore, impaired mitochondrial function is considered to be a driving factor of AD. The timely removal of damaged mitochondria plays a vital role in maintaining the normal physiological function of neurons and is essential for the survival and health of neurons. The level of mitochondrial autophagy is impaired in the brains of AD patients, and mitochondrial autophagy is impaired in various models such as transgenic mice of AD patients.

[0004] Mitochondria are organelles within cells that are responsible for the production of adenosine triphosphate and most of the cell's metabolic processes. Mitochondrial dysfunction is a core feature of neuropsychiatric diseases. The brain has the highest energy consumption, and biological conditions involving subtle mitochondrial changes can have a significant negative impact on brain function and increase vulnerability to brain diseases. Multiple studies have confirmed that patients with mitochondrial diseases have a significantly higher rate of psychiatric symptoms or psychiatric diagnoses than the general population. However, there are few mitochondrial markers in the clinical diagnosis and treatment of AD patients, and it is difficult to assess their role in disease progression. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a mitochondrial protein marker associated with anxiety disorder in Alzheimer's disease, and a method for screening the mitochondrial protein marker.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A mitochondrial protein marker associated with anxiety disorder in Alzheimer's disease, wherein the mitochondrial protein marker comprises Acot9, GHC2, NU3M and THIOM.

[0008] Preferably, the expression level of the mitochondrial protein marker in the samples of the Alzheimer's disease anxiety disorder group is significantly higher than that in the samples of the healthy group.

[0009] Another object of the present invention is to provide a method for screening mitochondrial protein markers, comprising the following steps: constructing an Alzheimer's disease anxiety disorder animal model group and a control group, obtaining brain tissue samples from the two groups respectively, labeling proteins using tandem mass tag technology, and separating the protein group by high performance liquid chromatography, comparing the expression differences of mitochondrial proteins in brain tissues of the model group and the control group by mass spectrometry analysis of the separated proteins, screening out differentially expressed proteins by bioinformatics analysis, and determining mitochondrial proteins associated with Alzheimer's disease-related anxiety disorders as biomarkers.

[0010] Preferably, the screening criteria for screening out differentially expressed proteins are up-regulated expression, differential expression fold ≥ 1.3 and p < 0.01.

[0011] Preferably, the bioinformatics analysis includes analyzing the abundance of differentially expressed proteins using a volcano plot, analyzing protein clusters using the R software Mfuzz package, performing GO enrichment analysis on the differentially expressed proteins, and performing KEGG enrichment analysis on the differentially expressed proteins.

[0012] Preferably, the differentially expressed proteins are analyzed according to KEGG and GO databases and show that functions related to fatty acid metabolism and degradation, sulfide group metabolism and amino acid degradation are significantly enriched.

[0013] Preferably, the Alzheimer's disease anxiety disorder animal model group is 5×FAD mice.

[0014] Another object of the present invention is to provide the use of the mitochondrial protein marker or the screening method in the preparation of a product for assisting the diagnosis of anxiety disorders in Alzheimer's disease.

[0015] Another object of the present invention is to provide the use of the mitochondrial protein marker or the screening method in constructing an animal model for screening drugs for anxiety disorders in Alzheimer's disease.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides mitochondrial protein markers related to anxiety disorders in Alzheimer's disease and screening methods and applications thereof. The present invention screens and obtains differentially expressed proteins through proteomics analysis, which can be used as mitochondrial biomarkers for detecting anxiety disorders in Alzheimer's disease caused by amyloid β protein, and provides potential targets for the development of new therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Experimental flow for protein mapping of 5×FAD mice and wild-type (WT) mice;

[0019] Figure 2 It is a heat map of mitochondrial differentially expressed proteins in the BLA of 5×FAD mice and WT mice, and the ordinate indicates the animal groups (n=3 per group);

[0020] Figure 3 Volcano plot of differentially expressed mitochondrial proteins in aBLA of 5×FAD mice and WT mice (n=3 for each group);

[0021] Figure 4 GO enrichment analysis of differential mitochondrial proteins in aBLA of 5×FAD mice and WT mice;

[0022] Figure 5 Heat map of differentially expressed mitochondrial proteins in aBLA of 5×FAD mice and WT mice (n=3 for each group);

[0023] Figure 6 Volcano plot of differentially expressed mitochondrial proteins in pBLA of 5×FAD mice and WT mice (n=3 for each group);

[0024] Figure 7 GO enrichment analysis of differential mitochondrial proteins in pBLA between 5×FAD mice and WT mice;

[0025] Figure 8 Heat map of differentially expressed mitochondrial proteins in pBLA of 5×FAD mice and WT mice (n=3 for each group);

[0026] Fig. 9 This is the mitochondrial localization pattern of the expression levels of mitochondrial functional proteins in aBLA and pBLA of 5×FAD mice and WT mice. DETAILED DESCRIPTION

[0027] The present invention provides a mitochondrial protein marker associated with anxiety disorder in Alzheimer's disease, wherein the mitochondrial protein marker comprises Acot9, GHC2, NU3M and THIOM.

