Use of a formulation that enhances ctl1 activity in the manufacture of a medicament for treating a neurodegenerative disease

Abstract

The present application relates to the technical field of biopharmaceutical, and particularly relates to application of a preparation for enhancing CTL1 activity in preparation of a medicament for treating neurodegenerative diseases, wherein the CTL1 is a kind of mitochondrial autophagy receptor.The present application identifies that CTL1 is a kind of mitochondrial autophagy receptor in SH-SY5Y and MN9D neuron models.CTL1 interacts with LC3 in a LIR-dependent manner, and it is observed that CTL1 phosphorylation induced by mitochondrial damage can trigger selective mitochondrial autophagy.Dopaminergic neuron-specific CTL1 knockout mice show parkinson's disease-like motor dysfunction and neurodegenerative lesions.Induced CTL1 in neurons can improve MPTP-induced death of dopaminergic neurons in mice.These results confirm that CTL1 is a kind of phosphorylation-regulated mitochondrial autophagy receptor, and its dysfunction can drive neurodegenerative lesions, thereby providing a direct molecular mechanism for the pathological mechanism of parkinson's disease.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and more specifically to the use of formulations that enhance CTL1 activity in the preparation of drugs for treating neurodegenerative diseases. Background Technology

[0002] Impaired mitochondrial clearance is a hallmark of many age-related human pathologies, including neurodegenerative diseases and cardiovascular diseases. Mitophagy, the selective autophagic degradation of damaged or unwanted mitochondria, maintains cellular homeostasis by eliminating reactive oxygen species (ROS) and suppressing inflammation. This quality control mechanism is particularly important for neurons, long-lived and energy-intensive cells, as they depend on optimal mitochondrial fitness for synaptic activity and survival.5,6 Mitophagy deficiency is closely associated with neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS). Enhancing mitophagy is widely considered a potential therapeutic strategy to halt the progression of neurodegenerative diseases, but insufficient understanding of the mechanisms of neuronal mitophagy has hindered its clinical translation.

[0003] The PINK1 / Parkin pathway is the most extensively studied and well-understood mechanism in ubiquitin-dependent mitophagy. However, PINK1 / Parkin signaling is not essential for basal mitophagy, which mediates constitutive mitochondrial turnover to maintain neuronal homeostasis under normal or chronic pathological conditions. In fact, basal mitophagy remains highly active in neurons and other brain cells. Its core mechanism is thought to involve mitophagy receptors, which typically contain LC3-interacting region LIR motifs and can directly bind to autophagosome markers; their physiological importance in peripheral tissues and specific developmental settings is well-established. However, strong evidence is currently lacking to suggest they play a fundamental role in maintaining long-term brain health, particularly in chronic progressive neurodegenerative diseases. Although some known receptors, such as BNIP3, BNIP3L / NIX, and FUNDC1, have shown protective effects in acute brain injury models of mitophagy, the transient protection under acute stress is fundamentally different from the requirements for maintaining neuronal homeostasis over decades in chronic diseases. Although supraphysiological levels of BNIP3 can induce neuronal mitophagy and delay brain aging, endogenous expression of BNIP3 and BNIP3L / NIX in the adult brain is significantly lower than in peripheral tissues.

[0004] Therefore, given the current lack of endogenous receptors that can effectively mediate long-term, basal mitophagy in the adult brain, the search for unrecognized mitophagy receptors within neurons and the development of a therapeutic agent that enhances the activity of these receptors to promote sustained neuronal mitochondrial clearance may play a crucial role in neurodegenerative diseases. Summary of the Invention

[0005] To address the above problems, this invention provides the application of formulations that enhance CTL1 activity in the preparation of drugs for treating neurodegenerative diseases.

[0006] This invention is achieved through the following technical solution: The application of formulations that enhance CTL1 activity in the preparation of drugs for treating neurodegenerative diseases, wherein CTL1 is a mitochondrial autophagy receptor; the formulation comprises recombinant viral particles that overexpress the CTL1 gene.

[0007] The specific method for constructing the recombinant viral particles overexpressing the CTL1 gene is as follows: the CTL1 gene fragment is ligated to the backbone plasmid to form a recombinant plasmid; the recombinant plasmid, packaging plasmid and helper plasmid are co-transfected into host cells for culture, and the recombinant viral particles overexpressing the CTL1 gene are obtained after purification.

[0008] Preferably, the backbone plasmid is pAAV-MCS; the packaging plasmid is pAAV2 / 9 Rep-Cap; and the helper plasmid is pHelper.

[0009] Preferably, the formulation is a liquid formulation.

[0010] Preferably, the drug comprises a formulation that enhances CTL1 activity and pharmaceutically acceptable excipients.

[0011] Preferably, the pharmaceutically acceptable excipient is one or more of the following: diluent, disintegrant, precipitation inhibitor, flow aid, binder, dispersant, suspending agent, isotonic agent, thickener, emulsifier, preservative, and stabilizer.

[0012] Preferably, the acceptable dosage forms of the drug include tablets, capsules, granules, injections, pills, powders, or ointments.

[0013] Preferably, the neurodegenerative disease includes one or more of Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS).

