Application of CARNS1 in Parkinson's disease

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of CARNS1 in Parkinson's disease (PD). The research finds that CARNS1 is highly expressed in striatum and nigra brain regions in C57BL / 6 mouse brains, is positioned in dopaminergic neurons in the nigra brain regions, and is down-regulated in MPTP and alpha-syn animal models and MPP + cell models; the nigra region CARNS1 knocks down and aggravates MPTP-induced dyskinesia and neurodegeneration, and the exogenous supplement carnosine can partially reverse phenotypes; the overexpression of the wild type CARNS1 can be used for relieving the neurodegeneration in MPTP and alpha-syn models. The invention clarifies the regulation role of CARNS1 in PD, provides a new perspective for understanding the pathogenesis of PD, and prompts that the CARNS1-carnosine metabolic axis may become an important target for intervening the pathological process of PD.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the application of CARNS1 in Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is a chronic, progressive neurological disorder characterized by four cardinal clinical features: resting tremor, rigidity, bradykinesia, and abnormal posture and gait, with or without associated non-motor deficits such as cognitive, psychiatric, and behavioral impairments, autonomic dysfunction, sleep disturbances, and hyposmia. The primary pathological changes in PD include the selective loss of dopamine (DA) neurons in the substantia nigra compacta (SNpc) with the formation of Lewy bodies, a significant decrease in the activity of tyrosine hydroxylase (TH), the key rate-limiting enzyme in DA biosynthesis, and progressive damage to the dopaminergic neuronal projection pathway from the SNpc to the dorsal striatum. The incidence and prevalence of PD increase with age. Although PD is currently more common in the elderly, studies have found that some patients develop the disease at an age of onset (AAO) earlier than 50 years old, which is defined as early-onset Parkinson's disease (EOPD), accounting for 5% to 10% of PD. EOPD has an earlier age of onset, a slower course of disease, highly heterogeneous clinical manifestations, and relatively atypical symptoms, leading to an increased risk of missed and misdiagnosis. Current PD treatments can partially alleviate motor symptoms but cannot slow the progression of neurodegeneration. This treatment dilemma places multiple burdens on patients, families, and society. Focusing on the molecular pathological mechanisms of PD, analyzing key molecular events and genetic risk factors, constructing a multidimensional mechanism network, and developing neuroprotective agents and specific targets to regulate the disease process are the keys to breaking through the bottleneck in PD treatment.

[0003] Carnosine, also known as β-alanine-L-histidine, is widely distributed in mammalian tissues, with particularly high expression in the brain, heart, and skeletal muscle. Carnosine exhibits multimodal mechanisms of action in the nervous system, including anti-inflammatory, antioxidant, anti-glycation, anti-protein aggregation, metal ion chelation, wound healing, and free radical scavenging activities. This has given it significant therapeutic potential in neurobiology and has garnered widespread attention in recent years. Several age-related neurological disorders, such as Alzheimer's disease (AD), multiple sclerosis (MS), cancer, and diabetic complications, may benefit from carnosine supplementation. Despite significant advances in carnosine research in neurobiology, research on enzymes and proteins involved in carnosine metabolism is relatively limited. The CARNS1 gene specifically encodes carnosine synthase 1, a member of the ATP-grasp enzyme family and an ATP-dependent carboxylic acid-amine ligase that catalyzes the production of carnosine from β-alanine and L-histidine. As a key enzyme encoding carnosine synthesis, whether CARNS1 and the CARNS1-carnosine metabolic axis play an important role in the pathological mechanism of PD, the specific mechanism of action has not yet been fully elucidated. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of CARNS1 in Parkinson's disease, systematically analyze the expression regulation mechanism of CARNS1 in PD models and its neuroprotective effect, and provide a new theoretical basis and therapeutic target for the diagnosis and treatment of PD.

[0005] To achieve the above object, the present invention adopts the following technical solutions: Use of reagents targeting CARNS1 and / or the CARNS1-carnosine metabolic axis in the preparation of drugs for treating Parkinson's disease; Furthermore, the reagent is carnosine with CAS accession number 305-84-0 or a reagent that promotes CARNS1 expression; the reagent that promotes CARNS1 expression is a CARNS1 overexpression adeno-associated virus vector; Furthermore, the drug alleviates the motor dysfunction and neurodegeneration of Parkinson's disease.

[0006] A drug for treating Parkinson's disease, comprising an agent targeting CARNS1 and / or the CARNS1-carnosine metabolic axis; Furthermore, the reagent is carnosine with CAS accession number 305-84-0 or a reagent that promotes CARNS1 expression; the reagent that promotes CARNS1 expression is a CARNS1 overexpression adeno-associated virus vector.

