Polypeptides for treating parkinson's disease

Abstract

The application discloses a kind of polypeptides for treating Parkinson's disease.In the application, it is first found that transient receptor potential M2 type (TRPM2) channel is more expressed in fragile DA neuron subgroup, and its expression amount is positively correlated with age in Parkinson's disease (PD) patient, and the mechanism of action is determined through rigorous experiment, i.e.TRPM2 channel is preferentially activated in ADE neuron through PARP-1 / PARG / ADPR axis, drives Mfn2 / Bcl-2 complex dependent mitochondrial superfusion, causes ADE neuron death.In addition, TRPM2 channel also drives MPTP treatment mouse SNc DA neuron susceptibility through Mfn2 / Bcl-2 mediation.Firstly, it is found that blocking PARP-1 / PARG / ADPR and Mfn2 / Bcl-2 mediated DA neuron death pathway can prevent the death of induced pluripotent stem cell (iPSC) derived DA neuron specific to spontaneous Parkinson's disease (SPD) patient.As susceptible SNc DA neuron loss is the main symptom of PD, therefore, PARP-1 / PARG / ADPR and Mfn2 / Bcl-2 pathway can become a new therapeutic target for PD.

Description

Technical Field

[0001] This invention relates to biomedicine, and in particular to polypeptides for the treatment of Parkinson's disease. Background Technology

[0002] Dopaminergic (DA) neurons play a crucial role in the regulation of voluntary movement, emotion, and reward in vertebrates (humans, rodents) (Schultz, 2007; Ungless and Grace, 2012). More than 70% of DA neurons located in the ventral midbrain (VM) are generated in the basal midbrain region during embryonic development. Midbrain DA neurons can be divided into three anatomically and functionally distinct clusters: the distal red nucleus (RR), the substantia nigra pars compacta (SNc), and the ventral tegmental area (VTA), with the SNc and VTA containing the majority of DA neurons (Anderegg et al., 2015). *C. elegans*, a classic model organism commonly used in human disease research, similarly exhibits DA neurons composed of three anatomically distinct subtypes: two lateral forebrain neurons (ADE), four head sensory neurons (CEP), and two lateral hindbrain neurons (PDE). These three ciliated DA neuron subtypes play a redundant role in regulating deceleration behavior in response to food and mechanosensory responses (Sawin et al., 2000). Therefore, the diversity among DA neuron subpopulations is conserved from model organisms to humans.

[0003] DA neuronal subtypes share common neural markers such as tyrosine hydroxylase (TH) and dopamine decarboxylase (DDC, used for dopamine synthesis), but certain DA neuronal populations are more susceptible to stress-induced damage than others. One well-known example is the most significant degeneration of SNc neurons, rather than VTA neurons, in Parkinson's disease patients (Damier et al., 1999; Hirsch et al., 1988). Exposure to the metabolite 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that can induce a PD-like phenotype, leads to selective loss of SNc neurons in mice (Di Salvio et al., 2010; Liss et al., 2005). Previous studies have also noted that not all DA neurons degenerate in transgenic Drosophila expressing α-synuclein (Feany and Bender, 2000), suggesting that varying degrees of susceptibility among DA neuronal subtypes are conserved across species. Caenorhabditis elegans is a widely used model organism for research on human neurodegenerative diseases (Fang et al., 2019; Sorrentino et al., 2017), but it is unclear whether there is differential susceptibility of DA neurons in this model.

[0004] Research over the past few decades has suggested that the selective vulnerability of SNc neurons is primarily due to their enormous bioenergy demands, such as pacemaker activity (Surmeier, 2007), high dopamine (DA) content and iron-related toxicity (Hare and Double, 2016; Segura-Aguilar et al., 2014), and complex axonal branching (Bolam and Pissadaki, 2012; Matsuda et al., 2009; Pacelli et al., 2015; Parent and Parent, 2006). However, the molecular mechanisms mediating differential susceptibility among DA neuron subsets remain largely unclear. Therefore, further research into the mechanisms mediating susceptible DA neurons is crucial for guiding the treatment of Parkinson's disease. Summary of the Invention

[0005] The purpose of this invention is to provide a polypeptide.

[0006] Another object of the present invention is to provide a peptide derived from a polypeptide.

[0007] Another object of the present invention is to provide a pharmaceutical composition.

[0008] Another object of the present invention is to provide a method for preventing and / or treating Parkinson's disease.

[0009] Another object of the present invention is to provide the use of a PARP-1 inhibitor or PARG inhibitor and / or a medicament capable of inhibiting the formation of the Mfn2 and Bcl-2 complex.

[0010] In a first aspect, the present invention provides an isolated polypeptide capable of specifically blocking the interaction between Mfn2 and Bcl-2.

[0011] In some preferred embodiments, the polypeptide has a coiled-coil domain of Mfn2 (aa391-434).

[0012] In some preferred embodiments, the polypeptide comprises a sequence selected from the following:

[0013] (i) The amino acid sequence as shown in SEQ ID NO:23;

[0014] (ii) A sequence formed by substitution, deletion or addition of one or more amino acids in SEQ ID NO:23;

[0015] (iii) Having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 23.

[0016] In some preferred embodiments, the length of the polypeptide is 20-100 bp, preferably 22-63 bp.

[0017] In some preferred embodiments, the amino acid sequence of the polypeptide is selected from any of the following:

[0018] (i) The amino acid sequence as shown in SEQ ID NO:1-33;

[0019] (ii) Sequences formed by substitution, deletion or addition of one or more amino acids in SEQ ID NO:1-33;

[0020] (iii) Having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequences of SEQ ID NO: 1-33.

[0021] In some preferred embodiments, the amino acid sequence of the polypeptide is selected from any of the following:

[0022] (i) an amino acid sequence as shown in SEQ ID NO:1; or

[0023] (ii) A sequence formed by substitution, deletion or addition of one or more amino acids in SEQ ID NO:1;

[0024] (iii) It has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0025] In a second aspect, the present invention provides a derivative peptide of the polypeptide described in the first aspect of the present invention, the derivative peptide comprising the polypeptide described in the first aspect of the present invention and a functional peptide linked to the polypeptide, the functional peptide being linked to the N-terminus and / or C-terminus of the polypeptide.

[0026] In some preferred embodiments, the functional peptide is a transmembrane peptide or a protein tag.

[0027] In some preferred embodiments, there are two transmembrane peptides.

[0028] In some preferred embodiments, the transmembrane peptide is linked to the N-terminus and C-terminus of the aforementioned polypeptide.

[0029] In some preferred embodiments, the membrane-penetrating peptide is a pH-responsive cell membrane-penetrating peptide, such as octaarginine.

[0030] In some preferred embodiments, the protein tag is biotin or a peptide derived therefrom.

[0031] In some preferred embodiments, in the derivative peptide of the polypeptide, the N-terminus of the polypeptide is linked to biotin or a derivative peptide thereof; and the C-terminus of the derivative peptide of the polypeptide is linked to a pH-responsive cell-penetrating peptide.

[0032] In some preferred embodiments, the amino acid sequence of the derivative peptide of the polypeptide is shown in SEQ ID NO:34.

[0033] SEQ ID NO:34:

[0034] Biotin-EEMREERQDRLKFIDKQLELLAQDYKLRIKQITEEVERQVSTAM-RRRRRRRR.

[0035] A third aspect of the present invention provides a pharmaceutical composition comprising a polypeptide as described in the first aspect of the present invention or a peptide derived from a polypeptide as described in the second aspect of the present invention; and a pharmaceutically acceptable carrier.

[0036] A fourth aspect of the present invention provides a method for preventing and / or treating Parkinson's disease, the method comprising the step of administering to a subject a therapeutically effective amount of the polypeptide described in the first aspect of the present invention or a derivative peptide of the polypeptide described in the second aspect of the present invention.

[0037] A fifth aspect of the invention provides the use of the polypeptide described in the first aspect of the invention or a derivative peptide of the polypeptide described in the second aspect of the invention for:

[0038] (i) Inhibit DA neuron death or enhance DA neuron activity;

[0039] (ii) Reduces the susceptibility of DA neurons;

[0040] (iii) Inhibit the formation of the Mfn2 / Bcl-2 complex or downregulate the interaction between Mfn2 and Bcl-2 or act as an Mfn2 / Bcl-2 inhibitor;

[0041] (iv) Inhibit excessive mitochondrial fusion in DA neurons;

[0042] (v) Prevention and / or treatment of Parkinson's disease;

[0043] (vi) To prepare medicines for the prevention and / or treatment of Parkinson's disease; and / or

[0044] (vii) Inhibit TRPM2 activation.

[0045] A sixth aspect of the present invention provides a method for in vitro, non-therapeutic inhibition of dopaminergic (DA) neuron death under stress, the method comprising the steps of:

[0046] Add the polypeptide described in the first aspect of the present invention or a derivative peptide of the polypeptide described in the second aspect of the present invention to a dopaminergic (DA) neuron cell culture system under stress.

[0047] A seventh aspect of the present invention provides the use of a medicament capable of inhibiting the formation of the Mfn2 and Bcl-2 complex, for:

[0048] (i) Inhibit DA neuron death or enhance DA neuron activity;

[0049] (ii) Reduce the susceptibility of DA neurons (preferably reduce the susceptibility of DA neurons under stress);

[0050] (iii) Inhibit Ca in DE neurons 2+ And an increase in ROS levels.

[0051] (iv) Inhibit excessive mitochondrial fusion in DA neurons;

[0052] (v) Prevention and / or treatment of Parkinson's disease; and / or

[0053] (vi) To prepare medicines for the prevention and / or treatment of Parkinson's disease.

[0054] In some preferred embodiments, the drug capable of inhibiting the formation of the Mfn2 and Bcl-2 complex is selected from at least one of compounds, nucleic acids, proteins, peptides, and enzymes.

[0055] In some preferred embodiments, the inhibition of DA neuron death includes reducing the death of SNc DA neurons under stress conditions.

[0056] In some preferred embodiments, the reduction of susceptibility to DA neurons is described as selectively reducing susceptibility to ADE neurons. This selective reduction of ADE neuron susceptibility is achieved by inhibiting Ca2+ in DE neurons. 2+ This is achieved through an increase in ROS levels.

[0057] An eighth aspect of the invention provides the use of a PARP-1 inhibitor for:

[0058] (i) Inhibit DA neuron death or enhance DA neuron activity;

[0059] (ii) Reduce the susceptibility of DA neurons (preferably reduce the susceptibility of DA neurons under stress);

[0060] (iii) Inhibit Ca in DE neurons 2+ And an increase in ROS levels.