[0028] In the present invention, the expression level of the mitochondrial protein marker in the samples of the Alzheimer's disease anxiety disorder group is significantly higher than that in the samples of the healthy group.

[0029] The present invention also provides a method for screening the mitochondrial protein marker, comprising the following steps: constructing an Alzheimer's disease anxiety disorder animal model group and a control group, obtaining brain tissue samples of the two groups respectively, labeling proteins using tandem mass tag technology, and separating the protein group by high performance liquid chromatography, comparing the expression differences of mitochondrial proteins in brain tissues of the model group and the control group by mass spectrometry analysis of the separated proteins, screening out differentially expressed proteins by bioinformatics analysis, and determining mitochondrial proteins associated with Alzheimer's disease-related anxiety disorders as biomarkers.

[0030] The Alzheimer's disease anxiety disorder animal model group described in the present invention is 5×FAD mice purchased from GENEAND Pence (Yangzhou, Jiangsu, China), and the 5×FAD mice carry three APP mutations and two PSEN1 mutations, which can simulate AD pathological characteristics; the protein labeled with tandem mass tag technology is preferably labeled with tandem mass tags (TMT, purchased from ThermoFisher).

[0031] In the present invention, the screening criteria for screening out differentially expressed proteins are up-regulated expression, differential expression fold ≥ 1.3 and p < 0.01, preferably the differential expression fold ≥ 1.5.

[0032] In the present invention, the bioinformatics analysis includes analyzing the abundance of differentially expressed proteins using a volcano plot, analyzing protein clusters using the R software Mfuzz package, performing GO enrichment analysis on the differentially expressed proteins, and performing KEGG enrichment analysis on the differentially expressed proteins.

[0033] In the present invention, analysis based on the KEGG and GO databases showed that the differentially expressed proteins were involved in fatty acid metabolism and degradation, sulfide group metabolism and amino acid degradation-related functions.

[0034] The present invention also provides the use of the mitochondrial protein marker or the screening method in preparing a product for assisting in the diagnosis of anxiety disorders in Alzheimer's disease.

[0035] The present invention also provides the use of the mitochondrial protein marker or the screening method in constructing an animal model for screening drugs for anxiety disorders in Alzheimer's disease.

[0036] The differentially expressed proteins obtained by screening the method provided by the present invention can be used as mitochondrial biomarkers for detecting anxiety disorders caused by amyloid β protein in Alzheimer's disease, and provide potential targets for the development of new therapeutic drugs.

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] Example 1

[0039] 1. Adult male C57BL / 6 mice (6 months old, denoted as WT mice) and 5×FAD mice (6 months old) were purchased from GENEAND Pence (Yangzhou, Jiangsu, China) (denoted as 5×FAD mice). The animals were grouped (5-4 / cage) and raised at a stable temperature (23-25°C) with a 12h 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 on animal experiments and research).

[0040] 2. Protein extraction and digestion: After the onset of the disease, 5×FAD mice and WT mice were sacrificed and dissected, and proteomic experiments were performed using tandem mass tags (TMT): mass spectrometry trypsin (Promega, V5072) was used to digest each sample into peptides according to the instructions, and then labeled with different TMTs (ThermoFisher 90406). The labeled samples were mixed together, and the labeled proteins after operation were dissolved in 0.1% formic acid (FA) according to the instructions, and captured with a silica capillary column filled with C18 resin (Varian, Lexington, MA, USA). The fractions were separated by UltiMate 3000 UHPLC liquid chromatography at a flow rate of 120 min and a flow rate of 0.30 μL / min. The separated fractions were used for subsequent Q Exactive (ThermoScientific, NJ, USA) mass spectrometer analysis was performed with the following parameters: 400-1800 m / z, 70,000 resolution, MS / MS scan (100-1800 m / z). Proteome Discoverer 2.1 software (Thermo Scientific) was used to search the MS / MS data against the Uniport human database.