[0014] Compared with the prior art, the present invention has the following beneficial effects: Mitophagy is a key mitochondrial quality control mechanism for maintaining neuronal homeostasis, and its dysfunction is associated with neurodegenerative diseases such as Parkinson's disease. However, the mechanisms of neuronal mitophagy are not fully understood, and there are no mitophagy receptors associated with neurodegenerative diseases. This invention identifies choline transporter-like protein 1, or CTL1, as a novel mitophagy receptor in neuronal models. Experiments have demonstrated that CTL1, located in mitochondria, interacts directly with LC3B / GABARAP through a conserved N-terminal LC3 interaction region LIR motif. Ectopic expression of CTL1 induces Parkin-independent mitophagy, which is independent of choline transport function and depends on ATG5 rather than Beclin1. Endogenous CTL1 deficiency leads to MPP (mitochondrial autophagy). + Mitochondrial clearance function is impaired during stimulation. Mechanistically, MPP + Activation of CaMKII, which phosphorylates serine residue 269 of CTL1, drives selective mitophagy. Dopaminergic neuron-specific CTL1 knockout mice exhibit Parkinson's disease-like motor dysfunction and neurodegenerative changes. Neuronal induction of CTL1 ameliorate MPTP-induced dopamine neuron death in mice. These results confirm that CTL1 is a phosphorylation-regulated mitophagy receptor whose dysfunction drives neurodegenerative changes, thus providing a direct molecular mechanism for the pathogenesis of Parkinson's disease and offering a potential drug target. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a diagram showing the binding results of GST-LC3B of the present invention with the mitochondrial protein CTL1; Figure 1In the diagram, A shows the results of immunoblotting analysis of SH-SY5Y cell lysates with purified GST or GST-LC3B protein; B shows the results of immunoblotting analysis of MN9D cell lysates with purified GST or GST-LC3B protein; C shows the results of immunoprecipitation analysis of the interaction between Flag-CTL1 and GFP-LC3B; D shows the results of immunoprecipitation analysis of the interaction between Flag-CTL1 and GFP-LC3B; E shows the results of in vitro interaction assay of recombinant GST-LC3B and 8*His-MBP-Flag-CTL1 protein; and F shows the results of in vitro interaction assay of recombinant GST-GABARAP and 8*His-MBP-Flag-CTL1 protein.

[0017] Figure 2 The diagrams show the results of this invention demonstrating that CTL1 overexpression induces mitophagy; A is the result of flow cytometry analysis of MitoTracker DeepRed staining to assess mitochondrial content; B is the quantitative analysis result of normalized MitoTracker DeepRed levels in A, mean ± SEM; C is the result of immunoblotting analysis of TIM23, TOM20, LC3B, and Flag in cells treated under specified conditions; D is the result of flow cytometry analysis of MitoTracker DeepRed staining to assess mitochondrial content; E is the result of quantitative analysis of normalized MTDR levels in D, mean ± standard error; F is the result of immunoblotting analysis of TIM23, TOM20, LC3B, and Flag; G is the result of flow cytometry analysis of cells transfected with Flag-CTL1; H is the result of quantitative analysis of normalized MTDR levels in G, mean ± SEM.

[0018] Figure 3 This is a diagram illustrating the results of CTL1 overexpression inducing mitophagy that is dependent on ATG5 rather than Beclin1. Figure 3Figure A shows the results of flow cytometry analysis of control cells and ATG5 knockdown cells transfected with the vector or Flag-CTL1. ATG5 knockdown is represented by ATG5-KD in the figure. Figure B shows the results of quantitative analysis of normalized MTDR levels in group A, mean ± SEM. Figure C shows the results of Western blotting analysis of ATG5, Flag, TIM23, and TOM20. Figure D shows the results of quantitative analysis of normalized TIM23 protein levels in group C, mean ± SEM. Figure E shows the results of quantitative analysis of normalized TOM20 protein levels in group C. The results are shown in Figure 1, mean ± SEM; F is the result of flow cytometry analysis of control cells transfected with the vector or Flag-CTL1 and cells transfected with Beclin1-KD; G is the result of quantitatively standardized MTDR levels, mean ± SEM; H is the result of Western blotting analysis of Beclin1, Flag, TIM23 and TOM20; I is the result of quantitatively standardized TIM23 protein levels in H, mean ± SEM; J is the result of quantitatively standardized TOM20 protein levels in H, mean ± SEM.

[0019] Figure 4 To prove MPP of this invention + The results show that activated CaMKII phosphorylation of CTL1 is crucial for mitophagy; A shows the results of Western blotting detection of CTL1, TIM23, TOM20, and LC3B; B shows the results using vector or MPP. + Figure 1 shows the flow cytometry results of the control group and CTL1-KD SH-SY5Y cells; C is the quantitative graph of the standardized MitoTracker DeepRed level in B, mean ± SEM; D is the LC-MS / MS spectrum. E is the result of immunoprecipitation analysis of phosphorylated Serine levels to verify the input protein; F is the result of immunoblotting analysis; G is the result of immunoprecipitation of endogenous Thr286 phosphorylated CaMKII with CTL1; H is the result of immunoprecipitation analysis of phosphorylated serine levels and verification of the input protein by immunoblotting; I is the result of immunoblotting detection of phosphorylated serine levels.

[0020] Figure 5 The dopaminergic neuron-specific CTL1 gene knockout mice of this invention exhibit a Parkinson's disease-like phenotype. A is a schematic diagram showing the exon 6 resection of CTL1 driven by the DAT promoter; B is a diagram showing the results of detecting the 152bp DAT-Cre targeting allele; C is CTL1 fl / fl and CTL1 fl / fl Immunofluorescence analysis results of CTL1 expression in dopaminergic neurons of DAT-Cre mice. Scale bar: 20 μm; D represents the expression of CTL1 in DAT-Cre mice. fl / fland CTL1 fl / fl Rotor testing was performed on DAT-Cre mice, with n=9 mice in each group; E represents the number of mice in CTL1. fl / f l and CTL1 fl / fl The bar test was performed in DAT-Cre mice, and the data are expressed as mean ± standard error; F represents CTL1. fl / fl The open field test was conducted on mice, with n=9 mice in each group. The figure shows the representative movement trajectories during the 10-minute test; G represents CTL1. fl / fl Open field test diagrams for DAT-Cre mice, n=9 mice per group. The diagrams show representative movement trajectories during a 10-minute test; H is the open field behavioral parameter diagram: total distance traveled; data are expressed as mean ± standard error; I is the open field behavioral parameter diagram: time spent in the central region; data are expressed as mean ± standard error; J is the open field behavioral parameter diagram: distance traveled in the central region; data are expressed as mean ± standard error; K is the open field behavioral parameter diagram: number of times the mouse entered the central region; data are expressed as mean ± standard error; L is CTL1. fl / fl Representative TH staining images of mouse SNpc dopaminergic neurons, n=5 mice per group, scale bar: 200μm; M represents CTL1. fl / fl ; Representative TH staining of SNpc dopaminergic neurons in DAT-cre mice, n=5 mice per group, scale bar: 200μm; N is the stereotactic count of TH-positive cells in L and M; O is CTL1 fl / fl and CTL1 fl / fl : Dopamine concentration in the striatum of DAT-Cre mice, 6 mice per group. P represents CTL1. fl / fl and CTL1 fl / fl : DOPAC concentration in the striatum of DAT-Cre mice, 6 mice per group.