[0061] (iv) Inhibit excessive mitochondrial fusion in DA neurons;

[0062] (v) Prevention and / or treatment of Parkinson's disease; and / or

[0063] (vi) To prepare medicines for the prevention and / or treatment of Parkinson's disease.

[0064] In some preferred embodiments, the PARP-1 inhibitor is as shown in Formula I:

[0065]

[0066] A ninth aspect of the present invention provides the use of PARG inhibitors for:

[0067] (i) Inhibit DA neuron death or enhance DA neuron activity;

[0068] (ii) Reduce the susceptibility of DA neurons (preferably reduce the susceptibility of DA neurons under stress);

[0069] (iii) Inhibit Ca in DE neurons 2+ And an increase in ROS levels.

[0070] (iv) Inhibit excessive mitochondrial fusion in DA neurons;

[0071] (v) Prevention and / or treatment of Parkinson's disease; and / or

[0072] (vi) To prepare medicines for the prevention and / or treatment of Parkinson's disease.

[0073] In some preferred embodiments, the PARG inhibitor is as shown in Formula II:

[0074]

[0075] Compared with the prior art, the present invention has at least the following advantages:

[0076] (1) This invention is the first to discover that transient receptor potential M2 (TRPM2) channels are highly expressed in vulnerable DA neuronal subsets, and their expression level is positively correlated with age in Parkinson's disease (PD) patients. The mechanism of action was rigorously elucidated through experiments: TRPM2 channels are preferentially activated in ADE neurons via the PARP-1 / PARG / ADPR axis, driving FZO-1 / CED-9 complex-dependent mitochondrial hyperfusion, leading to ADE neuronal death. Furthermore, TRPM2 channels also mediate susceptibility of SNc DA neurons in MPTP-treated mice through the mammalian homologs Mfn2 / Bcl-2 of nematode FZO-1 / CED-9.

[0077] (2) In this invention, it was discovered for the first time that blocking the PARP-1 / PARG / ADPR and Mfn2 / Bcl-2-mediated DA neuron death pathways can prevent the death of induced pluripotent stem cell (iPSC)-derived DA neurons specific to patients with spontaneous Parkinson's disease (SPD). In summary, these findings indicate that these pathways play a conserved role in mediating DA neuron susceptibility. Since the loss of susceptible SNcDA neurons is a major symptom of PD, the TRPM2-related pathway may become a new therapeutic target for PD.

[0078] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0079] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0080] Figure 1A This is the UMAP of cells separated after differentiation of -ipsc-dopaminergic neurons according to the embodiments of the present invention;

[0081] Figure 1B This refers to the percentage of different neuron clusters in the ctrl group and rotenone group according to embodiments of the present invention;

[0082] Figure 1C It is the UMAP expressed by KCNJ6 according to the embodiments of the present invention;

[0083] Figure 1D It is the UMAP expressed by CALB1 according to the embodiments of the present invention;

[0084] Figure 1E The expression abundance of human TRPM2, TRPC6 and TRPA1 in different neuronal clusters of the Ctrl group according to the embodiments of the present invention;

[0085] Figure 1F This refers to the expression of neuronal TRPM2 in single-cell transcriptome data of PD patients (SNc) according to an embodiment of the present invention.

[0086] Student t-test;

[0087] Figure 1G According to an embodiment of the present invention, the above figure is a scatter plot showing the correlation between age and TRPM2 expression level in SNc neurons of PD patients;

[0088] Figure 1H According to an embodiment of the present invention, UMAP displays 10 distinct transcriptional subsets based on snRNA-seq of human SNc DA neuron nuclei. The pink-marked subsets represent the highly vulnerable DA neuron cluster “SOX6_AGTR1”.

[0089] Figure 1I The scatter plots and box plots shown in the embodiments of the present invention represent the expression of KCNJ6(Girk2);

[0090] Figure 1J The scatter plots and box plots shown in the embodiments of the present invention represent the expression of Calbindin (CALB1);

[0091] Figure 1K The scatter plots and box plots shown in the embodiments of the present invention represent the expression of TRPM2;

[0092] Figure 1L These are representative fluorescent RNA-scope images of TRPM2 (red dots) expression in 10-week and 40-week-old mouse SNc and VTA ventral layer TH+ (green) neurons, as described in this embodiment of the invention. Blue represents DAPI. Scale bar = 20 μm;

[0093] Figure 1M According to the embodiments of the present invention, the mRNA of (M)TRPM2 was quantified based on the number of red dot-like spots in each TH-positive neuron. A two-way ANOVA was performed using the Holm-Sidak multiple comparison test (n=3). The average number of dot-like spots per mouse was calculated from six intervals of brain slices (each six slices spanning the entire SNc from -2.7 mm to -4.04 mm from front to back).

[0094] Figure 2A According to embodiments of the present invention, the expression of human TRPM2 in the substantia nigra pars compacta (SNc) and ventral tegmental region (VTA) regions from the human protein map database is used. The RNA expression of TRPM2 is represented by the consistent normalized expression (“nTPM”) in the SNc and VTA regions, using an unpaired t-test.

[0095] Figure 2B According to the embodiments of the present invention, the expression of human TRPM2 in the SNc and VTA regions of the human protein atlas database was analyzed by Holm-Sidak multiple comparison test in a two-way ANOVA.

[0096] Figure 2C The sample age is as described in the embodiments of the present invention;

[0097] Figure 3A This describes the distribution of dopaminergic neurons in nematodes according to an embodiment of the present invention;

[0098] Figure 3B This is a schematic diagram of the structure of wild-type (WT) and htrpm2-expressing worms under the control of the Pdat-1 promoter according to an embodiment of the present invention;

[0099] Figure 3C This describes the gross morphology of wild-type and htrpm2-expressing nematodes according to embodiments of the present invention;

[0100] Figure 3D This is the genotyping identification of the hTRPM2 transgenic nematode integrated according to an embodiment of the present invention;

[0101] Figure 3E The images shown are representative of embodiments of the present invention, depicting PDE neurons of wild-type and htrpm2-expressing nematodes on day 7 of adulthood, scale bar = 20 μm.

[0102] Figure 3F This is the quantitative expression of PDE neurons in nematodes by WT and htrpm2 in the embodiments of the present invention, and the P value is calculated using the Mann-Whitney test;

[0103] Figure 3G This is the complete morphology of CEP neurons in nematodes expressing htrpm2 on day 10 of adulthood according to an embodiment of the present invention, scale bar = 50 μm;

[0104] Figure 3H According to the embodiments of the present invention, ADPR (1mM) and Ca2+ (10μM) induced TRPM2 current in ADE and CEP neurons of WT and htrpm2-expressing nematodes (on day 2 of adult worms). (I) Panel current statistical analysis, the data are expressed as mean ± semn = 4-7, and Holm-Sidak multiple comparison test was used for two-way ANOVA.

[0105] Figure 3I This is a schematic diagram illustrating the changes in PA values ​​of ADE and CEP neurons in WT and htrpm2 nematodes according to an embodiment of the present invention;

[0106] Figure 3J This is the quantification of ADE and CEP neurons in WT and TRPM2 worms treated with 2 mM MPP for 7 days according to an embodiment of the present invention. The p-value is from a two-way ANOVA of Holm-Sidak multiple comparison test.

[0107] Figure 4A These are representative confocal images of ADE and CEP dopaminergic neurons in WT and hTRPM2-expressing nematodes on days 1, 4, and 7 of adulthood, according to embodiments of the present invention. Scale bar = 20 μm.

[0108] Figure 4B This is a quantification of ADE and CEP dopaminergic neurons in WT and hTRPM2 animals according to the embodiments of the present invention. The error bars represent the mean ± sem, and the P value is from the two-way ANOVA of Holm-Sidak multiple comparison test.

[0109] Figure 4C This is a schematic diagram of the hTRPM2 channel structure according to an embodiment of the present invention. The inactivated E960Q mutation is located in the pore region between S5 and S6 of the transmembrane (TM) segment, and the inactivated mutation R1433A is located in the c-terminal ADPR binding domain.

[0110] Figure 4D These are representative confocal images of ADE and CEP DA neurons in *C. elegans* expressing the mutant hTRPM2 according to embodiments of the present invention;

[0111] Figure 4E This is a quantitative analysis of ADE dopaminergic neurons in TRPM2, TRPM2(E960Q) and TRPM2(R1433A) animals according to the embodiments of the present invention. The error bars represent the mean ± sem, and the p-values ​​are from the Dunnett multiple comparison test after one-way ANOVA.

[0112] Figure 5A This is a bar graph of Ca2+ in the cytoplasm of different transgenic strains measured using CaMP3 and an ultraprobe according to an embodiment of the present invention. The P-value is from a one-way ANOVA using Dunnett's multiple comparison test.

[0113] Figure 5B This is a bar graph of cytoplasmic ROS of different transgenic strains measured using CaMP3 and an ultraprobe according to an embodiment of the present invention. The P-value is from a one-way ANOVA using Dunnett's multiple comparison test.

[0114] Figure 5C These are typical confocal images according to embodiments of the present invention, showing the effects of 100 μM BAPTA-AM, 1 mM NAC and 1 mM CsA on htrpm2-expressing animals, scale bar = 20 μm;

[0115] Figure 5D The quantitative analysis of ADE neurons in panel A experiment according to the embodiment of the present invention uses the Kruskal-Wallis test to perform one-way ANOVA on the P-value.

[0116] Figure 5E These are representative confocal images of the effects of egl-1 (n1086n3082) and cyn-1 (tm4171) mutations on htrpm2 expression animals according to embodiments of the present invention, with a scale bar of 20 μm.

[0117] Figure 5F The quantitative analysis of ADE neurons in group C experiment according to the embodiment of the present invention uses the Kruskal-Wallis test to perform one-way ANOVA on the P-value.

[0118] Figure 6A The images show the expression of TRPM2 in TH-positive DA neurons of SNc and VTA regions of WT and TRPM2 knockout mice after MPTP or solvent treatment according to embodiments of the present invention. Scale bar = 50 μm; scale bar = 20 μm for magnified view.

[0119] Figure 6B This is the quantitative analysis of th-positive neurons in the SNc region according to the embodiments of the present invention. The scale bar is 50μm or 200μm. The Holm-Sidak multiple comparison test was used for two-way ANOVA. The data are expressed as mean ± semn = 6-9.