[0007] Furthermore, the drug alleviates the motor dysfunction and neurodegeneration of Parkinson's disease.

[0008] The use of agents that inhibit CARNS1 expression in establishing mouse models of Parkinson's disease with increased motor dysfunction and / or neurodegeneration; Furthermore, the reagent for inhibiting CARNS1 expression is mouse CARNS1 interfering shRNA or an adeno-associated virus vector containing a mouse CARNS1 interfering shRNA sequence; Furthermore, the DNA sequence of the mouse CARNS1 interfering shRNA is as follows: 5′-CCAACTGGTACAGACCTTT-3′; Furthermore, the Parkinson's disease mouse model includes an MPTP-induced Parkinson's disease mouse model and an α-synuclein-induced Parkinson's disease mouse model.

[0009] The significant advantages of the present invention are: The present invention constructs two PD models: one is a neurotoxic model, including an MPTP-induced mouse model and an MPP-induced mouse model. + The first is an SH-SY5Y cell model for intervention; the other is a model based on α-synuclein (α-syn) to detect the expression of CARNS1. Secondly, the present invention studied the effects of CARNS1 expression regulation in the substantia nigra on the susceptibility of mice to PD-like behavior and neurodegenerative pathology, as well as the potential mechanism of carnosine-mediated PD protection. The results showed that CARNS1 expression was significantly downregulated in the substantia nigra and striatum tissues of PD patients, and in a variety of PD models (such as MPTP-induced mouse model and MPP-induced mouse model). + A decrease in CARNS1 protein levels was observed in both the cell model treated with CARNS1 and the mouse model overexpressing SNAC A53T in the substantia nigra. In order to further explore the effect of CARNS1 expression level regulation on the behavioral phenotype and neuropathology of PD model mice, the present invention further constructed a CARNS1 knockdown and overexpression mouse model in the substantia nigra region with the help of brain stereotactic virus injection technology, and used MPTP and α-synuclein to induce PD phenotype, respectively. Combined with behavioral assessment and histopathological analysis, the effect of CARNS1 expression level regulation on the susceptibility of mice to PD-like behavior and neurodegenerative pathology was analyzed. The results showed that CARNS1 knockdown in the substantia nigra region aggravated MPTP-induced motor dysfunction and neurodegeneration, while carnosine supplementation could partially reverse the phenotype; overexpression of wild-type CARNS1 could alleviate neurodegeneration in MPTP and α-syn models. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1: Expression of CARNS1 in mouse brain regions under physiological conditions. Str: striatum; Sn: substantia nigra; Ob: olfactory bulb; Cor: cortex; Hip: hippocampus; Ht: hypothalamus; Cb: cerebellum.

[0011] Figure 2 :The expression of CARNS1 in the substantia nigra region of the midbrain of wild-type C57BL / 6 mice and its colocalization with different neural cell types.

[0012] Figure 3 : Expression of CARNS1 in brain tissue of PD patients.

[0013] Figure 4 : Expression of CARNS1 in different PD models.

[0014] Figure 5 : Schematic diagram of the experimental process of the effect of knocking down CARNS1 in the substantia nigra on PD model mice.

[0015] Figure 6 :Identification of PD model mice with CARNS1 knockdown in the substantia nigra.

[0016] Figure 7 :Effects of CARNS1 knockdown in the substantia nigra and exogenous carnosine on the results of rotarod test and pole climbing test in PD model mice.

[0017] Figure 8 :Effects of CARNS1 knockdown in the substantia nigra and exogenous carnosine on the open field test results in PD model mice.

[0018] Figure 9 :Effects of CARNS1 knockdown and exogenous carnosine in the substantia nigra on neuronal loss in PD model mice.

[0019] Figure 10 :Effects of CARNS1 knockdown in the substantia nigra on carnosine biosynthesis in PD model mice.

[0020] Figure 11 :Effects of CARNS1 knockdown in the substantia nigra and exogenous carnosine on neuronal apoptosis in PD model mice.

[0021] Figure 12 :Schematic diagram of the experimental process of the effect of upregulating CARNS1 in the substantia nigra on PD model mice.

[0022] Figure 13 : Identification of PD model mice with upregulation of CARNS1 in the substantia nigra brain region.

[0023] Figure 14 :The effect of up-regulation of CARNS1 in the substantia nigra on the results of rotarod test and pole climbing test in PD model mice.

[0024] Figure 15 :The effect of up-regulation of CARNS1 in the substantia nigra on the open field test results in PD model mice.

[0025] Figure 16 :The effect of upregulation of CARNS1 in the substantia nigra on neuronal loss in PD model mice.