[0041] Bioinformatics analysis: The abundance of all differentially expressed proteins in each group was estimated by volcano plot analysis, with FC values ​​as the calculation standard. The R software Mfuzz package was used for protein cluster analysis to determine the biological processes and pathways of the expressed proteins in the animal groups. The images were visualized using Rstudio software.

[0042] 3. Major differentially expressed proteins

[0043] 5×FAD mice and WT mice (n=3 / group) were anesthetized and killed at the same time. Proteins were characterized by non-targeted proteomics, that is, brain samples of each group of mice were labeled with TMT and detected by LC-MS / MS ( Figure 1A total of 772 mitochondrial-related proteins were identified and quantified, with 595 mitochondrial-related differentially expressed proteins in aBLA and 562 differentially expressed proteins in pBLA ( Figure 2 ).

[0044] like Figure 3 , Figure 5 As shown in the data, the upregulated proteins in aBLA include ACOT9, IFIT3, CLUS, RIDA, CC90B, GHC2, ARL8A, NU3M, HCLS1, THIOM, PLSL, etc.; the downregulated proteins include COX7C, MTCH1, NDUB6, MAON, BABA1, KPCA, GLRX5, MAIP1, SYEM, ATIF1, etc.

[0045] like Figure 6 , Figure 8 As shown, the upregulated proteins in pBLA include: M4K3, CC90B, THEM4, ARL2, KS6A5, RIDA, ACO11, TACO1, RCC2, etc.; the downregulated proteins include: DHSO, NNRD, KIF2A, HMCS2, NDUA8, MAON, CALX, CCSAP, RT30, COX7C, etc.

[0046] 4. GO functional annotation and KEGG enrichment analysis of differentially expressed proteins

[0047] like Figure 4 , Figure 7 As shown in the figure, through GO functional annotation, three BP, GC, and MF entries were enriched respectively, indicating that in BLA, 5×FAD model mice and WT mice showed obvious functional enrichment in fatty acid metabolism, sulfur compound metabolism, regulation of mitochondrial organization, mitochondrial matrix, mitochondrial outer membrane, mitochondrial inner membrane, metal cluster binding, iron-sulfur cluster binding, 2-iron, 2-sulfur cluster binding, fatty acid degradation, valine, leucine, and isoleucine degradation-related proteins. Among them, KEGG enriched fatty acid metabolism and degradation, sulfide group metabolism, and some amino acid degradation-related functions in aBLA and pBLA. This shows that mitochondrial material metabolism-related pathways play an important role in the occurrence of the disease.

[0048] According to the localization of mitochondrial proteins, the differential proteins were marked on the mitochondria with a heat map, and the mitochondrial functions were specifically divided into 12 mitochondrial inner membrane proteins, 9 mitochondrial outer membrane proteins, 5 mitochondrial inner membrane proteins, 3 mitochondrial matrix proteins, and 11 cytochrome transport chains as candidate differential protein markers. (e.g. Fig. 9 (shown)

[0049] The differentially expressed proteins involved in the function are as follows:

[0050] Acot9 (Acyl-CoA thioesterase 9): Acyl-CoA thioesterases are a group of enzymes that catalyze the hydrolysis of acyl-CoA to free fatty acids and coenzyme A (CoASH), with the potential to regulate the intracellular levels of acyl-CoA, free fatty acids, and CoASH. Active towards long-chain acyl-CoAs.

[0051] fit3 (interferon-inducible protein with tetratricopeptide repeats 3): an IFN-induced antiviral protein that inhibits cellular and viral processes, cell migration, proliferation, signaling, and viral replication. It enhances MAVS-mediated host antiviral responses by acting as an adaptor bridging TBK1 to MAVS, leading to TBK1 activation and IRF3 phosphorylation, which translocates to the nucleus and promotes antiviral gene transcription. It inhibits antiproliferative activity by upregulating the negative cell cycle regulators CDKN1A / p21 and CDKN1B

[0052] CLUS (CLU, Clusterin): is a secreted chaperone that can also be found in the cytosol of cells under certain stress conditions. It is believed to be involved in several fundamental biological events, such as cell death, tumor progression, and neurodegenerative diseases. Alternative splicing produces coding and noncoding variants.