[0120] Figure 6C This is a schematic diagram and experimental design of a PD mouse model treated with rotenone according to an embodiment of the present invention;

[0121] Figure 6D According to the embodiments of the present invention, in the first and second weeks after Rot treatment, TRPM2 knockout reduced apo-induced rotational behavior. The data are expressed as mean ± sem, n = 9-13, and a two-way ANOVA was performed using the Holm-Sidak multiple comparison test.

[0122] Figure 6E According to the embodiment of the present invention, the results of PD mice treated with rot in the rot bar test are shown in the figure. The data are expressed as mean ± semn = 9-13. Holm-Sidak multiple comparison test was used for two-way ANOVA.

[0123] Figure 6FThese are representative confocal images of TH immune response (TH+) fibers in the striatum after Rot treatment in four groups according to embodiments of the present invention. The magnified area is 20 μm, and the scale bar of the central image is 1000 μm.

[0124] Figure 6G This is the quantitative analysis of th positive fibers on the injured and uninjured sides of the striatum according to the embodiment of the present invention. The data are expressed as mean ± semn = 5, 7, and a two-way ANOVA was performed using the Holm-Sidak multiple comparison test.

[0125] Figure 7A These are key regulatory genes for apoptosis in *C. elegans* according to embodiments of the present invention; red indicates human homologous genes.

[0126] Figure 7B These are representative confocal images of WT and hTRPM2-expressing nematodes carrying ced-3 (n717), ced-4 (n1162), ced-9 (n1950, gain of function) and ced-9 (n2812, loss of function) mutations according to embodiments of the present invention, with a scale bar of 20 μm.

[0127] Figure 7C This is the quantitative analysis of ADE neurons in Group B experiment according to the embodiment of the present invention, using one-way ANOVA and Dunnett's multiple comparison test;

[0128] Figure 7D These are representative confocal images, scanning electron microscope (SEM) images, and three-dimensional reconstructed SEM images of mitochondria in ADE and CEP neurons of wild-type and hTRPM2-expressing animals according to embodiments of the present invention. Confocal images: scale bar = 2 μm, SEM: scale bar = 500 nm, three-dimensional scanning electron microscope: scale bar = 1 μm.

[0129] Figure 7E According to the embodiments of the present invention, the statistical analysis of the mitochondrial major axis / minor axis ratio based on confocal image data includes one-way ANOVA and Dunnett's multiple comparison test.

[0130] Figure 7F The confocal images in this embodiment of the invention show the effect of the ced-9 inactivation mutation on the mitochondrial morphology of wild-type and hTRPM2 nematodes ADE and CEP neurons, scale bar = 2 μm;

[0131] Figure 7G The confocal images shown in the embodiments of the present invention illustrate the effects of overexpression of drp-1RNAi, fzo-1RNAi, DRP-1 (280-502) and FZO-1 (346-595) on WT and hTRPM2 strains, with scale bar = 20 μm.

[0132] Figure 7H The quantitative analysis of ADE neurons in the panel G experiment according to the embodiment of the present invention was performed by Holm-Sidak multiple comparison test using a two-way ANOVA.

[0133] Figure 7I According to the embodiments of the present invention, positive PLA signals (red spots) are shown to indicate the interaction between Bcl-2 and Mfn2 in WT and TRPM2-knockout mice after MPTP or solvent treatment, scale bar = 5 μm;

[0134] Figure 7J This is a quantification of the number of PLA spots according to an embodiment of the present invention. These spots indicate the interaction strength of Mfn2 / Bcl-2 in the SNc and VTA regions. The data are expressed as mean ± semn = 4,5. Holm-Sidak multiple comparison test was used for two-way ANOVA.

[0135] Figure 8A This is a schematic diagram of the key enzymes involved in ADPR synthesis and metabolism according to an embodiment of the present invention. The TRPM2 channel is activated by a co-agonist, ADPR, and Ca2+.

[0136] Figure 8B These are representative confocal images of WT and hTRPM-2 nematodes treated with parp-1 overexpression, parp-1 knockdown, parg-1 knockdown, ndx-6 overexpression, and PJ-34 / PDD according to embodiments of the present invention, with a scale bar of 20 μm.

[0137] Figure 8C The quantitative analysis of ADE and CEP neurons in Group B experiment according to the embodiment of the present invention was performed by Holm-Sidak multiple comparison test using two-way ANOVA.

[0138] Figure 8D This is the quantitative analysis of ADE neurons in experiment B (PJ-34 and PDD treatment) according to the embodiment of the present invention, using one-way ANOVA and Dunnett's multiple comparison test.

[0139] Figure 9A The immunofluorescence of iPSC markers OCT-4, Nanog, Tra-1-60 and Sox-2 according to embodiments of the present invention is shown in scale bar = 50 μm.

[0140] Figure 9B These are micrographs of DA neurons differentiated from ipsc in healthy control groups and SPD patients according to embodiments of the present invention. The DA neurons were cultured in vitro for 15 days after maturation, and the differentiated DA neurons were all TH / MAP2 / Girk2 positive. Scale bar = 20 μm.

[0141] Figure 9C This is the quantification of differentiated TH+ neurons in dapi-stained cells relative to healthy controls and SPD patients according to embodiments of the present invention, where the dots represent individual values ​​from independent experiments.

[0142] Figure 9D This is a representative trace of action potential discharge induced by 4s overthreshold current recorded by the DA neurons derived from ipsc in the embodiment of the present invention.

[0143] Figure 9E This is a voltage-clamp recording of sodium current in DA neurons derived from ipsc according to an embodiment of the present invention;

[0144] Figure 10A This invention relates to an embodiment of the present invention, which describes the effect of CTG detection on ACA (0.5 μM) and A10 (0.6 μM) treatment on the activity of ipsc-derived DA neurons from SPD patients. The data are expressed as mean ± sem; the p-values ​​are from a one-way ANOVA using the Holm-Sidak multiple comparison test.

[0145] Figure 10B According to the embodiments of the present invention, the effect of PJ-34 (2μM) treatment on the activity of ipsc-derived DA neurons from SPD patients was detected by CTG. The data are expressed as mean ± sem, and the P value was obtained from Student's t test.

[0146] Figure 10C According to the embodiments of the present invention, the effect of PDD (1 μM) on the activity of ipsc-derived DA neurons from SPD patients was determined by CTG experiment. The data are expressed as mean ± sem, and the P value is from Student's t test.

[0147] Figure 10D This is a statistical analysis of the relative length of mitochondrial branches according to an embodiment of the present invention. The data are expressed as mean ± sem; the p-value is from a one-way ANOVA of Dunnett's multiple comparison test.

[0148] Figure 10E The representative confocal images in this embodiment of the invention show the mitochondrial network of ipsc-derived DA neurons in PD patients treated with PJ-34, PDD, A10, ACA or PEP, scale bar = 20 μm.

[0149] Figure 10F According to the embodiments of the present invention, the interaction between Bcl-2 and Mfn2 in human ipsc-derived DA neurons was determined by PLA experiments using positive PLA signals (red spots), scale bar = 5 μm.

[0150] Figure 10GThis invention relates to the quantification of the number of PLA spots in human ipsc-derived DA neurons, which indicate the strength of Mfn2 / Bcl-2 interaction. Data are expressed as mean ± sem and analyzed using a one-way ANOVA with Holm-Sidak multiple comparison test.

[0151] Figure 11A The present invention describes the use of Western blot to show the polymerization (ADPR) in ipsc-derived dopamine neurons in healthy controls (WT) and SPD patients, as well as the effects of PJ-34 (2 μM) or PDD (1 μM) on ipsc-derived dopamine neurons in SPD patients.

[0152] Figure 11B According to embodiments of the present invention, flow cytometry was used to analyze the Ca2+ levels of ipsc-derived DA neurons from healthy controls and SPD patients.

[0153] Figure 11C The standardized intensity of the calcium ion level measured in b is used to quantitatively analyze the data according to the embodiments of the present invention. The data are expressed as mean ± SEM Student's t-test.

[0154] Figure 11D According to the embodiments of the present invention, flow cytometry was used to detect the Ca2+ level of DA neurons derived from ipsc in SPD patients after treatment with PJ-34 (2μM), PDD (0.5μM), A10 (0.6μM), ACA (0.5μM) or synthetic peptide (PEP, 2μM).

[0155] Figure 11E The standardized intensity of calcium ion levels measured by d is used to quantitatively analyze the data according to the embodiments of the present invention. The data are expressed as mean ± sem, and the one-way ANOVA is performed using Dunnett's multiple comparison test.

[0156] Figure 12A This is a localization diagram of the coiled colonic structural domains of nematode FZO-1 and human Mfn2 according to an embodiment of the present invention;

[0157] Figure 12B The representative confocal images in the embodiments of the present invention show that the synthetic polypeptide PEP (10 μM) inhibited the interaction between Bcl-2 and Mfn2 in SH-SY5Y cells after MPP+ or solvent treatment. The red fluorescent dotted areas indicate that PLA detected the interaction between Mfn2 and Bcl-2. Scale bar = 5 μm.

[0158] Figure 12CThe quantitative analysis of the red dot in Figure b according to the embodiment of the present invention is expressed as mean ± semn = 6-9, and a two-way ANOVA is performed using Holm-Sidak multiple comparison test.

[0159] Figure 12D According to the embodiments of the present invention, the activity of ipsc-derived DA neurons from SPD patients and PEP treatments was detected by CTG. The data are expressed as mean ± semP values ​​from a one-way ANOVA of Holm-Sidak multiple comparison test.

[0160] Figure 13 This is a schematic diagram of an experiment in which TRPM2 mediates the vulnerability of DA neurons by sensing ROS and calcium homeostasis imbalance according to an embodiment of the present invention. In different models, the TRPM2 channel is preferentially activated in vulnerable DA neurons. PARP-1 / PARG / ADPR activates TRPM2 under stress, leading to further calcium overload, oxidative stress, and subsequent Mfn2 / Bcl-2 (FZO-1 / CED-9 in nematodes)-dependent mitochondrial hyperfusion, ultimately leading to the death of vulnerable DA neurons. Detailed Implementation

[0161] Studies have reported that certain dopaminergic (DA) neuron populations are more susceptible to stress-induced damage than other subpopulations, and the varying degrees of susceptibility among DA neuron subpopulations are conserved across species. However, the molecular mechanisms mediating these differential susceptibility remain largely unclear. Through extensive and in-depth research, the inventors have discovered that transient receptor potential M2 (TRPM2) plays a conserved role in mediating DA neuron susceptibility. They have also elucidated for the first time the mechanism by which TRPM2 channels are preferentially activated in ADE neurons via the PARP-1 / PARG / ADPR axis, driving FZO-1 / CED-9 complex-dependent mitochondrial hyperfusion and leading to ADE neuron death. Furthermore, they found that TRPM2 channels also mediate susceptibility of SNc DA neurons in MPTP-treated mice through the nematode FZO-1 / CED-9 homolog Mfn2 / Bcl-2. Since the loss of susceptible SNc DA neurons is a major symptom of PD, novel drugs for treating PD can be developed targeting multiple potential targets within these mechanisms.