[0021] Figure 6 To demonstrate that overexpression of CTL1 can prevent MPP in vivo and in vitro +The results of the induced pathology are shown in Figure 1. A shows the cell morphology and cell viability under bright field as detected by the MTT assay. B shows the quantitative statistical graph of cell survival rate in MN9D cells after corresponding treatment. C shows the quantitative statistical graph of cell survival rate in SH-SY5Y cells after corresponding treatment. D is a summary diagram of the experimental procedure. The IF experiment demonstrates that rAAV2 / 9-CTL1-hsyn-EGFP-injection into the SNpc region is effective. Image scale bar: 200 μm. E shows the results of immunoblotting analysis of CTL1 and TH expression in mouse midbrain tissue. F shows the representative TH staining results of mouse SNpc dopaminergic neurons, with 3 mice per group. Image scale bar: 200 μm. G shows the quantitative results of stereoscopic counting of TH-positive cells in Figure F. H shows the dopamine concentration in the striatum of mice, with 5 mice per group. I shows the DOPAC concentration in the striatum of mice, with 5 mice per group. Detailed Implementation

[0022] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0024] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0025] Example 1 I. Experimental Methods 1. Cell Culture and Transfection SH-SY5Y, MN9D, HeLa, and HEK-293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences. SH-SY5Y cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, purchased from Gibco; MN9D cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, purchased from Gibco; HeLa and HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, purchased from Gibco; all cell lines were cultured at 37°C and 5% CO2.

[0026] Transfection was performed using Lipofectamine 3000, purchased from Thermo Fisher Scientific or according to the manufacturer's instructions. CTL1-KOSH-SY5Y cells were constructed using CRISPR / Cas9. The construction of stable lentiviral cell lines was performed as described above. Guide RNA was cloned into the lentiviral vector pLenti-Puro containing Cas9. The sequence of the guide RNA used in CRISPR / Cas9 CTL1-KOSH-SY5Y cells is shown in SEQ ID NO.1: 5'-gtgctacagacttaatcttc-3'.

[0027] The following steps were used to construct a plasmid for cell transfection: First, using a plasmid containing human CTL1 cDNA as a template, primers were designed to amplify its complete coding sequence (CDS) by PCR, with HindIII and XhoI restriction sites and protective bases introduced into the upstream and downstream primers, respectively. The PCR product was double-digested with the pcDNA3.1-C-Flag vector using the same HindIII and XhoI enzymes. Subsequently, the purified CTL1 fragment was directionally cloned into the C-terminal Flag tag sequence of the vector using T4 DNA ligase, ensuring correct fusion of the reading frames. The cells were then transformed into competent cells, and clones were selected for sequencing verification to obtain a plasmid overexpressing wild-type CTL1, namely the overexpressing WT Flag-CTL1 plasmid. Using this plasmid as a template, overlapping extension PCR was performed to induce site-directed mutagenesis by introducing primers specific to the S269A mutation site. The clone was then digested with HindIII / XhoI and cloned into the vector. After sequencing confirmation, the overexpressing mutant S269A Flag-CTL1 plasmid was obtained.

[0028] 2. Flow cytometry determination of mitochondrial autophagy flux In short, the method for measuring mitophagy flux involved digesting cells with 0.25% trypsin (EDTA-free) at 37°C for 5 minutes, followed by resuspending in complete medium containing 10 nM MitoTracker Deep Red and 1 µg / mL propidium iodide (Sigma-Aldrich) for 15 minutes at 37°C. Data were collected from 10,000 cells using a BD FACSCanto II flow cytometer equipped with FL3 and FL4 channels. MitoTracker Deep Red was purchased from Invitrogen; propidium iodide was purchased from Sigma-Aldrich; and the BD FACSCanto II flow cytometer was purchased from San Jose, California, USA.

[0029] 3. Immunofluorescence For immunofluorescence assays (IF), cells were seeded in confocal culture dishes with a glass bottom and treated according to instructions. Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature. After rinsing with PBS, cells were permeabilized in PBS containing 0.1% saponin (w / v) for 3 minutes, and then blocked with PBS containing 5% BSA for 30 minutes at room temperature. Cells were then incubated overnight at 4°C with primary antibody diluted in PBS containing 1% BSA. After washing three times with PBS, cells were incubated with the corresponding fluorescent secondary antibody in PBS at room temperature in the dark for 1 hour. The nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI) for 10 minutes at room temperature in the dark. Cells were washed twice with PBS and once with deionized water, and then mounted. Images were acquired using a confocal microscope equipped with a 60x oil immersion microscope and analyzed using ImageJ software. Saponins were purchased from Sigma; the 60x oil immersion microscope was purchased from OLYMPUS, Spin SR.

[0030] 4. Immunoprecipitation and Western blotting For co-immunoprecipitation (Co-IP), cells were lysed for 30 minutes in ice-cold lysis buffer composed of 25 mM pH 7.4 HEPES-HCl, 1 mM EDTA, 150 mM NaCl, 10% glycerol, a 1×Combolete protease inhibitor mixture, a 1×HALT phosphatase inhibitor mixture, and 0.3% sodium deoxycholate. The lysate was centrifuged at 20,000 rcf for 10 minutes, and the supernatant was collected as the total lysate. Protein concentration was determined using the BCA method. From the total 500 µL of lysate, 40 µL was retained as input. The remaining 460 µL was incubated overnight at 4°C with primary antibody and Protein A / G agarose beads. The beads were washed three times with washing buffer composed of 25 mM pH 7.4 HEPES-HCl, 1 mM EDTA, 150 mM NaCl, and 0.5% Tween 20. Elute the binding protein with low pH elution buffer and neutralize immediately. Add 4×SDS loading buffer to the sample and incubate at 37°C for 30 minutes. Then perform Western blot analysis on the sample.