[0026] Figure 17 :The effect of up-regulation of CARNS1 in the substantia nigra on neuronal apoptosis in PD model mice. DETAILED DESCRIPTION

[0027] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0028] Example 1: 1. Physiological expression of CARNS1 in mouse brain To clarify the homology of the amino acid sequence of carnosine synthase 1 (CARNS1) across different species, a global comparison was performed using the Uniprot protein database and EMBOSS Needle. The results showed that the amino acid sequence of CARNS1 is highly conserved between humans (GeneID: 79571, data from NCBI) and mice (Gene ID: 67845, data from NCBI), with a similarity of 91.2%. Its key functional domain is the ATP-grasp domain, which is responsible for catalyzing the biosynthesis of carnosine. The functional domain of the two species is 95% identical, and key catalytic residues (such as His-178 and Glu-201) are completely conserved, supporting cross-species functional consistency. Therefore, subsequent experiments using mouse models will further explore its physiological role, its role in the Parkinson's disease brain, and its potential molecular mechanisms.

[0029] 1.1CARNS1 is relatively highly expressed in the substantia nigra of the midbrain To study the expression of CARNS1 in the mouse brain under physiological conditions, we first used immunohistochemistry to detect the expression level of CARNS1 in brain slices of 3-month-old wild-type male C57BL / 6 mice. The results showed that all brain regions showed positive signals, especially the striatum and substantia nigra, which are closely related to the pathogenesis of Parkinson's disease. Figure 1A). Then, qPCR technology was used to measure the expression levels of CARNS1 mRNA in different brain regions of 3-month-old wild-type male C57BL / 6 mice. The results showed that CARNS1 mRNA was widely expressed in multiple brain regions, including the olfactory bulb, cortex, hippocampus, hypothalamus, striatum, substantia nigra, and cerebellum. The expression level of CARNS1 mRNA in the substantia nigra was relatively high ( Figure 1 B).

[0030] 1.2CARNS1 is mainly expressed in neurons and oligodendrocytes Immunofluorescence was used to detect the expression of CARNS1 in the substantia nigra of the midbrain of 3-month-old wild-type C57BL / 6 mice and its co-localization with different neural cell types. NeuN antibody was used to label neurons, GFAP antibody to label astrocytes, Iba-1 antibody to label microglia, and Oligo2 antibody to label oligodendrocytes. Laser confocal microscopy was used for fluorescence co-localization analysis. The results showed that CARNS1 was mainly expressed in neurons (CARNS1 + NeuN + / NeuN + About 68%) and oligodendrocytes (CARNS1 + Oligo2 + / NeuN + About 24%) Figure 2 A). Further, by using TH antibody to label dopaminergic neurons in the substantia nigra and verifying it on brain slices of 3-month-old wild-type C57BL / 6 mice and 2-month-old wild-type SD rats, it was found that CARNS1 in the substantia nigra region of the midbrain showed a high degree of co-localization with dopaminergic neurons ( Figure 2 B).

[0031] Expression of CARNS1 in 2PD patients 2.1 Expression of CARNS1 in brain tissue of PD patients To explore the expression differences of CARNS1 in the brains of PD patients, the transcriptome dataset of PD patient autopsy specimens in the GEO database (ID: GSE205450) was used for in-depth analysis. This dataset covers the caudate nucleus and putamen samples of 40 healthy controls and the corresponding samples of 35 PD patients. The mRNA expression data of the CARNS1 gene and TH gene were extracted to further analyze their expression differences. The analysis results revealed that compared with the healthy control group, the expression levels of CARNS1 mRNA (both p < 0.0001) and TH mRNA (both p < 0.05) in the caudate nucleus and putamen of the striatum of PD patients were decreased; further correlation analysis showed that the expression levels of CARNS1 mRNA and TH mRNA in the caudate nucleus and putamen of the striatum of PD patients showed a positive correlation trend, but the correlation was not significant ( Figure 3 A). In addition, referring to the 2024 joint analysis of single-nucleus transcriptome sequencing (snRNA-seq) and single-nucleus transposase-accessible chromatin sequencing (snATAC-seq) of the substantia nigra tissue of the midbrain by the Levi Adams research team (Adams, L., et al., A single-nuclei paired multiomic analysis of the human midbrain reveals age-and Parkinson's disease-associated glial changes. NatAging, 2024.4(3): p.364-378.), it was suggested that CARNS1 mRNA was significantly downregulated in the substantia nigra of the midbrain of aging people and PD patients ( Figure 3 B) The above results show that CARNS1 expression is reduced in both the striatum and substantia nigra of PD patients, which may indirectly indicate that downregulation of CARNS1 expression plays a key role in the onset and progression of PD.