[0162] polypeptide

[0163] This invention also relates to an isolated polypeptide that can specifically block the interaction between human Mfn2 and Bcl-2.

[0164] In some preferred embodiments, the polypeptide has a coiled-coil domain of Mfn2 (aa391-434).

[0165] In a preferred embodiment of the present invention, the polypeptide comprises a sequence selected from the following: (i) an amino acid sequence as shown in SEQ ID NO:23;

[0166] (ii) A sequence formed by substitution, deletion or addition of one or more amino acids in SEQ ID NO:23, but retaining the same biological activity as SEQ ID NO:23 (activity of binding to the coiled-helix domain of human Mfn2);

[0167] (iii) Having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 23, but maintaining the same biological activity as SEQ ID NO: 23 (blocking the interaction between human Mfn2 and Bcl-2).

[0168] In a preferred embodiment of the present invention, the length of the polypeptide is 20-100 bp, preferably 22-63 bp.

[0169] In a preferred embodiment of the present invention, the amino acid sequence of the polypeptide is selected from any of the following:

[0170] (i) The amino acid sequence as shown in SEQ ID NO:1-33;

[0171] (ii) Sequences formed by substitution, deletion or addition of one or more amino acids in SEQ ID NO:1-33;

[0172] (iii) Having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequences of SEQ ID NO: 1-33.

[0173] In a preferred embodiment of the present invention, the amino acid sequence of the polypeptide is selected from any of the following:

[0174] (i) an amino acid sequence as shown in SEQ ID NO:1; or

[0175] (ii) A sequence formed by substitution, deletion or addition of one or more amino acids in SEQ ID NO:1;

[0176] (iii) It has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0177] In this invention, sequences formed by substitution, deletion, or addition of one or more amino acids in the target sequence and sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the target sequence all have the same biological activity as the target sequence.

[0178] peptide derivatives

[0179] This invention also relates to derivatives of the aforementioned polypeptides, wherein the polypeptide derivatives include the aforementioned polypeptides and functional peptides linked to the aforementioned polypeptides, such as membrane-penetrating peptides, wherein the functional peptides are linked to the N-terminus and / or C-terminus of the aforementioned polypeptides. In this invention, the membrane-penetrating peptides are conventionally described in the art, as long as they can assist in delivering the polypeptides into cells to exert their effects. Generally, the membrane-penetrating peptides are short peptide molecules composed of 10-30 amino acids. In a preferred embodiment of this invention, the membrane-penetrating peptide is a pH-responsive membrane-penetrating peptide, such as octameric arginine. In a preferred embodiment of this invention, the functional peptide may also include a tag peptide, wherein the tag peptide is a biotin or a derivative thereof. For example, a polypeptide as shown in SEQ ID NO:34:

[0180] SEQ ID NO:34:

[0181] Biotin-EEMREERQDRLKFIDKQLELLAQDYKLRIKQITEEVERQVSTAM-RRRRRRR (PEP polypeptide).

[0182] As used herein, the term "peptide" refers to a molecule comprising a sequence of amino acids linked by peptide bonds, regardless of length, post-translational modifications, or function. As used herein, the term "polypeptide" refers to a protein that is naturally occurring or produced or altered chemically or otherwise through recombinant processes, and is essentially conceived as a three-dimensional protein that has undergone post-translational processing in the same manner as the native protein.

[0183] The polypeptides in this invention can be prepared by conventional methods in the art, such as by chemical synthesis if the polypeptide sequence is known.

[0184] Pharmaceutical Composition

[0185] This invention also relates to a pharmaceutical composition comprising the polypeptide or polypeptide-derived peptide of this invention and a pharmaceutically acceptable carrier.

[0186] In this invention, the pharmaceutically acceptable carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned polypeptide or a derivative of the polypeptide, and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0187] Uses of polypeptides or their derivatives

[0188] This invention also relates to the use of peptides for: (i) inhibiting DA neuron death or enhancing DA neuron activity; (ii) reducing DA neuron susceptibility; (iii) inhibiting the formation of the Mfn2 / Bcl-2 complex or downregulating the interaction between Mfn2 and Bcl-2; (iv) inhibiting excessive mitochondrial fusion in DA neurons; (v) preventing and / or treating Parkinson's disease; (vi) preparing medicaments for the prevention and / or treatment of Parkinson's disease; and / or (vii) inhibiting TRPM2 activation. The peptides of this invention can be used alone or prepared into pharmaceutical compositions. Of the foregoing uses, (i)-(iv) and (vii) can all be for in vitro non-therapeutic purposes, such as use as research reagents. When used as medicaments for the prevention and / or treatment of Parkinson's disease, the peptides of this invention can alleviate DA neuron degeneration / death in Parkinson's disease patients, or prevent or reduce nerve damage, or slow the progression of Parkinson's disease, or alleviate Parkinson's disease symptoms.

[0189] Treatment

[0190] This invention also relates to methods for preventing and / or treating Parkinson's disease, comprising the steps of administering to a subject a therapeutically effective amount of a polypeptide or a polypeptide derivative thereof as described in this invention.

[0191] The term "subject" as used herein is defined as including animals, such as mammals, including but not limited to primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In a particular embodiment, the subject is a human.

[0192] As used herein, "therapeutic effective amount" refers to the quantity of a drug sufficient to provide a therapeutic effect in the treatment or control of a disease or disorder, or sufficient to delay or minimize one or more symptoms associated with that disease or disorder. Therapeutic effective amount of a drug refers to the quantity of a therapeutic agent, used alone or in combination with other therapies, that provides a therapeutic effect in the treatment or control of a disease or disorder. The term "therapeutic effective amount" may also include the quantity that improves overall therapy, reduces or avoids symptoms or causes of the disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent.

[0193] In this invention, the route of administration for the drug or drug composition is a conventional route of administration for polypeptide drugs, preferably by injection or oral administration. The injection route preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The drug composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, a non-aqueous solution, or a suspension, more preferably in tablets, capsules, granules, injections, or infusions.

[0194] In addition to the polypeptides or polypeptide-derived peptides provided by this invention, this invention also provides other drugs for treating diseases related to DA neuronal death (e.g., Parkinson's disease), such as other drugs that can inhibit the formation of the Mfn2 and Bcl-2 complex, RARP-1 inhibitors, or PARG inhibitors.

[0195] This invention also relates to the use of a medicament capable of inhibiting the formation of the Mfn2 and Bcl-2 complex, for:

[0196] (i) Inhibit DA neuron death or enhance DA neuron activity;

[0197] (ii) Reduce the susceptibility of DA neurons (preferably reduce the susceptibility of DA neurons under stress);

[0198] (iii) Inhibit Ca in DE neurons 2+ And an increase in ROS levels.

[0199] (iv) Inhibit excessive mitochondrial fusion in DA neurons;

[0200] (v) Prevention and / or treatment of Parkinson's disease; and / or

[0201] (vi) To prepare medicines for the prevention and / or treatment of Parkinson's disease.

[0202] As used in this invention, the term "susceptibility" refers to the sensitivity of neurons to stress stimuli such as aging, oxidative stress, calcium homeostasis imbalance, and other cellular physiological homeostasis imbalances. It is measured by the survival rate of dopamine neurons under stress stimuli. Substantia nigra dopamine neurons are susceptible to Parkinson's disease, which is manifested by a large number of deaths in Parkinson's patients.

[0203] In this invention, the degree of mitochondrial fusion can be determined by the following method: mitochondria are labeled with mitochondrial-specific dyes and imaged using fluorescence confocal microscopy. The images are then processed using ImageJ software to analyze the mitochondrial length. In dopaminergic neurons differentiated from pluripotent stem cells derived from Parkinson's patients, the mitochondrial length is significantly increased compared to differentiated neurons derived from normal individuals, which can be considered as excessive mitochondrial fusion.

[0204] This invention also relates to the use of PARP-1 inhibitors and / or PARG inhibitors for:

[0205] (i) Inhibit DA neuron death or enhance DA neuron activity;

[0206] (ii) Reduce the susceptibility of DA neurons (preferably reduce the susceptibility of DA neurons under stress);

[0207] (iii) Inhibit Ca in DE neurons 2+ And an increase in ROS levels.

[0208] (iv) Inhibit excessive mitochondrial fusion in DA neurons;

[0209] (v) Prevention and / or treatment of Parkinson's disease; and / or

[0210] (vi) To prepare medicines for the prevention and / or treatment of Parkinson's disease.

[0211] In a preferred embodiment of the present invention, the PARP-1 inhibitor is shown in Formula I:

[0212]

[0213] In a preferred embodiment of the present invention, the PARG inhibitor is shown in Formula II:

[0214]

[0215] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0216] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0217] Unless otherwise specified, the term “or” means the term “and / or” and is used interchangeably with the term “and / or”.

[0218] As used herein, including the appended claims, unless the context clearly indicates otherwise, the singular forms of words such as “an,” “a,” and “the” include their respective plural referents.

[0219] Materials Methods

[0220] Caenorhabditis elegans

[0221] Rearing of *C. elegans*: Nematodes were cultured under standard conditions on nematode growth medium (NGM) plates at 20°C and fed OP50 as previously described (Corsi et al., 2015). The strains used in this study are listed in the reagents and tools table.