[0031] For Western blotting, cells or mouse tissue were washed with ice-cold PBS and lysed on ice with RIPA buffer. Protein samples (60 µg) were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk or BSA, then incubated overnight at 4°C with primary antibody, followed by incubation at room temperature with a suitable HRP-conjugated secondary antibody for 1 hour. Target proteins were detected using ECL reagent. PVDF membranes were purchased from Bio-Rad.

[0032] 5. In vitro kinase assay Immunoprecipitation of Flag-CTL1 protein expressed in cells was performed using Protein A / G agarose beads conjugated with anti-Flag antibody. The beads were washed with a wash buffer consisting of 25 mM pH 7.4 HEPES-HCl, 1 mM EDTA, 150 mM NaCl, and 0.5% Tween 20. The bead-bound Flag-CTL1 was incubated with 3.3 μM ATP and 200 ng of active GST-CaMKII or GST control protein in 1× kinase buffer at 30°C for 30 min. The reaction was terminated by adding SDS loading buffer. Phosphorylation was analyzed by Western blotting using an anti-phosphorylated serine antibody. ATP was purchased from CST Biotechnology (catalog number 9804); active GST-CaMKII was purchased from MCE Biotechnology; 1× kinase buffer was purchased from CST Biotechnology (catalog number 9802); and the anti-phosphorylated serine antibody was purchased from Abcam Biotechnology.

[0033] 6. Animals CTL1flox / flox mice and DAT-Cre mice were manufactured by Cyagen Biosciences (Guangzhou) Co., Ltd. To achieve selective knockout of CTL1 in dopamine neurons, CTL1flox / flox mice were crossed with DAT-Cre transgenic mice. Offspring that were positive for both the fluxed allele and the Cre recombinase gene were backcrossed with CTL1flox / flox mice to obtain littermates. Mice from these littermates that were homozygous for the fluxed allele and positive for Cre were named CTL1flox / flox:DAT-Cre as the selective dopamine neuron-specific CTL1 gene knockout group. CTL1flox / flox:DAT-Cre will be referred to as CTL1 in the following text and results figures. fl / fl DAT-Cre; A littermate mouse that was Cre-negative and homozygous for the fluxed allele was named CTL1flox / flox as a control. CTL1flox / flox will be referred to as CTL1 in the following text and results figures. fl / fl All mice, four per cage, were housed under specific pathogen-free conditions, with free access to pelleted feed and distilled water. The ambient temperature was maintained at 22°C, humidity at 60%, and a 12-hour light / dark cycle. All experimental mice were 12-month-old males and females.

[0034] Adult C57BL / 6 mice were purchased from the Vital River Laboratory in Beijing, China. Starting 14 days before MPTP administration, adeno-associated virus (AAV) 2 / 9-hSyn-Control-GFP (represented as AAV-vector in the results image) was injected bilaterally into the substantia nigra pars compacta (SNpc). Recombinant viral particles overexpressing the CTL1 gene, rAAV2 / 9-hSyn-CTL1-GFP (represented as AAV-CTL1 in the results image), were also injected bilaterally into the SNpc. Injection coordinates relative to the anterior fontanelle were: AP -3.1 mm, ML ± 1.2 mm, DV -4.3 mm. Mice were administered 25 mg / kg intraperitoneally daily for 5 consecutive days. -1 MPTP. Three days after the last injection of MPTP, mice were sacrificed under deep isoflurane anesthesia and tissues were collected. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, was purchased from Sigma-Aldrich, catalog number M0896.

[0035] The method for constructing rAAV2 / 9-hSyn-CTL1-GFP according to the present invention includes the following steps: First, a target fragment containing an hSyn promoter, a CTL1 coding sequence, a P2A self-cleaving peptide sequence, a GFP reporter gene, and a polyA signal is obtained through gene synthesis, with specific restriction endonuclease sites, such as EcoRI and XbaI, introduced at both ends; then, this synthesized fragment is used as raw material and a pAAV-MCS backbone plasmid that has been linearized by double digestion with the same restriction endonucleases EcoRI and XbaI, and ligation is performed under the action of T4 DNA ligase to directionally clone the target expression cassette into the AAV2 ITR sequence; the ligation product is transformed into DH5α competent cells, positive clones are screened on ampicillin-resistant plates, and verified by plasmid extraction, restriction enzyme digestion analysis, and DNA sequencing to obtain the correct recombinant plasmid pAAV2 / 9-hSyn-CTL1-P2A-GFP; finally, this recombinant plasmid is combined with pAAV2 / 9... Rep-Cap packaging plasmid and pHelper helper plasmid were co-transfected into HEK293T cells. After culturing, collecting, and lysing the cells, high-titer recombinant viral particles, namely rAAV2 / 9-hSyn-CTL1-GFP, were obtained by gradient centrifugation with iodixanol.

[0036] All animal procedures were approved by the Animal Care and Use Committee of Wenzhou Medical University (reference number wydw2023-0560) and conducted in accordance with Chinese national standards for laboratory animal welfare. During data collection and, where feasible, during analysis, the experimental staff were unaware of the grouping arrangements.

[0037] 7. Behavioral Testing Pole test: Mice were placed head-up on approximately 7.5 cm from the top of a vertical metal pole wrapped in gauze; the pole was 9 mm in diameter and 75 cm high. Mice underwent two days of training, five times per day, prior to formal evaluation. On the test day, each mouse underwent three tests. Recorded parameters included flip latency and total descent time; flip latency was the time it took for the mouse to flip downwards; total descent time was the total time it took to reach the bottom.