[0032] 2.2 Expression of CARNS1 in PD models To explore the expression of CARNS1 in PD models, two PD models were constructed: a neurotoxic model, including an MPTP-induced mouse model and an MPP-induced mouse model. + The other is an α-synuclein (α-syn)-induced SH-SY5Y cell model, including the SNCA A53T mutant α-synuclein (α-syn) mouse model and the SNCA gene-overexpressing SH-SY5Y cell model. The construction methods are as follows: MPTP-induced mouse model: SPF male C57BL / 6 mice (8-10 weeks old) were selected and housed in a standard housing environment for 7 days, with 4-5 mice per cage and free access to food and water. MPTP hydrochloride (Sigma-Aldrich, USA, Cat No. M0896) was dissolved in sterile saline to prepare a 1 mg / mL solution. The model group received intraperitoneal injection (ip) of 30 mg / kg daily at a fixed time (9:00-11:00 a.m.) for 5 consecutive days. The control group received an equal volume of saline.

[0033] MPP + Induced SH-SY5Y cell model: SH-SY5Y cells were seeded in complete cell culture medium containing 10% fetal bovine serum and cultured in a 5% CO2 cell culture incubator. The medium was changed every 2 days. SH-SY5Y cells in good condition were digested and then plated at 2×10 4 Cells / ml were inoculated into a new culture plate, and MPP+ at a final concentration of 1000, 1400, and 1800 μmol / L was added to the culture medium and gently shaken. The culture plate was placed in a cell culture incubator and cultured for 24 hours to induce a PD cell model. The cells in the control group were cultured conventionally without MPP. + deal with.

[0034] SNCA A53T mutant α-syn mouse model: SPF male C57BL / 6 mice (8-10 weeks old) were selected and housed in a standard housing environment for 7 days, with 4-5 mice per cage and free access to food and water. Mice were randomly divided into two groups: the control group received stereotactic injection of an empty vector virus (pAAV-hSy-MCS-WPRE, Shanghai Heyuan Biotechnology Co., Ltd.) into the right substantia nigra; the model group received stereotactic injection of a recombinant virus carrying the human A53T mutant α-syn (pAAV-hSy-SNCA(A53T)-WPRE, Shanghai Heyuan Biotechnology Co., Ltd.) into the right substantia nigra.

[0035] SH-SY5Y cell model of SNCA gene overexpression: SH-SY5Y cells were cultured in complete cell culture medium containing 10% fetal bovine serum in a 5% CO2 cell culture incubator, with the medium changed every 2 days. SH-SY5Y cells in good condition were digested and then plated at 2×10 4Cells / ml were inoculated into new culture plates. For the modeling group, the SNCA gene overexpression lentiviral plasmid pHBLV-CMV-SNCA-3×FLAG-EF1-ZsGreen-T2A-PURO (Shanghai Hanheng Biotechnology Co., Ltd.) was added to the culture medium. The amount of virus added to each well (μL) = MOI × number of cells at infection / titer (TU / mL) × 1000 was adjusted. The cells were gently shaken and cultured in a cell culture incubator for 12-16 hours to induce the PD cell model. For the control group, the empty vector virus pHBLV-CMV-3×FLAG-EF1-ZsGreen-T2A-PURO (Shanghai Hanheng Biotechnology Co., Ltd.) was added to the culture medium.

[0036] Western-Blot analysis of CARNS1 expression showed that in the MPTP-induced mouse model, CARNS1 expression in the substantia nigra was decreased (p<0.05), accompanied by decreased TH expression (p<0.05); + In the SH-SY5Y cell model induced by MPP + With the gradual increase of concentration, the level of CARNS1 protein showed a significant trend of gradual decrease ( Figure 4 A). In addition, nigral tissue protein samples were collected 2 months after the A53T mutant α-syn mouse model was established, and it was found that the expression of CARNS1 in the substantia nigra brain region was reduced (p<0.05), accompanied by an increase in α-syn expression (p<0.05); in the SH-SY5Y cell model with overexpression of the SNCA gene, the expression of CARNS1 showed a downward trend, but it was not statistically significant (p>0.05) ( Figure 4 B).

[0037] Example 2: This study aims to establish CARNS1 gene knockdown and overexpression mouse models in the substantia nigra by using stereotactic viral injection technology, and to induce PD phenotype using MPTP. Combined with behavioral assessment, histopathological analysis, and molecular biological testing, this study systematically analyzes the effects of regulating CARNS1 expression levels on PD-like behavioral susceptibility and neurodegenerative pathology in mice, in order to provide new perspectives for the study of the pathogenesis of PD and the development of therapeutic targets.