[0222] Succession and Construction of Caenorhabditis elegans Strains

[0223] The coding sequence for human TRPM2 was cloned into the plasmid Pdat-1::sl2::DsRed2b, regulated by the Pdat-1 promoter, retaining the sl2 sequence and DsRed2b as positive markers for hTRPM2 expression. Transgenic *C. elegans* var. *hTRPM2* and WT (DsRed2b specifically expressed only in DA neurons) were constructed. 50 ng / μL of the plasmid and 10 ng / μL of the co-labeled Punc-122-GFP were injected into the gonads of young adult nematodes. DsRed2b-expressing progeny were isolated for integration strain construction. The array was integrated into the genome via trimethylpsoralen / ultraviolet (TMP / UV) mutagenesis. Extrachromosomal microarray transgenic animals were prepared by gonadal injection of 40–50 ng / μL of the constructed plasmid and 10 ng / μL of the co-labeled marker (Plin-44-mCherry).

[0224] Neurodegeneration test

[0225] Animals were fixed on 2% agarose pads in M9 buffer containing 12 mM levamisole (TCI, T1215). Representative images were obtained at 60x magnification using an Olympus FV1000 confocal microscope. The nematode heads were observed and captured using a 546 nm excitation laser to better image ADE and CEP neurons. Layer Z was processed using an FV10-ASW 4.2 Viewer. ADE and CEP neurons with visible cell bodies and continuous processes were counted as viable neurons, and each nematode was examined by two independent researchers.

[0226] Quantitative analysis of neuronal calcium ion and reactive oxygen species levels

[0227] Under the regulation of the Pdat-1 promoter, the coding sequence of the probe was cloned into a plasmid, and calcium ion and ROS levels were detected using genetically modified GCaMP3 and Hyper fluorescent probes, respectively. The probe plasmid and linker (Plin-44-mCherry) were then injected into the gonads of young adult worms. Images were acquired using a 60x objective (Olympus, FV1000) under a confocal microscope excited at 488 nm and 546 nm. Calcium ion and ROS levels (F488) were quantified by measuring the mean fluorescence of neuronal cell bodies. The baseline fluorescence f488 was obtained by taking the average fluorescence value of the head epidermis. Standardized calcium ion and ROS levels were calculated as (F488 - F488 - background) / (F546 - F546 - background), respectively.

[0228] Electrophysiology

[0229] Day 2 adult worms were attached to Sylgard-coated coverslips using cyanoacrylate adhesive. A dorsal incision was made using a sharp glass pipette to expose dopamine neurons located in the head. Whole-cell current recordings were performed using an EPC-10 amplifier and Patchmaster software (HEKA) on a fluorescence microscope (Olympus, BX51W1). The extracellular solution (ECS) consisted of 145 mM NaCl, 2.5 mM KCl, 5 mM CaCl2, 1 mM MgCl2, and 20 mM glucose, pH 7.3. The intracellular solution (ICS) contained 145 mM k-gluconate, 2.5 mM KCl, 0.25 mM CaCl2, 5 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 10 mM glucose, 5 mM Na2ATP, and 0.5 mM NaGTP, pH 7.2. The membrane potential was maintained at -70 mV. Ca2+ co-activation of TRPM2 was detected by adding 1 mM ADPR and 10 μM CaCl2 to ICS, and 20 μM MACA was added to ECS. Data were processed using Igor software (WaveMetrics).

[0230] Neuronal RNAi

[0231] Since the neurons of *Caenorhabditis elegans* are not easily affected by systemic RNAi ingestion (Calixto et al., 2010; Firmhaber and Hammarlund, 2013), we injected a mixture of sense and antisense gene fragments under the Phsp-16.2 promoter to obtain time-specific initiation and neuronal RNAi effects (Esposito et al., 2007; Hamakawa and Hirotsu, 2017; Tavernarakis et al., 2000). 40 ng / μL of the sense and antisense fragments of the target gene, and 10 ng / μL of the marker Plin-44-mCherry were injected. Young L4 worms were subjected to heat shock at 35°C for 30 min daily, followed by incubation at 20°C until day 7 of adulthood, at which point neuron counting was performed.

[0232] Drug treatment

[0233] The reagents and tools list details of the antibodies, drugs, and kits used in this study. All drugs were dissolved in high concentrations as stock solutions. PJ-34 (Sigma, P4365), PDD (Adipogen Life Sciences AG-CR1-3646-M005), NAC (Sigma, A9165), and BAPTA-AM (Sigma, A1076) were dissolved in DMSO or H2O to prepare 10 mM stock solutions. The drugs were added to the bacterial lawn inoculated with NGM plates, and the NGM plates were allowed to dry for several hours. L4 parasites were transferred to the drug-treated plates for treatment. Considering the decrease in drug efficacy, the parasites were transferred to another fresh drug-treated dish every 2 days.

[0234] Scanning electron microscopy and 3D reconstruction

[0235] The SEM preparation method for nematodes was as previously described (Shu et al., 2011), with some modifications. To better determine the location of ADE and CEP neurons, each nematode was first observed using a confocal microscope (Olympus FV1000), and the distance from the front to the target neuron cell body was determined based on DsRed2b fluorescence. The nematodes were then removed into PCR tubes for SEM sample preparation. The nematodes were fixed for 2 hours in 0.1M sodium cacodylate (CAS) buffer at pH 7.4 with 2% PFA and 1% glutaraldehyde. The cuticle was then sharply cut to allow the 4% glutaraldehyde fixation buffer to diffuse for better overnight fixation. After washing three times with CAS buffer, the nematodes were removed and placed in diaminobenzidine (DAB) buffer for co-oxidation with gaseous oxygen at 480 nm for 5 min. After washing three times with CAS buffer, the nematodes were fixed on ice in 0.1M CAS buffer with 2% diammonium tetroxide for 30 min, and then washed three times with ddH2O. The samples were stained with 1% uranium acetate aqueous solution at 4°C for 1 h, followed by washing three times with ddH2O. Dehydration was performed for 30 min at each step using a series of graded ethanol solutions (30%, 50%, 70%, 90%, 100%). The samples were washed twice with 1:1 acetone and methanol, and then with pure acetone. The samples were then soaked in 3:7, 7:3, and 1:0 resin-acetone mixtures for 12 h, 24 h, and 48 h, respectively. Finally, the samples were placed in a 65°C vacuum oven for 48 h. The positions of the samples were aligned parallel to the sample wells to locate the neurons. Based on previously recorded distances, the resin samples were cut to the desired regions at the micrometer level, and images of each slice (10 nm) were acquired on a Nova Nano 450 (Thermo, FEI). The sequence images were aligned and reconstructed using Amira software (ThermoFisher Scientific). Finally, the mitochondrial boundaries marked with photooxidative dab staining were manually segmented for three-dimensional reconstruction.

[0236] mouse

[0237] TRPM2- / - male mice (8–10 weeks old, 22–25 g) were introduced from the University of Leeds and then bred at Zhejiang University. Transgenic mice were produced at the University of Leeds (Zou et al., 2013). They were collectively housed in a controlled environment (21±2℃, 12-hour light / dark cycle) with free access to food and water. Experiments followed the ethical standards established by the Animal Protection and Utilization Committee of Zhejiang University. Efforts were made to minimize the number of animals used and their discomfort.

[0238] MPTP-induced mouse PD model

[0239] Mice were randomly assigned to either a solvent group or an MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) group. The MPTP group received intraperitoneal injections of MPTP 25 mg / kg / day for 5 consecutive days (Jackson-Lewis and Przedborski, 2007; Wei et al., 2020), while control mice were injected with an equal volume of standard suspension carrier (0.9% NaCl, w / v) (Jackson-Lewis and Przedborski, 2007; Tatton and Kish, 1997). Six days after the last injection, mice were euthanized after completing the rotarod experiment. 20 μM thick coronal frozen sections were prepared in 0.1 M phosphate buffer (pH 7.4) with 4% paraformaldehyde for immunofluorescence staining.

[0240] Stereotactic brain surgery (rotenone-induced PD mouse model)

[0241] We used the animal surgical method described in detail in our previous study, with minor modifications (Fang et al., 2021). Briefly, mice were anesthetized with 1.5% pelltobarbitalum natricum and placed in a stereotaxic apparatus (Refworld, Shenzhen, China). 2.5 μg of rotenone (R8875, Sigma-Aldrich, St. Louis, MO, USA) (1 μg / μl, dissolved in 5% DMSO + 5% cremoophor + 90% solvent) was injected into the left SNc and MFB; this was performed using a microinfusion set at a flow rate of 1 μl / min. Sham-operated mice received only the same volume of solvent.

[0242] Tyrosine hydroxylase (TH) immunofluorescence staining

[0243] The midbrain subcutaneous stromal (SNc) and striatum were coronally cut at a height of 20 μm on a cryostat (Leica, Wetzlar, Germany). Sections were collected for TH immunofluorescence staining. Briefly, brain slices were blocked for 2 hours at room temperature in 3% BSA and 1% TBST. After the blocking step, the slices were incubated at 4°C with anti-tyrosine hydroxylase antibody (clone LNC1; MAB318, Sigma) (1:500) in 0.3% PBST with 1% BSA added. The next day, the brain slices were washed three times in PBS for 5 min each time and then incubated for 2 h in fluorescently labeled goat anti-mouse IgG-H&L antibody. We analyzed multiple slices (one per four slices of the entire SNc from -2.7 mm to -4.04 mm) for TH-stained neuron counting. Fluorescent images were captured at 10x and 20X objectives using an Olympus VS120 microscope. The images were subsequently quantified and analyzed in CellSens Dimension.

[0244] Rotarod test

[0245] Motor coordination in mice was assessed using a rotarod apparatus (Bieri et al., 2019). Mice were trained for 300 s at a constant speed of 5 rpm / min for 3 consecutive days to adapt to the rotarod apparatus. Rotarod tests were conducted under uniform acceleration (5–30 rpm / min) for 300 s, and the fall latency was recorded for 3 tests. The rotarod and chamber were wiped with 70% ethanol between tests.

[0246] Apomorphine-induced rotation

[0247] Mice were administered apomorphine (0.5 mg / kg; on days 7 and 14) 30 min after acclimatization in the environment. Consistent with our previous standard (Zhou et al, 2019), they were rotated (360°) for counting. Video recording lasted 60 minutes.