[0053] GHC2 (SLC25A18, Solute Carrier Family 25 Member): 18 L-aspartate transmembrane transporter activity and L-glutamate transmembrane transporter activity. Expected to be involved in L-glutamate transmembrane transport, i.e. aspartate transmembrane transport-aspartate shuttle. Expected to be located in the inner mitochondrial membrane. Responsible for the transport of glutamate from the cytosol to the mitochondrial matrix and the import of protons (symport system).

[0054] Arl8a (ADP-ribosylation factor-like protein 8A): Plays a role in lysosomal motility. In neurons, mediates anterograde axonal long-distance transport of presynaptic lysosome-associated vesicles required for presynaptic biogenesis and synaptic function. May play a role in chromosome segregation. Belongs to the small GTPase superfamily, the Arf family.

[0055] NU3M (MT-ND3, Mitochondrially Encoded NADH:Ubiquinone Oxidoreductase Core Subunit 3): NADH dehydrogenase (ubiquinone) activity. Participates in mitochondrial electron transfer, NADH to ubiquinone. Part of the mitochondrial respiratory chain complex

[0056] Hcls1 (hematopoietic lineage cell-specific protein): substrate of antigen receptor-coupled tyrosine kinase. Plays a role in antigen receptor signaling in lymphocyte clonal expansion and deletion.

[0057] THIOM (TXN2, Thioredoxin 2): Important for controlling mitochondrial reactive oxygen species homeostasis, apoptosis regulation, and cell viability. Has dithiol reducing activity.

[0058] PLSL (LCP1, Lymphocyte Cytosolic Protein 1): PLSL is a family of actin-binding proteins that are conserved throughout eukaryotic evolution and are expressed in most tissues of higher eukaryotes.

[0059] MTCH1 (Mitochondrial Carrier 1): A member of the mitochondrial carrier family. The encoded protein is localized to the inner membrane of mitochondria and induces apoptosis, independently of the pro-apoptotic proteins Bax and Bak. It catalyzes the insertion of proteins with α-helical transmembrane regions, such as signal anchors, tail anchors, and multipass membrane proteins. It does not mediate the insertion of β-barrel transmembrane proteins. It may play a role in apoptosis.

[0060] NDUB6 (NDUFB6, NADH:Ubiquinone Oxidoreductase Subunit B6): is a subunit of the multi-subunit NADH:ubiquinone oxidoreductase (complex I). Mammalian complex I consists of 45 different subunits. It is located in the inner mitochondrial membrane. This protein has NADH dehydrogenase activity and oxidoreductase activity. It transfers electrons from NADH to the respiratory chain. The direct electron acceptor of this enzyme is thought to be ubiquinone.

[0061] BABA1 (BABAM1, BRISCAndBRCA1 A Complex Member 1): A component of the BRCA1-A complex, a complex that specifically recognizes the "Lys-63"-linked ubiquitinated histones H2A and H2AX at DNA damage sites, resulting in the targeting of the BRCA1-BARD1 heterodimer to DNA damage sites at double-strand breaks (DSBs). The BRCA1-A complex also has deubiquitinase activity that specifically removes ubiquitin linked to "Lys-63" on histones H2A and H2AX. In the BRCA1-A complex, the integrity of the complex and its localization at DSBs are essential.

[0062] KPCA (PRKCA, Protein Kinase C Alpha): Protein kinase C (PKC) refers to a family of serine / threonine protein kinases grouped by their activation mechanism. Conventional PKC (cPKCα-, betaI-, betaII-, and γ-) are activated by phosphatidylserine in a calcium-dependent manner and can bind diacylglycerol. Calcium-activated, phospholipid- and diacylglycerol (DAG)-dependent serine / threonine protein kinases participate in the positive and negative regulation of cell proliferation, apoptosis, differentiation, migration and adhesion, tumorigenesis, cardiac hypertrophy, angiogenesis, platelet function, and inflammation by directly phosphorylating targets such as RAF1, BCL2, CSPG4, TNNT2 / CTNT, or activating signaling cascades involving MAPK1 / 3 (ERK1 / 2) and RAP1GAP.

[0063] Glrx5 (pentanedioxin-related protein 5): A monothiol pentanedioxin localized in mitochondria that is involved in the biogenesis of iron-sulfur clusters. Involved in protein lipoylation, plays a role in the pathway that provides iron-sulfur clusters to lipoate synthase. Required for normal iron homeostasis. Required for normal regulation of hemoglobin synthesis by the iron-sulfur protein ACO1. May protect cells from apoptosis caused by reactive oxygen species and oxidative stress.