[0038] Effects of knockdown of CARNS1 in the substantia nigra on PD model mice 1.1 Knockdown of CARNS1 in the substantia nigra increases the susceptibility of MPTP-induced PD-like behavior in mice, and carnosine supplementation can improve the susceptibility. AAV2 / 9 viruses carrying shNC (negative control) or mouse CARNS1 interference shRNA, i.e., shCARNS1 (5'-CCAACTGGTACAGACCTTT-3'), were injected into the bilateral substantia nigra of C57BL / 6 mice using stereotaxic injection. After 3 weeks of viral expression, the following experimental procedures were carried out in sequence ( Figure 5 ): (1) Baseline Parkinson's disease-related behavioral assessment; (2) Intraperitoneal injection of MPTP for 5 consecutive days to construct a subacute Parkinson's disease model, as described in Example 1; (3) Systematic motor function testing (including rotarod test, pole climbing test, and open field exercise) was performed on days 1, 3, 5, and 7 after MPTP injection. Five groups were set up in the experiment: AAV-shNC+Saline, AAV-shNC+MPTP, AAV-shCARNS1+Saline, AAV-shCARNS1+MPTP, and AAV-shCARNS1+MPTP+Car (carnosine intervention group). Among them, the carnosine intervention group was continuously given sterile drinking water containing 2g / L carnosine (CAS accession number 305-84-0) starting 3 weeks before virus injection, and the intervention covered the entire process to evaluate its neuroprotective potential. The total carnosine intake was 1.1mg / mouse.

[0039] Rotarod test method: (1) Adaptation training: The C57BL / 6 mice in the group were ear-tagged and rotarod was used. Mice were acclimated using a fatigue instrument for three consecutive days, with two groups trained each day. Method: Mice were placed on a rotating beam at 4 rpm and trained in two sets according to a schedule of "4 rpm for 10 minutes, then accelerating to 20 rpm from 4 pm," with a 30-minute rest period between sets. Mice that showed agitation or jumped off the rod at the start of training were eliminated and replaced. If a mouse fell from the rod during training, it was returned to its cage and allowed to rest for 15 minutes before continuing training.

[0040] (2) Rotarod test: The rats were acclimated to a speed of 4 rpm for 10 min. Starting at 4 rpm, the acceleration was increased to 8 rpm / min over 5 min to 40 rpm. The time the rats spent on the rod, the number of turns on the rod, and the speed at which they landed were recorded. The speed at which the rats did not land was recorded as 40 rpm. The test was repeated three times, with at least 1 hour between tests, and the average value was calculated.

[0041] Pole climbing experiment method: The pole climbing test is often used to detect the degree of motor delay and limb coordination and balance ability of PD animals. Three days of adaptive training should be conducted in advance, with three sets of adaptive training performed each day, with a 30-minute interval between two sets of experiments. A simple climbing pole with a height of 55 cm and a diameter of 1 cm is made by hand, with a 2-cm diameter ball on the top. To prevent slipping, a layer of gauze is wrapped around the surface of the wooden pole. The experimental animal is placed head-up on the ball, and a stopwatch is used to record the time it takes for the mouse to turn head-up and start climbing to the middle of the pole, which is recorded as the half-pole time T-half. The time it takes for the feet to touch the ground after climbing to the bottom of the pole is recorded as T-Total. Each animal is tested three times, and the average value is used for statistical analysis.

[0042] Open field test method: (1) Familiarization stage The mouse test chamber used was an open field chamber from Shanghai Xinruan, measuring 50 cm × 50 cm × 40 cm (length × width × height). The chamber's bottom was divided into 16 grids. Before the experiment began, the mice were allowed to acclimate to the experimental environment for 2 hours to relieve their nervousness.

[0043] (2) Testing phase Remove the animal from its cage and place it in the center of the open field, facing away from the experimenter. The experimenter should quickly leave the room and allow the animal to move freely in the experimental box. Start the video capture software or the video recording system, and record the animal's activities in the open field for 10 minutes. After the experimental monitoring period, remove the mouse and return it to its cage. Thoroughly clean the open field to remove any residual animal information (such as urine, feces, and odor) to avoid affecting the next test results. After wiping it clean, replace the mouse and repeat the above experiment.

[0044] (3) Data statistics and analysis Computers were used to process and analyze data and to detect indicators such as the mice's athletic ability, tension, and anxiety levels.

[0045] Western Blot analysis confirmed that the expression of CARNS1 protein in the substantia nigra of the AAV-shCARNS1 group was significantly downregulated compared with the AAV-shNC+Saline group (p<0.0001), indicating that the CARNS1 gene knockdown model was successfully established ( Figure 6 ).