[0248] iPSC

[0249] Dopamine neuron differentiation

[0250] We performed DA neuron induction based on basal plate (FP) cells as previously described (Fedele et al., 2017; Ke et al., 2020). In short, human iPSCs were first digested with acutase and seeded onto matrix gel-coated plates, then cultured from day 0 to day 5 in FP cell induction medium N1 (containing SB431542 (10 μM), LDN193189 (100 nM), SHH-C24 (100 ng / mL), FGF8 (100 ng / mL), and purmorphamine (2 μM)). The medium was replaced with N2 medium on days 5, 6, 7, and 8 at ratios of 75% (N1):25% (N2), 50% (N1):50% (N2), on days 9 and 10 at ratios of 25% (N1):75% (N2), and on day 11 at ratios of 100% (N2). N1 medium (50ml) contained 41ml knockout DMEM, 7.5ml knockout serum replacement, 0.5ml GlutaMAX, 0.5ml NEAAs, and 0.5ml penicillin / streptomycin. N2 medium (50ml) contained 48.5ml DMEM / F12 with HEPES buffer / Neural basal, 0.5ml N2 replenishment, 0.5ml GlutaMAX, and 0.5ml penicillin / streptomycin. On day 11, FP cells were passaged, expanded, and cryopreserved. After six generations of expansion, FP cells were lysis using acutase and seeded onto fresh Matrigel-coated plates. The culture medium was replaced with DA-inducing medium containing NB / B27, GDNF (PeproTech, 20 ng / mL), BDNF (PeproTech, 20 ng / mL), 0.2 mM ascorbic acid (Sigma-Aldrich), DAPT (10 nM; Tocris), cAMP (10 μM, Sigma-Aldrich), and transforming growth factor β3 (1 ng / mL; R&D). Cells were then lysis using acutase and diluted to 5 × 10⁶ cells / mL. 5 / mL, and then re-seed the cells into culture dishes pre-coated with poly-d-lysine hydrobromide (PDL) and laminin until the desired maturity stage.

[0251] Drug treatment of IPSC-derived dopaminergic neurons

[0252] All drugs (PJ-34, PDD, ACA, A10, and synthetic peptides (PEP, dendritic bacteria)) were dissolved in 10 mM stock solution. The appropriate drug concentrations were administered on day 15 after DA neurons matured, with the drug-containing culture medium changed every 4 days.

[0253] Western blot experiment

[0254] In short, cultured neurons were collected and lysed with 50 μL of pre-chilled RIPA buffer containing 1% PMSF and 1% ThermoFisher cocktail. Cells were resuspended and sonicated, then centrifuged at 12500g for 20 min at 4°C. The supernatant was collected, and the concentration of each sample was determined using a BCA assay kit (Beyotime Biotechnology). The supernatant was mixed with 5-fold loading buffer for sample preparation and electrophoresis. Band intensity was analyzed using Image Studio Lite software.

[0255] PLA Adjacency Linking Technology

[0256] To detect the direct interaction between Bcl-2 and Mfn2, PLA detection was performed according to the standard procedure using the Duolink™ In Situ Detection Kit (Sigma-Aldrich). Differentiated DA neurons, SH-SY5Y cells, and mouse brain sections were first fixed with 4% PFA on day 18. Briefly, the samples were incubated in blocking buffer at 37°C for 60 minutes and washed twice. Antibodies against Bcl-2 (Santa Cruz, sc-7382) and anti-mfn2 (Abcam, ab124773) were diluted with Duolink antibody diluent and added to the samples, incubated overnight at 4°C. Next, the samples were washed three times with wash buffer A, and the PLUS MINUS PLA probe, diluted 1:5 in the antibody diluent, was applied and incubated at 37°C for 1 hour. The probe was washed with wash buffer A, ligation buffer was added, and incubation was performed at 37°C for 30 minutes. After washing, amplification buffer was added, and incubation was performed at 37°C for 100 minutes. Samples were washed with washing buffer B and then fitted with Duolink in-situ mounting medium containing DAPI. Confocal imaging was performed using a Zeiss, LSM 880, and Airyscan.

[0257] Ca 2+ Flow cytometry

[0258] The calcium ion probe Fluo-4 (Invitrogen, Fluo-4, AM, F14201) was dissolved in extracellular working solution (containing 130 mM NaCl, 4.6 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM D-Glucose, and 100 μM EGTA). The probe solution was preheated to 37°C and added to wells for incubation for 60 min. The probe solution was discarded, and cells were washed twice with ECS. 300 μL of ECS was added and incubated for 30 min. The ECS was discarded again, and cells were digested with preheated Accutase at 37°C for 5 min. The ECS was removed, and cells were resuspended in ECS for flow cytometry (Beckman, DxFLEX). Data were processed using FlowJo 10 software.

[0259] Mitochondrial imaging

[0260] Mitochondrial morphology was observed using MitoTracker staining. In short, DA neurons differentiated on day 18 were stained with 500 nM MitoTracker Red (Thermo, M22425) in 37°C medium for 30 minutes. Cells were then washed with DPBS. To ensure observation of desired dopamine neurons, fragile DA neurons were labeled with Girk2 antibody (Proteintech, 21647-1-1AP). After PFA fixation, they were stained with Girk2 antibody for 12 hours, followed by staining with a second antibody for 12 hours. Samples were washed three times and protected with 50% glycerol for fluorescence. Neuronal staining was observed using a laser confocal microscope (Zeiss, LSM880+Airy scanning, NA 100 nm, 63x oil magnification). Mitochondrial branch length was analyzed using ImageJ software.

[0261] Immunofluorescence staining

[0262] In simple terms, the cells are attached to a culture dish and fixed with 4% paraformaldehyde for 20 minutes at room temperature. After washing with PBS, they are blocked for 1 hour at room temperature with 3% BSA and 0.5% TBST (PBS containing 0.5% Triton X-100). After the blocking step, the cells are co-incubated overnight at 4°C with anti-oct-4 antibody (ab19857, abcam) (1:1000), anti-tr-1-60 antibody (ab16288, abcam) (1:1000), anti-nanog antibody (ab109250, abcam) (1:1000), and anti-sox-2 antibody (ab97959, abcam) (1:500). The following day, cells were washed three times with PBS for 5 minutes each time, and then incubated with a diantibody [goat anti-rabbit IgG (H+L) highly cross-linked diantibody, Alexa Fluor Plus 488 (A32723, Invitrogen, 1:100) / goat anti-rabbit IgG (H+L) cross-linked diantibody, Alexa Fluor 546 (A11010, Invitrogen, 1:100)]. Finally, cells were washed three times with PBS and inoculated with DAPI-containing adherent medium for imaging. Fluorescence images were taken using a Zeiss LSM 880 and an Airyscan 20X objective lens.

[0263] Statistical analysis

[0264] Data were processed using GraphPad Prism 8 software and expressed as mean ± standard deviation (mean ± sem). The distribution of continuous data was analyzed using the Shapiro-Wilk test. Comparisons among multiple groups were performed using one-way or two-way ANOVA with appropriate post-hoc tests. Two-tailed t-tests were used to determine statistically significant differences between groups, and the Mann-Whitney test was used for data that did not conform to a normal distribution. Significance was defined as P < 0.05.

[0265] Data and code availability

[0266] The datasets used or analyzed in the current study may be obtained from the corresponding author upon reasonable request.

[0267] Example 1: TRPM2 enrichment in fragile DA neuronal clusters

[0268] In this embodiment, the inventors performed single-cell transcriptome sequencing on DA neurons differentiated from induced pluripotent stem cells (iPSCs), analyzed data from the untreated group and the rotenone-treated group, and divided the differentiated neurons into 6 clusters (DAn1-6). Figure 1A The DAn1 cluster showed significant neuronal loss. Figure 1BThis indicates greater vulnerability. It has been reported that the G protein-gated inward rectifier potassium channel subunit (Girk2, encoded by the KCNJ6 gene) is a marker gene for SNc DA neurons, and the calcium-binding protein (calbindin, encoded by the CALB1 gene) is a marker gene for VTA neurons in the mammalian brain (Thompson et al., 2005). Using these marker neurons, our results showed that SNc DA neurons, rather than VTA DA neurons, are primarily expressed in the vulnerable DAn1 cluster. Figure 1C and Figure 1D This indicates that the ipsc-derived DAn1 neuronal clusters are consistent with the vulnerability of fragile SNc DA neurons in the human brain. Furthermore, we examined the expression distribution of transient receptor potential (TRP) ion channels, including TRPM2, TRPC5, and TRPA1, which can detect changes in cellular redox state and mediate calcium influx. The results showed that only TRPM2 was preferentially enriched in the fragile DAn1 clusters. Figure 1E To further validate this finding, we also analyzed TRPM2 expression based on three other public databases. Midbrain TRPM2 expression profiles obtained from the Human Protein Atlas dataset (www.proteinatlas.org) showed that, regardless of sex, TRPM2 expression in the SNc region was higher than that in the VTA region. Figures 2A-2C We also analyzed TRPM2 expression in SNc neurons from single-cell transcriptome maps of PD patients. The results showed that TRPM2 expression was positively correlated with aging in PD patients. Figure 1F and Figure 1G We also found that Girk2-positive neurons and TRPM2, but not Calbindin neurons, were more abundant in this fragile cluster of DAn1 neurons. Figure 1I-Figure 1K To validate these RNA-seq-based data, we also examined TRPM2 expression in the mouse midbrain using RNA-scope. The results showed that TRPM2 mRNA levels were indeed significantly higher in the ventral layer of SNc DA neurons in aged mice than in the VTA layer. Figure 1L and Figure 1M This is consistent with the results based on RNA-seq mentioned above. Therefore, TRPM2 may be involved in mediating the susceptibility of DA neurons.

[0269] Example 2: Ectopic expression of hTRPM2 in nematodes induces selective dopaminergic neuronal degeneration.

[0270] No homolog of human TRPM2 exists in *C. elegans*, and previous studies have not observed preferential loss of DA neurons in various neurodegeneration models of *C. elegans* carrying gene mutations, α-synuclein overexpression, or toxin treatment (Chikka et al., 2016; Lakso et al., 2003; Nagarajan et al., 2014). To investigate whether human TRPM2 truly confers susceptibility to DA neurons in *C. elegans*, in this embodiment, the inventors constructed a *C. elegans* strain with TRPM2 that independently and specifically expresses human TRPM2 and DsRed fluorescent protein in DA neurons under the control of the Pdat-1 promoter. Figures 3A-3D Interestingly, ADE, PDE, and CEP neurons exhibit completely different phenotypes in TRPM2 expression. In TRPM2-expressing nematodes, both ADE and PDE neurons show neurodegeneration, characterized by gradual neuronal loss within 7 days from the first day of adulthood. Figures 4A-4B as well as Figure 3E and Figure 3F Conversely, even on days 7 and 10 of adulthood, CEP neurons from the same human TRPM2-expressing nematode remained unaffected. Figure 4A and Figure 4B as well as Figure 3G Therefore, only ADE and PDE in *C. elegans* are susceptible to TRPM2 expression. Since age-related increases in gut autofluorescence severely interfere with the quantification of PDE neurons, we will focus on ADE and CEP neurons in our subsequent studies.