[0064] Maip1 (m-AAA protease interacting protein 1): localized in mitochondria, promotes the sorting of SMDT1 / EMRE in mitochondria by ensuring mitochondrial maturation. It interacts with the transit peptide region of the SMDT1 / EMRE precursor protein in the mitochondrial matrix, protecting it from proteolytic degradation by YME1L1, thereby ensuring the maturation of SMDT1 / EMRE by mitochondrial processing peptidases (PMPCA and PMPCB).

[0065] SYEM (EARS2, Glutamyl-tRNA Synthetase 2): A member of the class I family of aminoacyl-tRNA synthetases. These enzymes play a key role in protein biosynthesis by charging tRNAs with their cognate amino acids. This protein is encoded by the nuclear genome but is likely imported into the mitochondria, where it is thought to catalyze the attachment of glutamate to tRNA molecules.

[0066] ATIF1 (ATP5IF1, ATP Synthase Inhibitory Factor Subunit 1): Enables multiple functions, including ATPase binding activity, angiostatin binding activity, and mitochondrial proton transport ATP synthase complex binding activity. Involved in multiple processes, including mitochondrial depolarization, negative regulation of ATPase activity, and regulation of protein targeting to mitochondria. Located on the cell surface and in mitochondria.

[0067] M4K3 (Mitogen-Activated Protein Kinase 3, serine / threonine kinase): Plays a role in the response to environmental stress. Appears to act upstream of the UN-N-terminal pathway. An activator of the Hippo signaling pathway, it plays a key role in organ size control and tumor suppression by limiting proliferation and promoting apoptosis. MAP4Ks parallel and partially duplicate STK3 / MST2 and STK4 / MST2 in the phosphorylation and activation of LATS1 / 2, establishing MAP4Ks as components of the extended Hippo pathway

[0068] CC90B (Coiled-Coil Domain Containing 90B): Located in mitochondria. Diseases associated with CCDC90B include oculomotor syndrome and osteochondrosis. An important paralog of this protein is MCUR1.

[0069] RPS6KA5 (Ribosomal Protein S6 KinaseA5): Enables ATP binding activity and protein serine / threonine kinase activity. Involved in multiple processes including histone-serine phosphorylation. Positive regulation of histone modifications and transcriptional regulation, DNA templating. Located in the cytoplasm and nucleoplasm.

[0070] Tim29 (mitochondrial import inner membrane translocase subunit): Tim29 is a component of the TIM22 complex, a complex that mediates the import and insertion of multipass transmembrane proteins into the mitochondrial inner membrane. The TIM22 complex forms a two-pore translocase that uses the membrane potential as an external driving force. It is required for the stability of the TIM22 complex and for the assembly of TIMM22 proteins into the TIM22 complex. It promotes cooperation between TIM22 and the TOM complex by interacting with TOMM40

[0071] Them4 (Acyl-CoA Thioesterase): Has acyl-CoA thioesterase activity towards medium- and long-chain (C14 to C18) fatty acyl-CoA substrates and may play a role in mitochondrial fatty acid metabolism. May play a role in apoptosis, possibly through its regulation of AKT1 activity.

[0072] Arl2 (ADP-ribosylation factor-like protein 2): Small GTP-binding protein that cycles between an inactive GDP-bound form and an active GTP-bound form, with the cycling rate regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GTP-binding protein that does not act as an allosteric activator of the catalytic subunit of cholera toxin. Regulates the formation of new microtubules and centrosome integrity. Prevents TBCD-induced microtubule disruption.

[0073] Rida (2-iminobutyrate / 2-iminopropionate deaminase) catalyzes the hydrolytic deamination of enamine / imine intermediates formed during normal metabolism. It may promote the release of ammonia from these potentially toxic reactive metabolites, reducing their impact on cellular components. It may act on enamine / imine intermediates formed by several types of pyridoxal-5'-phosphate-dependent dehydratases, including L-threonine dehydratase.

[0074] ACOT11 (Acyl-CoAThioesterase 11): has acyl-CoA thioesterase activity and prefers long-chain fatty acyl-CoA thioesters, hexadecanoyl-CoA / palmitoyl-CoA and tetradecanoyl-CoA / myristoyl-CoA, which are the main substrates in the mitochondrial β-oxidation pathway.