[0038] Rotary bar test: For two consecutive days, mice were acclimatized to the device at a constant speed of 10 rpm for 5 minutes each day. During the test, the mice were placed on the bar, and the rotation speed was increased from 4 rpm to 40 rpm within 5 minutes. The fall latency was recorded for each mouse in 3 tests. Between each test, the bar and chamber were cleaned with 75% ethanol.

[0039] Open field experiment: Mice underwent a 30-minute acclimatization period in an open field chamber measuring 50cm × 50cm × 50cm. Subsequently, each mouse was placed in the center and allowed to explore freely for 5 minutes. SMART v3.0 video tracking software was used to record and analyze the mice's movement. The total movement distance and the distance within the central region were calculated; the total movement distance was the sum of the distances of all 16 equally divided regions; the distance within the central region was the sum of the distances of the four central regions.

[0040] 8. Immunohistochemistry and immunofluorescence staining of tissue sections Serial 40-micrometer brain slices were subjected to immunohistochemical staining and immunofluorescence staining, with immunohistochemical staining referred to as IHC. The primary antibodies used and their dilutions are as follows: the antibody against CTL1 protein was purchased from Signalway and is suitable for both immunofluorescence and immunohistochemistry experiments, with a working concentration of 1:200; the antibody against TH was purchased from Santa Cruz, with a concentration of 1:200 for immunofluorescence and 1:50 for immunohistochemistry; the antibody against NeuN was purchased from Abcam and used for immunohistochemistry experiments, with a dilution of 1:200. In the immunohistochemistry experiments, the free-floating slices were first blocked with 5% goat serum prepared with PBS containing 0.2% Triton X-100, and then co-incubated with either anti-TH antibody or anti-CTL1 antibody. Detection was then performed using biotin-labeled goat anti-mouse IgG and goat anti-rabbit IgG, and the colorimetric reaction was carried out using DAB peroxidase substrate produced by Sigma. In immunofluorescence experiments, the slides were incubated with secondary antibodies labeled with Alexa Fluor 488 and Alexa Fluor 594 provided by Invitrogen, and nuclear counterstaining was performed using DAPI. Finally, images were acquired using an OLYMPUS VS200 confocal microscope, and the image data were subsequently analyzed using ImageJ software.

[0041] 9. Quantitative analysis of dopaminergic neurons TH-stained midbrain sections were analyzed, with one section taken from every five sections. Quantitative counting of TH-positive dopaminergic neurons in the right substantia nigra was performed at 40x magnification using optical fractionation in Stereo Investigator software from MBF Biosciences in Williston, Vermont. All stereoscopic counting was performed by researchers unaware of the experimental group assignments.

[0042] 10. HPLC-MS / MS quantitative analysis of dopamine and DOPAC The striatal lysates were analyzed using a SHIMADZU CBM-30A Lite UHPLC system coupled with an API 6500 Q-TRAP mass spectrometer in ESI-negative MRM mode. Separation was performed using a Waters Acquity HSS T3 column (2.1 × 100 mm, 1.8 μm); the mobile phase consisted of an aqueous solution containing 0.1% formic acid as phase A and acetonitrile as phase B. Quantitative analysis of dopamine and its metabolite DOPAC was performed using MultiQuant software, version 2.1, manufactured by AB SCIEX, located in California, with independent standards from GLPBIO as quantitative references.

[0043] 11. MTT Method Cell viability was determined using the MTT assay. Cells were seeded in 96-well plates at 5 × 10⁶ wells. 3 Cells / well were incubated overnight. Cells were then treated as instructed, with the addition of MTT, and cultured at 37°C for 3 hours until formazan crystals formed. The MTT-containing medium was then removed, and 100 μl of DMSO solution was added to dissolve the crystals. The absorbance of each well was recorded at 570 nm using a Thermo Fisher BIO-RAD680 microplate reader.

[0044] 12. Protein purification and in vitro binding assays LC3B was cloned into the pGEX-6P-1 vector, and GABARAP was cloned into the pGEX-4T-2 vector. Both were expressed as GST fusion proteins in *E. coli* strain BL21 DE3. The GST fusion proteins were purified using solid-phase glutathione (STP), purchased from Thermo Fisher Scientific, and dialysis was performed using a Slide-A-lyzer dialysis kit, also purchased from Thermo Fisher Scientific.

[0045] For the GST pull-down assay, cells were collected in lysis buffer, which consisted of 25 mM pH 7.4 HEPES-HCl, 1 mM EDTA, 150 mM NaCl, 10% glycerol, a 1×compolete protease inhibitor mixture, a 1×halt phosphatase inhibitor mixture, and 0.3% sodium deoxycholate, with 10% sodium deoxycholate added to a final concentration of 0.3%. The samples were incubated on ice for 30 minutes, centrifuged at 20,000 rcf for 10 minutes, and the supernatant was further diluted 2-fold with buffer A. The diluted lysate was pre-clarified with immobilized glutathione and incubated with magnetic beads bound to the GST fusion protein for 2 hours. The beads were washed five times with a wash buffer consisting of 25 mM pH 7.4 HEPES-HCl, 1 mM EDTA, 150 mM NaCl, and 0.5% Tween 20, and then boiled in Laemmli buffer containing β-mercaptoethanol for 5 minutes to elute the protein.

[0046] For in vitro binding assays, purified GST-LC3B and 8*His-MBP-Flag-CTL1 proteins were incubated with immobilized glutathione for 2 hours, washed five times with washing buffer, and then boiled for 5 minutes in Laemmli buffer containing β-mercaptoethanol.