[0271] To determine whether the selective susceptibility of DA neurons caused by hTRPM2 expression in *Caenorhabditis elegans* is due to functional changes in hTRPM2 channels in ADE and CEP neurons, we performed in vivo electrophysiological recordings of hTRPM2-mediated currents using saturation concentrations of ADPR and Ca2+ (see Singatulina et al., 2019; Vyas et al., 2014 for specific methods). ADE and CEP neurons in the transgenic nematode expressing hTRPM2 responded to ADPR / Ca2+ with similar current amplitudes. Figure 3H and Figure 3I This indicates that TRPM2 expression levels are similar in ADE and CEP neurons. Compared to WT-hTRPM2 expression, both integrated hTRPM2 mutants carrying the E960Q mutation in the pore region and the R1433A mutation in the C-terminal ADPR binding pocket significantly reduced ADE neuron death. Figures 4C-4EFurthermore, the expression of TRPM2 in these two mutant strains was similar, comparable to the intensity of DsRed, indicating that the selective induction of ADE-DA neuronal degeneration by ectopic TRPM2 expression depends on its channel activity.

[0272] We also treated nematodes with 2 mM MPP+, as MPTP can inhibit complex I of the electron transport system, thereby impairing mitochondrial function (see Nicklas et al., 1987). The results showed that, compared with WT, MPP+ treatment led to increased ADE death in TRPM2 nematodes. Figure 3J CEP neurons showed similar toxic effects after MPP+ treatment. Figure 3J These results indicate that it is ADE, rather than CEP, that carries the neuronal susceptibility.

[0273] Since Ca2+ and reactive oxygen species (ROS) are the main ligands for TRPM2 activation, to verify the role of Ca2+ in the hTRPM2-induced ADE neuronal degeneration pathway, we used GCaMP3 to measure the cytoplasmic Ca2+ levels of ADE and CEP neurons on day 1. The results showed that in hTRPM2-induced nematodes, the cytoplasmic Ca2+ level of ADE neurons was significantly higher than that of CEP neurons. Figure 5A The application of the Ca2+ chelator BAPTA-AM significantly inhibited ADE neurodegeneration in hTRPM2 nematodes. Figure 5C and Figure 5D This further demonstrates the role of Ca2+ overload in hTRPM2-induced ADE neuronal degeneration. Next, we examined ROS levels, which not only induce TRPM2 activation but also contribute to neuronal damage during pathogenesis. Using a ROS sensor, we observed that ROS levels in hTRPM2 nematode ADE neurons were significantly higher than in CEP neurons. Figure 5B Furthermore, treatment with the ROS scavenger n-acetylcysteine ​​(NAC) slightly inhibited the death of ADE neurons in hTRPM2 nematodes. Figure 5C and Figure 5D In summary, this indicates that the selective susceptibility of ADE neurons to hTRPM2 expression is partly due to elevated Ca2+ and ROS levels, which activate hTRPM2 and drive neuronal death.

[0274] Example 3: TRPM2 is involved in the susceptibility of mouse SNc DA neurons to stress.

[0275] To further verify the role of TRPM2 in the susceptibility of mammalian dopamine neurons, in this embodiment, the inventors used a mouse PD model induced by MPTP and rotenone (Rot). Our results indicate that MPTP treatment induces selective loss of SNc neurons, but not VTADA neurons, in WT mice. Figures 6A-6B Similar to our Celegans data, TRPM2 deficiency (TRPM2 knockout mice, TRPM2-KO) significantly suppressed MPTP-induced loss of mouse SNc DA neurons. Figures 6A-6B This confirms that TRPM2 is crucial in mediating DA neuron susceptibility. Furthermore, we found that stereotactic injection of rotenone to one side of the SNc induced apomorphine-induced rotation and reduced rotarod time in mice. Figures 6C-6E This suggests that TRPM2 alleviates PD-like behaviors and has a neuroprotective effect. Furthermore, in the WT group, progressive degeneration of dopaminergic neurons was observed in the striatum one month after injection. Figure 6C , Figure 6F and Figure 6G Consistently, these motor deficits and pathological changes were significantly reduced in TRPM2 ko mice. Figures 6D-6G These results further support the idea that TRPM2 mediates SNc DA neuron death. These findings confirm that the susceptibility of TRPM2-mediated SNc DA neurons is conserved across different species.

[0276] Example 4: TRPM2 induces ADE death through FZO-1 / ced-9 mediated mitochondrial hyperfusion.

[0277] In this embodiment, the inventors investigated whether hTRPM2 induces ADE neuronal death through a core apoptosis mechanism, including EGL-1, CED-9 (anti-apoptotic), CED-4, and CED-3 ( Figure 7A (Ellis and Horvitz, 1986). Researchers crossed hTRPM2 nematodes with mutants possessing loss-of-function ced-4(n1162) / apoptotic protease activator-1 (Apaf-1), egl-1(n1084n3082) / BH-3, ced-3(n717) / caspase or gain-of-function ced-9(n1950) / Bcl-2, which have been shown to prevent typical apoptosis (Hengartner and Horvitz, 1994). Among these mutants, only the loss-of-function ced-4 in hTRPM2 nematodes significantly reduced ADE neuron death ( Figure 7B and Figure 7C as well as Figure 5E and Figure 5FSurprisingly, the loss-of-function ced-9(n2812) mutant significantly suppressed ADE neuron degeneration in hTRPM2 nematodes, while the gain-of-function ced-9 mutant failed to protect ADE neurons. Figure 7B and Figure 7C These results indicate that hTRPM2 induces ADE neuronal death through ced-4 and ced-9, but not EGL-1 and CED-3.

[0278] Mitochondrial dysfunction is a crucial initiating factor for apoptosis and plays a significant role in stress-induced dopamine neurodegeneration. The Bcl-2 family of proteins, located in mitochondria, has been reported to play a key role in regulating mitochondrial dynamics (Lu et al., 2011; Rolland et al., 2009). Next, we explored the role of CED-9 by further investigating its contribution to mitochondrial dysfunction in ADE neurons of hTRPM2 nematodes. Using confocal microscopy, we found increased mitochondrial length in ADE neurons compared to CEP neurons in both TRPM2 and wild-type nematodes. Figure 7D and Figure 7E Three-dimensional reconstruction analysis of mitochondrial morphology based on scanning electron microscopy (SEM) results further confirmed this finding. Figure 7D This indicates a defect in mitochondrial excessive fusion or division in ADE neurons expressing hTRPM2. Furthermore, the loss-of-function ced-9 (n2812) mutation restored the normal mitochondrial morphology of hTRPM2-expressing nematodes. Figure 7E and Figure 7F This study confirmed that ced-9 is involved in hTRPM2-induced mitochondrial fusion / division defects in ADE neurons.

[0279] Previous studies have reported that in *C. elegans*, CED-9 promotes mitochondrial division through interaction with EGL-1 and DRP-1, or mediates mitochondrial fusion through interaction with CED-4 and FZO-1, but this remains controversial (Lu et al., 2011; Roland et al., 2009). This is because one study showed that CED-9 is not involved in mitochondrial dynamics (Breckenridge et al., 2009). In this study, our data indicate that when the EGL-1 mutant is hybridized with *hTRPM2* nematode, there is no protective effect. Figure 5E and Figure 5F This indicates that EGL-1 / CED-9-mediated mitochondrial division is not involved in ADE-related neuronal death. Conversely, knockdown of fzo-1, but not drp-1, alleviated ADE neuronal degeneration in hTRPM2 transgenic nematodes. Figure 7G and Figure 7HConsistently, expression of the fzo-1 ced-9 binding domain (AA346-595, containing a coiled-coil domain) also alleviated ADE neuronal degeneration in hTRPM2 nematodes. This may be due to competitive blocking of FZO-1 / CED-9-mediated mitochondrial fusion (…). Figure 7G and Figure 7H Therefore, our data suggest that mitochondrial hyperfusion mediated by the FZO-1 / CED-9 complex plays a crucial role in hTRPM2-induced ADE neuronal degeneration.

[0280] It has been reported that excessive mitochondrial fusion leads to mitochondrial permeability transition (MPT) (Papanicolaou et al., 2011), resulting in cell death. Therefore, we also investigated whether MPT is involved in TRPM2-mediated neuronal death. In *Caenorhabditis elegans*, deletion of the cyn-1 gene encoding a component of MPT regulation reduced ADE neuronal death in TRPM2-mediated nematodes. Figure 5E and Figure 5F Blocking the opening of the MPT pore with cyclosporine A (CsA) also improved the activity of ADE neurons. Figure 5C and Figure 5D These results indicate that the formation of the FZO-1 / CED-9 complex driven by TRPM2 activation further induces excessive mitochondrial fusion, and that MPT contributes to the death of ADE neurons in hTRPM2 nematodes.

[0281] To further determine whether the role of the FZO-1 / CED-9 complex in mediating dopaminergic neuronal death in mammals is conserved, we analyzed the interaction of Mfn2 / Bcl-2 (a mouse homolog of FZO-1 / CED-9) in an MPTP-treated mouse model. Using the proximity connection assay (PLA), we found that TRPM2 deficiency significantly attenuated MPTP-induced Mfn2 / Bcl-2 interaction in SNc region dopaminergic neurons, but not in VTA DA neurons. Figure 7I and Figure 7J This supports the view that TRPM2-induced Mfn2 / Bcl-2 interaction is crucial for selective SNc DA neuron death. These results further confirm that TRPM2 activation-mediated Mfn2 / Bcl-2 interaction does indeed conserve the susceptibility of mouse SNc DA neurons during stress.