[0075] Taco1 (translational activator of cytochrome c oxidase 1): a mitochondrial-encoded translational activator of cytochrome c oxidase 1 belonging to the TACO1 family

[0076] Rcc2 (RCC2 multifunctional protein): May affect the function of small GTPases such as RAC1 and RALA by regulating their activity. Required for normal progression of the cell cycle during interphase and mitosis. Required for the presence of normal levels of MAD2L1, AURKB, and BIRC5 at the inner centromere and for normal attachment of the centromere to the mitotic spindle during mitosis. Required for the normal organization of the microtubule cytoskeleton in interphase cells. Functions as a guanine nucleotide exchange factor (GEF) for RALA.

[0077] DHSO (SORD, Sorbitol Dehydrogenase): Sorbitol dehydrogenase (SORD) catalyzes the interconversion of polyols and their corresponding ketoses, and together with aldose reductase (ALDR1) constitutes the sorbitol pathway, which plays an important role in the development of diabetic complications. The first reaction of this pathway (also called the polyol pathway) is the reduction of glucose to sorbitol by ALDR1 with NADPH as a cofactor. SORD then oxidizes sorbitol to fructose using the NAD(+) cofactor.

[0078] NNRD (NAXD, NAD(P)HX Dehydratase): Enables ATP-dependent NAD(P)H-hydrate dehydratase activity. Expected to be involved in metabolite repair. Expected to be localized in the cytosol, endoplasmic reticulum, and mitochondria. Catalyzes the dehydration of the S form of NAD(P)HX at the expense of ATP, which is converted to ADP. Together with NAD(P)HX epimerase, which catalyzes the S and R diastereoisomerization, this enzyme can repair both diastereomers of NAD(P)HX, NAD(P)H) is a damaged form of NAD(P)H that is the result of enzyme- or heat-dependent hydration.

[0079] Kif2a (kinesin-like protein KIF2A): required for end-directed microtubule-dependent motility for normal brain development. May regulate microtubule dynamics during axon growth. Required for normal progression through mitosis. Required for normal chromosome meeting at the metaphase plate. Required for normal spindle dynamics during mitosis. Promotes spindle turnover. Involved in the formation of the bipolar mitotic spindle. Has microtubule depolymerizing activity

[0080] HMGCS2 (3-Hydroxy-3-Methylglutaryl-CoA Synthase 2): Belongs to the HMG-CoA synthase family. It is a mitochondrial enzyme that catalyzes the first reaction of ketosis, a metabolic pathway that provides lipid-derived energy to various organs during carbohydrate deprivation (e.g., fasting).

[0081] NDUFA8 (NADH: Ubiquinone Oxidoreductase Subunit A8): Belongs to the complex I 19kDa subunit family. Mammalian complex I consists of 45 different subunits. This protein has NADH dehydrogenase activity and oxidoreductase activity. It plays an important role in transferring electrons from NADH to the respiratory chain. The direct electron acceptor of this enzyme is believed to be ubiquinone

[0082] MAON (ME3, Malic Enzyme 3): Malic enzyme catalyzes the oxidative decarboxylation of malate to pyruvate using NAD+ or NADP+ as cofactor. Mammalian tissues contain three different isoforms of malic enzyme: a cytosolic NADP(+)-dependent isoform, a mitochondrial NADP(+)-dependent isoform, and a mitochondrial NAD(+)-dependent isoform. This gene encodes the mitochondrial NADP(+)-dependent isoform. Multiple alternatively spliced ​​transcript variants of this gene have been found, but the biological validity of some variants has not yet been determined.

[0083] RT30 (MRPS30, Mitochondrial Ribosomal Protein S30): Mammalian mitochondrial ribosomal protein encoded by nuclear genes that aids in protein synthesis within mitochondria. Mitochondrial ribosomes (mitochondrial ribosomes) consist of a small 28S subunit and a large 39S subunit. Their protein to rRNA composition is estimated to be 5% compared to prokaryotic ribosomes, where this ratio is reversed.

[0084] Zdhhc6 (palmitoyltransferase ZDHHC6): ER palmitoyl acyltransferase that mediates palmitoylation of proteins such as AMFR, CALX, ITPR1, and TFRC. Palmitoylates calnexin (CALX), which is required for its binding to the ribosome-transgenic complex and for the efficient folding of glycosylated proteins. Mediates palmitoylation of AMFR and promotes the distribution of AMFR to the peripheral ER. Together with SELENOK, palmitoylates ITPR1 in immune cells, thereby regulating ITPR1 stability and function.

