Polypeptide for treating Parkinson's disease

Abstract

The invention discloses a polypeptide for treating Parkinson's disease. In the application, it is found for the first time that a transient receptor potential M2 type (TRPM2) channel is expressed more in a fragile DA neuron subgroup, the expression quantity of the TRPM2 channel is positively correlated with age in a Parkinson's disease (PD) patient, and an action mechanism is defined through strict experiments, that is, the TRPM2 channel is preferentially activated in an ADE neuron through a PARP-1 / PARG / ADPR axis to drive Mfn2 / Bcl-2 complex-dependent mitochondrial hyper-fusion, so that the expression quantity of the TRPM2 channel is positively correlated with age; the ADE neuron death is caused. In addition, the TRPM2 channel also mediates the susceptibility of SNc DA neurons in the mouse treated by MPTP through Mfn2 / Bcl-2. It is found for the first time that blocking of PARP-1 / PARG / ADPR and Mfn2 / Bcl-2 mediated DA neuron death pathways can prevent DA neuron death of a specific induced pluripotent stem cell (iPSC) source of a patient suffering from the spontaneous Parkinson's disease (SPD). As susceptible SNc DA neuron loss is a main symptom of PD, PARP-1 / PARG / ADPR and Mfn2 / Bcl-2 pathways may become new therapeutic targets for PD.

Description

Technical Field

[0001] The present invention relates to biomedicine, and in particular to a polypeptide for treating Parkinson's disease. Background Art

[0002] Dopaminergic (DA) neurons play an important 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 region of the midbrain during embryonic development. Midbrain DA neurons can be divided into three anatomically and functionally distinct clusters, namely the distal red nucleus (RR), substantia nigra pars compacta (SNc), and ventral tegmental area (VTA), of which the SNc and VTA contain most DA neurons (Anderegg et al., 2015). Caenorhabditis elegans (C. elegans) is a classic model animal commonly used in the study of human diseases. Similarly, DA neurons in nematodes are composed of three anatomically distinct subtypes, including two lateral forebrain neurons (ADE), four head sensory neurons (CEP), and two lateral hindbrain neurons (PDE). These three ciliated DA neuron subtypes play redundant roles in regulating deceleration behavior in response to food and mechanosensation (Sawin et al., 2000). Thus, the diversity among DA neuron subpopulations is conserved from model animals to humans.

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

[0004] Studies over the past few decades have suggested that the selective vulnerability of SNc neurons is mainly due to their huge bioenergetic demands, such as pacemaker activity (Surmeier, 2007), high DA content and iron-related toxicity (Hare and Double, 2016; Segura-Aguilar et al., 2014), and complex axonal branches (Bolam and Pissadaki, 2012; Matsuda et al., 2009; Pacelli et al., 2015; Parent and Parent, 2006). However, the molecular mechanisms mediating the differential susceptibility of DA neuron subpopulations are largely unknown. Therefore, the mechanism of action of DA neurons mediating susceptibility needs to be studied in this field, which has great guiding significance for the treatment of Parkinson's disease. Summary of the invention

[0005] The object of the present invention is to provide a polypeptide.

[0006] Another object of the present invention is to provide a derivative peptide of the 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 a PARG inhibitor and / or a drug capable of inhibiting the formation of a complex between Mfn2 and Bcl-2.

[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 the coiled-coil domain (aa391-434) of Mfn2.

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

[0013] (i) the amino acid sequence 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) has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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 one of the following:

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

[0019] (ii) a sequence formed by substitution, deletion or addition of one or more amino acids in SEQ ID NOs: 1-33;

[0020] (iii) has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NOs: 1-33.

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

[0022] (i) the amino acid sequence 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) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0025] The second aspect of the present invention provides a derivative peptide of the polypeptide described in the first aspect of the present invention, wherein the derivative peptide of the polypeptide comprises the polypeptide described in the first aspect of the present invention and a functional peptide connected to the polypeptide, wherein the functional peptide is connected to the N-terminus and / or C-terminus of the above-mentioned 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 cell-penetrating peptides.

[0028] In some preferred embodiments, the cell-penetrating peptide is connected to the N-terminus and C-terminus of the above-mentioned polypeptide.

[0029] In some preferred embodiments, the cell-penetrating peptide is a pH-responsive cell-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 connected to Biotin or its derivative peptide; and the C-terminus of the derivative peptide of the polypeptide is connected 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] The third aspect of the present invention provides a pharmaceutical composition, which comprises 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; and a pharmaceutically acceptable carrier.

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

[0037] The fifth aspect of the present invention provides the use of the polypeptide of the first aspect of the present invention or the derivative peptide of the polypeptide of the second aspect of the present invention for:

[0038] (i) inhibiting DA neuron death or enhancing DA neuron activity;

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

[0040] (iii) inhibiting the formation of the Mfn2 / Bcl-2 complex or downregulating the interaction between Mfn2 and Bcl-2 or acting as an Mfn2 / Bcl-2 inhibitor;

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

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

[0043] (vi) preparing a medicament for preventing and / or treating Parkinson's disease; and / or

[0044] (vii) Inhibition of TRPM2 activation.