[0047] 13. Mass spectrometry Peptides were dissolved in mobile phase A of liquid chromatography and then separated using an EASY-nLC 1200 ultra-high performance liquid chromatography system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient settings were: 0 min–68 min, 6%–23% B; 68 min–82 min, 23%–32% B; 82 min–86 min, 32%–80% B; 86 min–90 min, 80% B, with the flow rate maintained at 500 nL / min. After separation by the ultra-high performance liquid chromatography system, the peptides were injected into an NSI ion source for ionization and then analyzed by an Orbitrap Exploris 480 mass spectrometer. The ion source voltage was set to 2300 V, 45 V, and -65 V. The peptide precursor ion and its secondary fragments were detected and analyzed using a high-resolution Orbitrap spectrometer. The primary mass spectrometry (MS / MS) scan range was set to 1200 m / z, and the scan resolution was set to 60,000 ions / s. The secondary MS / MS scan range had a fixed starting point of 110 m / z, and the secondary scan resolution was set to 15,000 ions / s. TurboTMT was set to off. Data acquisition mode used a data-dependent scanning program, abbreviated as DDA. This program selects the top 25 peptide precursor ions with the highest signal intensity after the primary scan and sequentially introduces them into the HCD collision cell for fragmentation at 27% fragmentation energy. Secondary MS / MS analysis is then performed sequentially. To improve the efficiency of the mass spectrometry, automatic gain control (AGC) was set to 100%, the signal threshold to 50,000 ions / s, the maximum injection time to Auto, and the dynamic exclusion time for tandem mass spectrometry scans was set to 20 s to avoid repeated scanning of precursor ions.

[0048] 14. Statistical Analysis Data were analyzed using GraphPad Prism 9 software and expressed as mean ± standard deviation, i.e., mean ± SEM. Significance was tested using unpaired Student's t-tests or analysis of variance. P A value <0.05 is considered statistically significant.* P <0.05,** P <0.01.

[0049] II. Experimental Results 1. CTL1 binds to ATG8 family proteins GST or GST-LC3B protein was isolated and purified from SH-SY5Y cell lysates. These lysates were treated with 200 nM BafA1 for 4 hours or with 100 μM MPP, respectively. +Combined treatment for 4 hours. Immunoblot analysis was performed to detect the interaction between CTL1 and LC3B; purified GST or GST-LC3B proteins were isolated from MN9D cell lysates, which were then treated with 200 nM BafA1 for 4 hours or with 100 μM MPP, respectively. + The cells were treated together for 4 hours. Immunoblot analysis was performed to detect the interaction between CTL1 and LC3B; Coomassie brilliant blue staining was used to show the abundance of GST and GST-LC3B proteins; the results indicated that purified GST-LC3B could be extracted from the MPP-treated cells. + In SH-SY5Y and MN9D cell lysates treated with BafA1, significantly more CTL1 was downregulated, such as Figure 1 As shown in A and B in the diagram. Cells were co-transfected with WT Flag-CTL1 plasmid and GFP-LC3B for 24 hours, and then the interaction between Flag-CTL1 and GFP-LC3B was analyzed by immunoprecipitation. Cells were also co-transfected with WT Flag-CTL1 plasmid and GFP-LC3B for 24 hours, and then the interaction between Flag-CTL1 and GFP-LC3B was analyzed by immunoprecipitation. The results showed that in 293T cells co-expressing Flag-CTL1 and GFP-LC3B, the interaction was confirmed by immunoprecipitation using anti-Flag and anti-GFP antibodies, as shown in Figure 1. Figure 1 As shown in C and D in the figure. The direct binding of CTL1 to LC3B was verified in vitro using purified 8×His-MBP-Flag-CTL1 and GST-LC3B, as shown in the figure. Figure 1 As shown in E. Similarly, CTL1 interacts with another Atg8 family member, GABARAP, as... Figure 1 As shown in F in the diagram.

[0050] 2. CTL1 induces mitophagy, independent of Parkin and choline. Transfection with the WT Flag-CTL1 plasmid is indicated as OE-CTL1 in the results image. Transfection with the empty vector is indicated as CON in the results image.

[0051] Cells were transfected with the WT Flag-CTL1 overexpression plasmid for 24 hours, and treated with 10 μM CCCP for 12 hours as a positive control (represented by CCCP in the figure). MitoTracker DeepRed (MTDR) staining was analyzed by flow cytometry to assess mitochondrial content; the results showed that CTL1 overexpression induced mitophagy in cells, at levels comparable to those induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Figure 2As shown in A and B. Cells were transfected with the WT Flag-CTL1 overexpressing plasmid for 6 hours in the presence or absence of 200 nM BafA1. Immunoblot analysis of TIM23, TOM20, LC3B, and Flag in cells treated under specified conditions showed that the CTL1-mediated reduction of OMM protein TOM20 and IMM protein TIM23 was blocked by BafA1. Figure 2 As shown in C. Furthermore, compared to the empty vector control, BafA1 treatment increased LC3B-II levels in CTL1-overexpressing cells, confirming that the CTL1-induced increase in LC3B-II reflects enhanced autophagy flux. These results collectively suggest that CTL1-driven mitochondrial clearance depends on a functional autophagy pathway.

[0052] This invention validates the function of CTL1 in HeLa cells lacking the functional Parkin gene. HeLa cells were transfected with the WTFlag-CTL1 overexpression plasmid for 24 hours. Cells were treated with 10 μM CCCP for 12 hours as a positive control. MTDR staining was analyzed by flow cytometry to assess mitochondrial content. HeLa cells were then transfected with the WT Flag-CTL1 overexpression plasmid and treated for 6 hours with or without 200 nM BafA1. Immunoblot analysis of TIM23, TOM20, LC3B, and Flag in cells treated under the specified conditions showed that ectopic CTL1 expression in HeLa cells also triggers mitophagy, and BafA1 can reverse this process. Figure 2 As shown in D~F, this indicates that Parkin is not necessary for CTL1-driven mitochondrial clearance.

[0053] To investigate whether the typical choline transport activity of CTL1 contributes to its mitophagy-inducing function, this invention systematically analyzed mitophagy levels under choline depletion and choline supplementation conditions. Flow cytometry analysis was performed on cells transfected with the WT Flag-CTL1 plasmid in both the presence and absence of choline. The results showed that regardless of choline supply, CTL1 overexpression induced a considerable level of mitophagy in cells. Figure 2 As shown in F and G in the figure. In summary, these results indicate that ectopic CTL1 expression induces mitophagy through a mechanism independent of its choline transport capacity.