[0085] Ccsap (centriole, cilium and spindle-associated protein): Plays a role in microtubule (MT) stabilization that involves the maintenance of NUMA1 at the spindle poles. Colocalizes with polyglutamylated MTs to promote MT stabilization and regulates bipolar spindle formation in mitosis. During mitosis, CCSAP binds to centrosomes and pericentrosome spindles to inhibit MT depolymerization, thereby stabilizing the mitotic spindle. May play a role in embryonic development.

[0086] Cox7c (cytochrome c oxidase subunit 7C): Located in mitochondria, it is a component of cytochrome c oxidase, the last enzyme in the mitochondrial electron transport chain, driving oxidative phosphorylation. The respiratory chain contains three multi-subunit complexes, succinate dehydrogenase (complex II, CII), ubiquinol-cytochrome c oxidoreductase (cytochrome b-c1 complex, complex III, CIII) and cytochrome c oxidase (complex IV, CIV), which cooperate to transfer electrons from NADH and succinate to molecular oxygen, generating an electrochemical gradient across the inner membrane that drives the transmembrane.

[0087] 5. Screening of mitochondrial protein markers associated with anxiety disorders in Alzheimer's disease

[0088] Screening was performed based on the criteria of up-regulated expression in the brain tissue of 5×FAD mice, differential expression fold (FC) ≥ 1.3 and p < 0.01. The results are shown in Table 1.

[0089] Table 1 Differentially expressed proteins

[0090] Differential Protein Is expression upregulated? FC p Acot9 Increase 2.629942248 p＜0.01 GHC2 Increase 1.55214447 p＜0.01 NU3M Increase 1.542098714 p＜0.01 THIOM Increase 1.507054297 p＜0.01

[0091] The present invention screens out key markers of mitochondrial differences by discovering the differential mitochondrial protein network between the BLA of 5×FAD mice and WT mice. The present invention can use the mitochondrial protein markers related to anxiety disorders in Alzheimer's disease obtained by screening as targets for drug treatment, construct relevant animal models, and be used for screening drugs for treating anxiety disorders in Alzheimer's disease.

[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mitochondrial protein marker associated with anxiety disorder in Alzheimer's disease, characterized in that: The mitochondrial protein markers include Acot9, GHC2, NU3M and THIOM.

2. The mitochondrial protein marker according to claim 1, characterized in that The expression level of the mitochondrial protein marker in the Alzheimer's disease anxiety disorder group samples was significantly higher than that in the healthy group samples.

3. The method for screening mitochondrial protein markers according to claim 1, characterized in that: The method comprises the following steps: constructing an animal model group and a control group for Alzheimer's disease anxiety disorder, obtaining brain tissue samples from the two groups respectively, labeling proteins using tandem mass tag technology, and separating the proteome by high performance liquid chromatography, comparing the expression differences of mitochondrial proteins in brain tissues of the model group and the control group by mass spectrometry analysis of the separated proteins, screening out differentially expressed proteins by bioinformatics analysis, and determining mitochondrial proteins associated with Alzheimer's disease-related anxiety disorders as biomarkers.

4. The screening method according to claim 3, characterized in that The screening criteria for screening out differentially expressed proteins are up-regulated expression, differential expression fold ≥ 1.3 and p < 0.

01.

5. The screening method according to claim 3, characterized in that The bioinformatics analysis includes analyzing the abundance of differentially expressed proteins using a volcano plot, analyzing protein clusters using the R software Mfuzz package, performing GO enrichment analysis on the differentially expressed proteins, and performing KEGG enrichment analysis on the differentially expressed proteins.

6. The screening method according to claim 3, characterized in that The differentially expressed proteins are significantly enriched in functions related to fatty acid metabolism and degradation, sulfide group metabolism and amino acid degradation.

7. The screening method according to claim 3, characterized in that The Alzheimer's disease anxiety disorder animal model is 5×FAD mice.

8. Use of the mitochondrial protein marker according to claim 1 or 2 or the screening method according to any one of claims 3 to 7 in the preparation of a product for assisting the diagnosis of anxiety disorders in Alzheimer's disease.

9. Use of the mitochondrial protein marker according to claim 1 or 2 or the screening method according to any one of claims 3 to 7 in constructing an animal model for screening drugs for anxiety disorders in Alzheimer's disease.

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