[0046] The results of the rotarod test showed that ( Figure 7), MPTP modeling can induce motor coordination disorders in mice: compared with the AAV-shNC+Saline group, the AAV-shNC+MPTP group had shorter rotarod time on the first day (p<0.01) and third day (p<0.05) after modeling, indicating that the Parkinson's disease model was successfully established. Further analysis found that knockdown of CARNS1 in the substantia nigra exacerbated MPTP-induced motor deficits - the AAV-shCARNS1+MPTP group had a further decrease in rotarod time on the fifth day (p<0.05) and seventh day (p<0.05) after modeling compared with the AAV-shNC+MPTP group. Carnosine intervention can partially reverse this phenotype. The AAV-shCARNS1+MPTP+Car group had longer rotarod time on the fifth day (p<0.05) and seventh day (p<0.05) compared with the untreated AAV-shCARNS1+MPTP group, suggesting that exogenous carnosine alleviates movement disorders by compensating for the loss of CARNS1 function.

[0047] The results of the pole climbing experiment showed that ( Figure 7 ), compared with the AAV-shNC+Saline control group, the half-pole time (from the top of the pole to the middle of the pole, both p<0.001) and full-pole time (from the top of the pole to the bottom of the pole, both p<0.001) of the AAV-shNC+MPTP group on the first and third days after modeling were significantly prolonged, and the half-pole time (p<0.01) and full-pole time (p<0.05) still maintained significant differences on the 7th day, indicating that MPTP successfully induced the characteristic bradykinesia and motor coordination disorders of Parkinson's disease. On the 7th day of the experiment, the half-rod time (p<0.01) and full-rod time (p<0.01) of the AAV-shCARNS1+MPTP group were further prolonged compared with the AAV-shNC+MPTP group, suggesting that knockdown of CARNS1 in the substantia nigra can enhance the neurotoxicity of MPTP, while carnosine intervention can partially reverse the phenotype: the half-rod time (p<0.01) and full-rod time (p<0.01) of the AAV-shCARNS1+MPTP+Car group were significantly shortened compared with the unintervention AAV-shCARNS1+MPTP group, suggesting that exogenous carnosine improves motor retardation by re-establishing the neurometabolic homeostasis in the substantia nigra region.

[0048] The open field test evaluated the anxiety-like behavior and exploration ability of mice by analyzing the central distance. Figure 8), on the 5th and 7th days after MPTP modeling, the central distance of the AAV-shCARNS1+MPTP group was further shortened than that of the AAV-shNC+MPTP group (5th day, p<0.01; 7th day, p<0.05), suggesting that knockdown of CARNS1 in the substantia nigra may aggravate anxiety-like behavior or inhibit exploratory behavior to a certain extent. The central distance of the carnosine intervention group (AAV-shCARNS1+MPTP+Car) showed a numerical recovery trend compared with the non-intervention group, but the difference did not reach statistical significance (p>0.05).

[0049] In summary, knockdown of CARNS1 in the substantia nigra increases the susceptibility of MPTP-induced Parkinson's disease-like motor phenotype in mice, while exogenous carnosine may effectively alleviate PD-like behavior by compensating for the loss of CARNS1 function, suggesting that targeted regulation of the CARNS1-carnosine metabolic axis may provide a new strategy for neuroprotective treatment of PD.

[0050] 1.2 Knockdown of CARNS1 gene in the substantia nigra induces TH neuron loss in MPTP model mice, and carnosine supplementation can improve Immunohistochemical analysis showed that ( Figure 9 A), MPTP modeling significantly induced substantia nigra TH neuron damage: compared with the AAV-shNC+Saline group, the number of TH-positive neurons in the substantia nigra region of the AAV-shNC+MPTP group was significantly reduced (p<0.001), confirming that the Parkinson's disease model was successfully constructed. Further analysis found that knockdown of CARNS1 in the substantia nigra could synergistically enhance MPTP neurotoxicity - the number of TH-positive neurons in the AAV-shCARNS1+MPTP group further decreased compared with the AAV-shNC+MPTP group (p<0.01). The carnosine intervention (AAV-shCARNS1+MPTP+Car) group can effectively reverse neuronal damage, and its TH-positive neuron number increased compared with the non-intervention (AAV-shCARNS1+MPTP) group (p<0.01). Western blot results showed ( Figure 9 B) TH protein expression in the substantia nigra was lower in the AAV-shNC+MPTP group compared with the AAV-shNC control group (p<0.05). Although TH protein expression in the AAV-shCARNS1+MPTP group showed a downward trend compared with the AAV-shNC+MPTP group, the difference was not significant. Furthermore, carnosine supplementation increased TH protein expression compared with the untreated group (AAV-shCARNS1+MPTP+Car vs. AAV-shCARNS1+MPTP, p<0.05).