[0054] 3. CTL1 induces ATG5-dependent, rather than Beclin1-dependent, mitophagy. To determine whether CTL1-induced mitophagy is a specific branch of typical autophagy or functions through an independent mechanism, this invention investigated the functional roles of two core autophagy regulators: ATG5 and Beclin1. ATG5 is essential for LC3 lipidation and autophagosome elongation / closure; Beclin1 is a key component of the PI3K complex I and is necessary for autophagosome nucleation.

[0055] Flow cytometry was used to analyze control and ATG5 knockdown cells transfected with the empty vector or overexpressing the WT Flag-CTL1 plasmid. The control group consisted of normal ATG5 cells (Control); ATG5 knockdown cells were treated with ATG5-KD. Results showed that ATG5 knockdown eliminated ectopic CTL1 expression-induced mitochondrial clearance in cells, a stark contrast to the control cells. Figure 3 A and B in the diagram. Control and ATG5-KD cells were transfected with either an empty vector or an overexpressing WT Flag-CTL1 plasmid for 24 hours. Western blot analysis of ATG5, Flag, TIM23, and TOM20 showed that ATG5 knockout reversed CTL1-mediated mitochondrial protein degradation, including the OMM marker TOM20 and the IMM protein TIM23. Figure 3 As shown in C~E. Conversely, Beclin1 knockout failed to inhibit CTL1-driven mitophagy. Flow cytometry analysis was performed on control and Beclin1-KD cells transfected with empty vector or overexpressing WT Flag-CTL1 plasmid. The control group consisted of normal Beclin1 cells (Control); Beclin1-KD cells were Beclin1 knockout cells. Control and Beclin1-KD cells were transfected with empty vector or overexpressing WT Flag-CTL1 plasmid for 24 hours. Beclin1, Flag, TIM23, and TOM20 were detected by Western blotting. Flow cytometry and Western blotting showed no significant differences between Beclin1-KD cells and control cells in the degradation levels of mitochondrial proteins such as TOM20 and TIM23, or in mitochondrial clearance efficiency. Figure 3 As shown in F~J.

[0056] 4. MPP + Mitochondrial autophagy is activated by phosphorylation of CaMKII, which in turn phosphorylates CTL1. Use PBS or 1000 μM MPP + Control and CTL1-knockdown cells were treated for 24 hours. Control cells were normal cells (represented as Control); CTL1-knockdown cells were represented as CTL1-KD. Western blotting was used to detect CTL1, TIM23, TOM20, and LC3B levels. Results showed that in MPP...+ Under treatment, the degradation of mitochondrial proteins almost disappeared in CTL1-KD cells, such as... Figure 4 As shown in A. Use PBS or 1000 μM MPP + Flow cytometry analysis of the control group and CTL1-KD cells after 24 hours further confirmed the absence of MPP in CTL1-deficient cells. + Induced mitophagy, such as Figure 4 As shown in B and C. These findings confirm that CTL1 is an MPP. + Triggered mitophagy is indispensable.

[0057] This invention further investigates MPP + How to activate CTL1. Mass spectrometry analysis shows that MPP + Treatment induces phosphorylation of serine (Ser) at position 269 of CTL1 in cells, such as... Figure 4 As shown in D in the diagram. Cells transfected with the specified plasmid were treated with PBS or 100 μM MPP 24 hours post-transfection. + Processing time was 30 minutes. Phosphorylated Ser levels were analyzed by immunoprecipitation, and the input protein was validated by Western blotting. Results showed that after MPP... + In cells expressing WT-CTL1, the p-Ser level of CTL1 was elevated, but not in the phosphorylation-deficient mutant S269A-CTL1. Figure 4 The E in the figure indicates that phosphorylation of serine Ser-269 at position 269 of CTL1 is caused by MPP. + Specifically triggered. Subsequently, this invention investigated whether phosphorylation of serine at position 269 affects MPP. + Induced mitophagy. CTL1-KO cells overexpressing WT Flag-CTL1 plasmid or the mutant S269AFlag-CTL1 plasmid were transfected with 1 mM MPP. + After 24 hours of treatment, immunoblotting analysis showed that MPP was present in CTL1-KO cells. + The degradation of mitochondrial proteins TIM23 and TOM20 was not induced. Reintroducing WT-CTL1, instead of S269A-CTL1, salvaged this degradation, as... Figure 4 As shown in F in the figure. These results collectively indicate that phosphorylation of serine at position 269 of CTL1 is crucial for MPP. + The resulting mitophagy is crucial.

[0058] Bioinformatics analysis predicted that CaMKII is a potential kinase responsible for CTL1 phosphorylation. CaMKII is a multifunctional kinase known to translocate to mitochondria and regulate mitophagy. Using 100 μM MPP...+ Cells were treated for 30 minutes, followed by immunoprecipitation of endogenous Thr286-phosphorylated CaMKII and CTL1. The input proteins were validated by Western blotting, and the results showed that in MPP... + In treated cells, the binding of CaMKII to phosphorylated CaMKII, i.e., p-CaMKII, increases, such as Figure 4 As shown in G, this indicates MPP + CaMKII phosphorylation was activated. Notably, this was achieved using 100 μM MPP. + Cells were treated with either the carrier or 10 μM CaMKII inhibitor KN-93 for 30 minutes. Phosphorylated serine levels were analyzed by co-immunoprecipitation, and the input protein was validated by Western blotting. Pretreatment with KN-93, an inhibitor of CaMKII Thr-286 phosphorylation, significantly inhibited MPP. + Induced CTL1 phosphorylation, such as Figure 4 As shown by H in the diagram. Cells were transfected with an empty vector, an overexpression of the WT Flag-CTL1 plasmid, or an overexpression of the mutant S269A Flag-CTL1 plasmid. Cell lysates were subjected to immunoprecipitation for in vitro kinase assays. The immunoprecipitated Flag-CTL1 was incubated with ATP, GST, or GST-CaMKII as instructed for 20 minutes. Phosphorylated serine levels were detected by Western blotting. The results showed that active CaMKII phosphorylated WT-CTL1 but did not phosphorylate the S269A mutant, as shown by H. Figure 4 As shown in Figure I. In summary, these results indicate that MPP + Activation of CaMKII phosphorylation at the Ser-269 site of CTL1 is a key step in initiating mitophagy.