[0282] Example 5: PARP-1 / PARG-1 / ADPR axis-mediated TRPM2 activation induces ADE-selective cell death

[0283] The results of the above embodiments indicate that human TRPM2 plays a novel role in dopamine neuron susceptibility. Therefore, in this embodiment, the inventors further investigated how TRPM2 selectively mediates ADE neuronal death using TRPM2-transgenic nematodes. ADPR is the main endogenous ligand of the TRPM2 channel, which is primarily synthesized by polyADPR polymerase-1 (PARP-1) and polyADPR glycolytic enzyme (PARG) in response to DNA damage. Figure 8A (Qi et al., 2019; Vyas et al., 2014). To determine whether preferential activation of TRPM2 is responsible for ADE death, we first investigated the potential roles of PARP-1 and PARG in hTRPM2-induced selective ADE neuronal death. Overexpression of nematode PARP-1 in DA neurons led to ADE cell death in WT animals and exacerbated ADE neuronal death in hTRPM2-induced animals (Qi et al., 2019; Vyas et al., 2014). Figure 8B -C). Notably, it also led to significant CEP neuron death ( Figures 8B-8C Similarly, knockdown of parp-1 or parg-1 significantly inhibited the death of ADE neurons in hTRPM2 animals. Figures 8B-8C Furthermore, treatment with the PARP-1 inhibitor PJ-34 (Szabo et al., 2002) or the PARP-1 inhibitor PDD (Raghunatha et al., 2020) both reduced the death of ADE neurons in hTRPM2 animals. Figure 8B and Figure 8D These results support the crucial role of TRPM2 activation in DA neuronal susceptibility. Currently, there are no probes for monitoring ADPR levels. To explore whether ADPR generation plays a role in TRPM2-mediated ADE death, we overexpressed nematode NDX-6 (a homolog of human NUDT9), which hydrolyzes ADPR to adenosine monophosphate (AMP) and 5'-phosphate ribose (AMP). Figure 8A The results showed that NDX-6 overexpression reduced ADE death in hTRPM2 nematodes. Figure 8B and Figure 8C This suggests the importance of ADPR generation in TRPM2-mediated ADE neuron death. In summary, these results support the importance of the PARP-1 / PARG-1 / ADPR axis for the vulnerability of hTRPM2-expressing ADE neurons in nematodes.

[0284] Example 6: In SPD patients, ipsc-derived dopamine neuron death is also mediated by TRPM2.

[0285] To further determine whether the role of TRPM2 in mediating dopamine susceptibility in *C. elegans* and mice is retained in the pathogenesis of Parkinson's disease, in this embodiment, the inventors utilized dopamine neurons differentiated from iPSCs, a mature cell model used in Parkinson's disease research. Employing a basal plate-based midbrain DA neuron differentiation strategy, we differentiated iPSCs from sporadic PD (SPD) patients and healthy controls into TH-positive DA neurons with an efficiency of approximately 80%. Figures 9A-9C These differentiated iPSC-DA neurons also express Girk2, while calcium-binding proteins are barely detectable. Figure 9B Evoked action potentials and voltage-dependent sodium channel currents were detected in differentiated DA neurons through whole-cell recording. Figure 9D and Figure 9E ).

[0286] Importantly, treatment with the TRPM2 inhibitors ACA or A10 improved the viability of SPD iPSC-derived DA neurons. Figure 10A This indicates that TRPM2 function promotes DA neuron death in our iPSC model. We then used a series of inhibitors to determine the role of the PARP-1 / PARG / ADPR axis in the viability of SPD ipsc-derived DA neurons. As expected, inhibiting PARP-1 with PJ-34 treatment reduced ADPR levels, while inhibiting PARG with PDD treatment increased ADPR levels. Figure 11A Both PJ-34 (a parp-1 inhibitor) and PDD (a PARG inhibitor) significantly improved the activity of DA neurons derived from SPDipsc. Figures 10B-10C Consistent with our results from the nematode PD model, we observed Ca2+ overload in DA neurons from SPD patient-specific iPSCs. Figure 11A and Figure 11C Treatment with inhibitors of the PARP-1 / PARG / TRPM2 signaling axis (including PJ-34, PDD, ACA, or A10) partially alleviated this condition. Figure 11D and Figure 11E We also observed insufficient mitochondrial fusion in iPSC-derived DA neurons from SPD patients, but not in healthy controls. The aforementioned inhibitors again treat PD through mitochondrial mechanisms. Figures 10D-10E These results demonstrate the role of the PARP-1 / PARG / ADPR axis in triggering TRPM2 and its downstream Ca2+ toxicity and mitochondrial excessive fusion-mediated DA neuronal death in human SPD cell models.

[0287] We further investigated whether the TRPM2 / FZO-1 / CED-9 axis is also conserved in ipsc-derived DA neurons of SPD patients and whether it participates in excessive mitochondrial fusion. It has been reported that CED-9 interacts with the coiled-coil domain of FZO-1 (Lu et al., 2011; Roland et al., 2009). We compared the *C. elegans* FZO-1 sequence with human Mfn2 and synthesized peptides corresponding to the coiled-coil domain (aa391–434) of human Mfn2, as shown in Table 1 below. Figure 12A Using PLA experiments, we found that PEP treatment strongly inhibited the interaction between Mfn2 and Bcl-2 in SH-SY5Y cells under basal conditions. Figure 12B and Figure 12C MPP+-treated cells exhibited enhanced Bcl-2 and Mfn2 interaction, manifested as an increase in the number of punctate clusters, which was inhibited by PEP treatment. Figures 12B-12C This is consistent with the effect of PEP on salvaging neuronal viability observed in ipsc-derived DA neurons in SPD patients. Figure 12D ), excessively fused mitochondrial morphology ( Figures 10D-10E ), downregulate Mfn2 / Bcl-2 interaction ( Figures 10F-10G The effects are consistent with those of intracellular Ca2+. Figure 11D and Figure 11E Compared with healthy controls, in DA neurons derived from ipsc in SPD patients, both PARP-1 and PARG activities significantly inhibited the interaction between Mfn2 and Bcl-2. Figures 10F-10G Therefore, our results from ipsc-derived dopamine neurons in SPD patients are consistent with those from nematode and mouse models, further supporting the role of Mfn2 / bcl-2-mediated mitochondrial hyperfusion in cross-species dopamine neuronal death.

[0288] Table 1

[0289]

[0290]

[0291] discuss

[0292] In this study, we provide clear evidence that TRPM2 plays a crucial role in mediating DA neuron susceptibility through a comprehensive cross-model analysis. We have demonstrated that TRPM2 and its novel associated signaling pathways contribute to ROS- and calcium-dependent mitochondrial hyperfusion, leading to selective DA neuron degeneration in Caenorhabditis elegans and mouse models, as well as dysfunction of iPSC-derived DA neurons in PD patients. We also reveal that both direct inhibition of TRPM2 and its associated signaling pathways conserve mitochondrial function, thereby contributing to the survival of susceptible DA neurons under stress challenges. Figure 13 These data suggest that TRPM2 acts as a key executor in susceptible DA neurons, tightly controlling the fate of DA neurons by sensing ROS and calcium levels.

[0293] In a PD mouse model, our data further confirm that TRPM2 deficiency protects susceptible SNc DA neurons from premature death. Furthermore, TRPM2 inhibitor treatment significantly improved the survival rate of iPSC-derived DA neurons in SPD patients. Therefore, TRPM2 is likely an oxidative stress sensor, and its selective activation via calcium permeability channels may be a key factor contributing to oxidative stress and calcium imbalance in susceptible dopaminergic neurons. Ultimately, this process leads to neuronal degeneration and death caused by persistent oxidative stress and calcium imbalance.

[0294] In our study, we found that both the cyn-1 inactivation mutation and the MPT pore blocker CsA strongly inhibited the death of ADE neurons in TRPM2 nematodes. The opening of the MPT is associated with neuronal death through mechanisms including excitotoxicity, neurotoxicity, apoptosis, and necrosis (Bonora et al., 2022; Perez-Pinzon et al., 2012).

[0295] Therefore, our findings suggest that TRPM2 is selectively activated by ROS and calcium during ADE, which further promotes CED-9 / CED-4-induced MPT, ultimately leading to ADE death in TRPM2 nematodes.

[0296] In the TRPM2-related pathway, loss of function of CED-4, rather than CED-3 or EGL-1, partially alleviated ADE neuron death in TRPM2-associated nematodes. Loss of function of CED-9, rather than enhancement, significantly suppressed ADE neuron death, suggesting that CED-4 and CED-9 mediate a cell death pathway. Our findings are the first to demonstrate a non-classical apoptosis pathway mediated by CED-4 / CED-9 under specific stress conditions.

[0297] Example 7: TRPM2 switching to CED-9(Bcl-2) / FZO-1(Mfn-2) composite enhances mitochondrial hyperfusion responsible for DA neuron susceptibility.

[0298] Using fluorescent probes and three-dimensional electron microscopy, we found an increase in hyperfusion mitochondria in TRPM2 nematodes, a phenomenon not observed in wild-type nematodes. Furthermore, FZO-1 knockout, rather than DRP-1 knockout, significantly reduced ADE neuron death in TRPM2 nematodes, confirming that TRPM2 promotes FZO-1-mediated hyperfusion. We observed that mitochondrial hyperfusion in TRPM2 nematodes can also cross-link with TRPM2 nematodes that are CED-9 inactivated or express a CED-9 binding domain in their DA neurons, thereby reducing Bcl-2 / Mfn2 interactions, supporting the role of CED-9 as a switch promoting mitochondrial fusion. This contradicts the findings of those skilled in the art who believe that CED-9 is not involved in regulating mitochondrial dynamics in *C. elegans* (Breckenridge et al., 2009).

[0299] We also observed mitochondrial hyperfusion in iPSC-derived DA neurons from SPD patients compared to healthy individuals, and that PARP-1 / PARG / ADPR antagonists could reverse mitochondrial hyperfusion in iPSC-derived DA neurons from SPD patients. More importantly, by intervening in the Mfn2 / Bcl-2 interacting peptide to disrupt mitochondrial hyperfusion in PD patient-specific iPSC-derived DA neurons, we also found increased Bcl-2 / Mfn2 interaction in the SNc region of PD mice in the MPTP mouse model, rather than in the control group. Furthermore, TRPM2 deficiency alleviated the Bcl-2 / Mfn2 interaction in susceptible SNc DA neurons of PD mice.

[0300] The data above suggest that antagonizing the PARP-1 / PARG / ADPR pathway, the interaction between Mfn2 and Bcl-2 can treat PD.

[0301] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. A peptide derived from an isolated polypeptide, characterized in that, The amino acid sequence of the derivative peptide of the polypeptide is shown in SEQ ID NO:

34.

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a peptide derived from the polypeptide as described in claim 1 and a pharmaceutically acceptable carrier.

3. Use of the polypeptide derivative of claim 1 in the preparation of a medicament for treating Parkinson's disease.

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