[0051] 1.3 Effects of CARNS1 deficiency in the substantia nigra on carnosine biosynthesis To clarify the effect of CARNS1 deficiency on carnosine biosynthesis, ELISA was used to detect the carnosine content in the substantia nigra of each group of mice ( Figure 10 A). The results showed that compared with the AAV-shNC+Saline group, the level of carnosine in the substantia nigra in the AAV-shNC+MPTP group was reduced (p<0.05), suggesting that MPTP may interfere with carnosine synthesis by inhibiting CARNS1 activity. Although the CARNS1 knockdown group showed a downward trend compared with the control, there was no statistical difference. It is speculated that this is related to the compensatory upregulation of the virus injection time or other synthesis pathways (such as non-enzymatic reactions). The level of carnosine in the substantia nigra in the carnosine intervention group (AAV-shCARNS1+MPTP+Car) was higher than that in the non-intervention group (AAV-shCARNS1+MPTP) (p<0.05), which indicates that exogenous carnosine can effectively penetrate the blood-brain barrier and accumulate in the target area ( Figure 10 B).

[0052] 1.4 CARNS1 knockdown in the substantia nigra promotes apoptosis of DA neurons in MPTP model mice, and carnosine supplementation can improve TUNEL staining results showed that MPTP modeling can induce apoptosis of dopaminergic neurons in the substantia nigra: compared with the AAV-shNC+Saline group, the proportion of TUNEL-positive neurons in the substantia nigra in the AAV-shNC+MPTP group increased (p<0.05). In addition, CARNS1 knockdown amplified the pro-apoptotic effect of MPTP - the proportion of TUNEL-positive cells in the AAV-shCARNS1+MPTP group was further increased compared with the AAV-shNC+MPTP group (p<0.05), indicating that CARNS1 deficiency can aggravate dopaminergic neuron apoptosis. Carnosine supplementation (AAV-shCARNS1+MPTP+Car group) can effectively inhibit this apoptotic cascade reaction, and its TUNEL-positive cell proportion is lower than that of the non-intervention group (AAV-shCARNS1+MPTP) (p<0.05), suggesting that exogenous supplementation of carnosine may play a neuroprotective role ( Figure 11 ).

[0053] Effects of upregulating CARNS1 in the substantia nigra on PD model mice 2.1 Overexpression of wild-type CARNS1 in the substantia nigra alleviates MPTP-induced PD-like behavioral phenotypes in mice. AAV viruses carrying empty control (AAV-V5) and CARNS1 (Gene ID: 67845, data from NCBI) wild-type group (AAV-CARNS1 WT-V5) were injected into the substantia nigra of C57BL / 6 mice bilaterally using stereotaxic injection technology. After 3 weeks of viral expression, the following experimental procedures were carried out in sequence ( Figure 12): (1) Baseline Parkinson's disease-related behavioral assessment; (2) Establishment of a subacute Parkinson's disease model by intraperitoneal injection of MPTP for 5 consecutive days, as described in Example 1; (3) Systematic motor function testing (including rotarod test, pole climbing test, and open field exercise) was performed on days 1, 3, 5, and 7 after MPTP injection, as described in Example 2. Four experimental groups were set up: Control, MPTP, AAV-V5 + MPTP, and AAV-CARNS1 WT + MPTP.

[0054] Western Blot analysis confirmed that the total protein level of CARNS1 in the substantia nigra of the wild-type overexpressing group was significantly increased compared with the empty virus control group (p<0.0001) ( Figure 13 ).

[0055] The results of the rotarod test showed that ( Figure 14 ), MPTP modeling can induce motor coordination disorders in mice: compared with the Control group, the MPTP group had shorter rotarod residence time on the 3rd day (p<0.01), 5th day (p<0.001) and 7th day (p<0.01) after modeling, indicating that the Parkinson's disease model was successfully established. Further analysis found that overexpression of wild-type CARNS1 (AAV-CARNS1 WT+MPTP group) significantly improved motor coordination ability, and its rotation rod residence time was longer than that of the mutant group and the empty virus group at all time points, as follows: on the first day after modeling, the time on the rod of the AAV-CARNS1 WT+MPTP group was longer than that of the AAV-V5+MPTP group (p<0.05); on the third day after modeling, the time on the rod of the AAV-CARNS1 WT+MPTP group was longer than that of the AAV-V5+MPTP group (p<0.05); on the fifth day after modeling, the time on the rod of the AAV-CARNS1 WT+MPTP group was longer than that of the AAV-V5+MPTP group (p<0.01).

[0056] The results of the pole climbing experiment showed that ( Figure 14), compared with the Control group, the MPTP group maintained significant differences in half-bar time (day 1, p<0.01; day 3, p<0.001; day 5, p<0.001; day 7, p<0.01) and full-bar time (day 1, p<0.05; day 3, p<0.01; day 5, p<0.05; day 7, p<0.01) on days 1, 3, 5, and 7 after modeling, suggesting that MPTP successfully induced characteristic bradykinesia and motor coordination disorders of Parkinson's disease. In terms of half-bar time performance, on day 7 after modeling, the AAV-CARNS1 WT+MPTP group was shorter than the AAV-V5+MPTP group (p<0.05). In the full-bar time assessment, on day 7 after modeling, the AAV-CARNS1 WT+MPTP group was shorter than the AAV-V5+MPTP group (p<0.01).