[0059] 5. CTL1 deficiency in dopaminergic neurons can induce Parkinson's disease-like symptoms in mice. To elucidate the in vivo function of CTL1 in dopaminergic neurons, this invention utilizes Cre recombinase expressed under the control of the DAT promoter to construct a dopaminergic neuron-specific conditional knockout mouse model, CTL1. fl / fl :DAT-Cre, such as Figure 5 As shown in A in the diagram. Two primers were used for PCR detection of the DAT-Cre mouse genotype. The upstream primer sequence is shown in SEQ ID NO.2: 5'-TGGCTGTTGGTGTAAAGTGG-3'; the downstream primer sequence is shown in SEQ ID NO.3: 5'-CCAAAAGACGGCAATATGGT-3'. These primers can detect the 152bp DAT-Cre target allele. The effectiveness of the conditional knockout was verified by detecting the expected 152bp recombinant allele using PCR. Figure 5As shown in B. Furthermore, co-immunofluorescence staining of CTL1 and TH in the substantia nigra, i.e., the SN, confirmed the presence of CTL1. fl / fl The selective ablation of CTL1 protein expression in TH-positive dopaminergic neurons of DAT-Cre mice successfully achieved cell type-specific knockout, such as... Figure 5 As shown in C.

[0060] At 12 months of age, CTL1 fl / fl DAT-Cre mice compared to CTL1 fl / fl The control group of littermates showed significant motor impairments. This deficiency was quantified by a significantly shortened fall latency on an accelerated rotating bar and an increased descent time in bar tests, such as... Figure 5 As shown in D and E in the diagram. Assessments of exploratory and anxiety-related behaviors in open spaces showed that CTL1, compared to the control group, [had lower scores]. fl / fl DAT-Cre mice exhibited a significantly reduced total walking distance, shorter coverage distance in the central region, reduced time spent in the center, and fewer entries into the center. Figure 5 As shown in F~K. This behavioral characteristic is consistent with decreased spontaneous motor ability and increased anxiety in the new environment. Neuropathological analysis confirmed the functional deficit, indicating that CTL1 fl / fl DAT-Cre mice show a significant loss of dopaminergic neurons in the substantia nigra, such as... Figure 5 The L~N values ​​are shown in the diagram. With neuronal degeneration, high-performance liquid chromatography (HPLC) revealed a significant decrease in striatal dopamine and its main metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), as shown in the diagram. Figure 5 The presence of O and P in the data suggests that the dopaminergic pathway in the substantia nigra striatum is undergoing progressive degradation.

[0061] Cell morphology and viability under bright field were detected by MTT assay. MN9D and SH-SY5Y cells were transfected with an empty vector and an overexpression of the WT Flag-CTL1 plasmid, respectively. Empty vector transfection was indicated by EV; overexpression of the WT Flag-CTL1 plasmid was indicated by OE-CTL1. The results showed that CTL1 overexpression could reverse MPP in MN9D and SH-SY5Y cells. + Induced cell death, such as Figure 6 As shown in A~C. Given that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, i.e., MPTP, is converted to MPP in vivo. + This leads to mitochondrial damage. An MPTP mouse model was used to evaluate whether neuron-specific CTL1 overexpression could protect dopaminergic neurons. Stereoscopic injection of rAAV2 / 9-hSyn-CTL1-GFP into the substantia nigra pars compacta (SNpc) significantly enhanced CTL1-GFP expression. Figure 6As shown in D in the figure. Immunoblot analysis confirmed that rAAV2 / 9-hSyn-CTL1-GFP injection significantly increased the levels of CTL1 and TH proteins in the midbrain of MPTP-treated mice, as shown in Figure D. Figure 6 As shown in E in the figure. Neuronal induction of CTL1 can inhibit MPTP-induced loss of substantia nigra dopaminergic neurons, such as... Figure 6 As shown in F~G. Furthermore, neuronal induction of CTL1 can reverse MPTP-induced reductions in striatal dopamine and DOPAC, as... Figure 6 As shown in H~I. These results indicate that pre-overexpression of CTL1 prior to MPTP exposure can mitigate the severity of dopaminergic neuron loss.

[0062] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0063] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. The application of formulations that enhance CTL1 activity in the preparation of drugs for treating neurodegenerative diseases, characterized in that, The CTL1 mentioned above is a mitochondrial autophagy receptor; The formulation comprises recombinant viral particles that overexpress the CTL1 gene.

2. The application according to claim 1, characterized in that, The specific method for constructing the recombinant viral particles overexpressing the CTL1 gene is as follows: the CTL1 gene fragment is ligated to the backbone plasmid to form a recombinant plasmid; the recombinant plasmid, packaging plasmid and helper plasmid are co-transfected into host cells for culture, and the recombinant viral particles overexpressing the CTL1 gene are obtained after purification.

3. The application according to claim 2, characterized in that, The backbone plasmid is pAAV-MCS; the packaging plasmid is pAAV2 / 9 Rep-Cap; and the helper plasmid is pHelper.

4. The application according to claim 1, characterized in that, The formulation is a liquid formulation.

5. The application according to claim 1, characterized in that, The drug includes formulations that enhance CTL1 activity and pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable excipients are one or more of the following: diluents, disintegrants, precipitation inhibitors, flow aids, binders, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, and stabilizers.

7. The application according to claim 5, characterized in that, The acceptable dosage forms of the drug include tablets, capsules, granules, injections, pills, powders, or ointments.

8. The application according to claim 1, characterized in that, The neurodegenerative diseases mentioned include one or more of Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis.

Images