[0057] The results of the open field experiment showed that ( Figure 15 ). On days 1, 3, and 5 after MPTP modeling, the central distance of the MPTP group was shorter than that of the control group (day 1, p<0.01; day 3, p<0.05; day 5, p<0.05), indicating that the model mice exhibited anxiety-like behaviors and decreased exploratory ability. On day 3 after modeling, the central distance of the AAV-CARNS1 WT+MPTP group was longer than that of the AAV-V5+MPTP group (p<0.01), suggesting that wild-type CARNS1 can alleviate the MPTP-induced anxiety phenotype to some extent.

[0058] In summary, overexpression of wild-type CARNS1 in the substantia nigra can alleviate the MPTP-induced PD-like phenotype in mice.

[0059] 2.2 Overexpression of wild-type CARNS1 in the substantia nigra improves TH neuron loss in MPTP model mice. Immunohistochemical analysis showed that ( Figure 16 ), MPTP modeling significantly induced TH neuron damage in the substantia nigra: compared with the Control group, the number of TH-positive neurons in the substantia nigra region in the MPTP group (p<0.01) and the AAV-V5+MPTP group was significantly reduced (p<0.001), confirming that the Parkinson's disease model was successfully constructed. Further analysis found that overexpression of wild-type CARNS1 in the substantia nigra can effectively alleviate MPTP neurotoxicity - the number of TH-positive neurons in the AAV-CARNS1 WT+MPTP group increased compared with the AAV-V5+MPTP group (p<0.05). Western blot results showed that this phenotype was further verified ( Figure 16C) Compared with the control group, TH protein expression in the substantia nigra was lower in the MPTP group (p<0.0001) and the AAV-V5+MPTP group (p<0.01), whereas TH protein levels were higher in the AAV-CARNS1WT+MPTP group (p<0.05). These results suggest that wild-type CARNS1 protects DA neurons by resisting MPTP neurotoxicity.

[0060] 2.3 Overexpression of wild-type CARNS1 in the substantia nigra improves DA neuron apoptosis in MPTP model mice. TUNEL staining results showed that ( Figure 17 MPTP-induced apoptosis of dopaminergic neurons in the substantia nigra: Compared with the control group, the proportion of TUNEL-positive neurons in the MPTP group (p<0.001) and the AAV-V5+MPTP group (p<0.001) increased. The AAV-CARNS1 WT+MPTP group effectively inhibited apoptosis, with a decreased proportion of TUNEL-positive cells compared with the AAV-V5+MPTP group (p<0.01).

Claims

1. CARNS1 and / or CARNS1 -Application of reagents targeting the carnosine metabolic axis in the preparation of drugs for the treatment of Parkinson's disease.

2. The use according to claim 1, characterized in that: The reagent is carnosine or a stimulating CARNS1 Expression of the agent; said promoting CARNS1 The reagents used for expression are CARNS1 Overexpression adeno-associated viral vector.

3. The use according to claim 1, characterized in that: The drug alleviates the motor dysfunction and neurodegeneration of Parkinson's disease.

4. A drug for treating Parkinson's disease, characterized in that: The drug contains CARNS1 and / or CARNS1 -Agents targeting the carnosine metabolic axis.

5. The drug according to claim 4, characterized in that: The reagent is carnosine or a stimulating CARNS1 Expression of the agent; said promoting CARNS1 The reagents used for expression are CARNS1 Overexpression adeno-associated viral vector.

6. The drug according to claim 4, characterized in that: The drug alleviates the motor dysfunction and neurodegeneration of Parkinson's disease.

7. Inhibition CARNS1 Application of the expressed reagents in constructing a mouse model of Parkinson's disease with exacerbated motor dysfunction and / or neurodegeneration.

8. The use according to claim 7, characterized in that: The inhibition CARNS1 The expression reagent is mouse CARNS1 Interfering shRNA or containing mouse CARNS1 Adeno-associated viral vectors containing interfering shRNA sequences.

9. The use according to claim 8, characterized in that: The rat CARNS1 The DNA sequence of the interfering shRNA is as follows: 5'-CCAACTGGTACAGACCTTT-3'.

10. The use according to claim 7, characterized in that: The Parkinson's disease mouse model includes an MPTP-induced Parkinson's disease mouse model and an α-synuclein-induced Parkinson's disease mouse model.

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