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

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

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

[0048] (i) inhibiting DA neuron death or enhancing DA neuron activity;

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

[0050] (iii) Inhibition of Ca2+ in DE neurons 2+ and increased ROS levels.

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

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

[0053] (vi) preparing a medicament for preventing and / or treating Parkinson's disease.

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

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

[0056] In some preferred embodiments, the susceptibility of DA neurons is reduced by selectively reducing the susceptibility of ADE neurons. The selective reduction of the susceptibility of ADE neurons is achieved by inhibiting Ca 2+ and an increase in ROS levels was achieved.

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

[0058] (i) inhibiting DA neuron death or enhancing DA neuron activity;

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

[0060] (iii) Inhibition of Ca2+ in DE neurons 2+ and increased ROS levels.

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

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

[0063] (vi) preparing a medicament for preventing and / or treating Parkinson's disease.

[0064] In some preferred embodiments, the PARP-1 inhibitor is represented by the following formula I:

[0065]

[0066] The ninth aspect of the present invention provides the use of a PARG inhibitor for:

[0067] (i) inhibiting DA neuron death or enhancing DA neuron activity;

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

[0069] (iii) Inhibition of Ca2+ in DE neurons 2+ and increased ROS levels.

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

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

[0072] (vi) preparing a medicament for preventing and / or treating Parkinson's disease.

[0073] In some preferred embodiments, the PARG inhibitor is represented by the following formula II:

[0074]

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

[0076] (1) In the present invention, it was discovered for the first time that transient receptor potential M2 (TRPM2) channels are more highly expressed in vulnerable DA neuron subpopulations, and their expression levels are positively correlated with age in Parkinson's disease (PD) patients. The mechanism of action was clarified through rigorous experiments, namely, TRPM2 channels are preferentially activated in ADE neurons through the PARP-1 / PARG / ADPR axis, driving FZO-1 / CED-9 complex-dependent mitochondrial superfusion, leading to ADE neuron death. In addition, TRPM2 channels also mediate the susceptibility of SNc DA neurons in MPTP-treated mice through Mfn2 / Bcl-2, the mammalian homolog of nematode FZO-1 / CED-9.

[0077] (2) In the present invention, it was found for the first time that blocking the 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 neurons specific to patients with spontaneous Parkinson's disease (SPD). In summary, these findings indicate that these pathways have 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 the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0080] Figure 1A It is the UMAP of the isolated cells after -ipsc-dopaminergic neuron differentiation according to the embodiment of the present invention;

[0081] Figure 1B is the percentage of different neuronal clusters in the ctrl group and the rotenone group according to an embodiment of the present invention;

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

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

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

[0085] Figure 1F It is the expression of neuronal TRPM2 in the single-cell transcriptome data of SNc of PD patients in an embodiment of the present invention.

[0086] Student's t-test;

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

[0088] Figure 1H It is a UMAP display of 10 transcriptionally distinct subpopulations based on snRNA-seq of human SNc DA neuron nuclei according to an embodiment of the present invention. The pink marker is the highly vulnerable DA neuron cluster "SOX6_AGTR1";

[0089] Fig. 1I According to the embodiment of the present invention) scatter plot and box plot show the expression of KCNJ6 (Girk2);

[0090] Figure 1J The scatter plot and box plot show the expression of Calbindin (CALB1) according to the embodiment of the present invention;

[0091] Figure 1K The expression of TRPM2 is shown in the scatter plot and box plot according to the embodiment of the present invention;

[0092] Figure 1L Representative fluorescent RNA-scope images of TRPM2 (red dots) expression in TH+ (green) neurons in the ventral layer of the SNc and VTA regions of 10-week and 40-week mice according to the embodiment of the present invention (L). Blue is DAPI. Scale bar = 20 μm;

[0093] Figure 1M The mRNA of (M) TRPM2 was quantified according to the number of red dots per TH-positive neuron according to the embodiment of the present invention. Two-way ANOVA was performed using the Holm-Sidak multiple comparison test (n=3). The average number of dots per mouse was calculated from 6 interval brain slices (each 6 slices spanned the entire SNc from -2.7mm to -4.04mm from front to back);

[0094] Figure 2A is the expression of human TRPM2 in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) regions from the human protein atlas database according to an embodiment of the present invention, the RNA expression of TRPM2 is represented by the consistent normalized expression ("nTPM") of the SNc and VTA regions, unpaired t test;

[0095] Figure 2B The expression of human TRPM2 in the SNc and VTA regions in the human Protein Atlas database according to the embodiment of the present invention was analyzed by two-way ANOVA using the Holm-Sidak multiple comparison test;

[0096] Figure 2C is the age of the sample according to the embodiment of the present invention;

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

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

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

[0100] Figure 3D It is the genotyping and identification of the hTRPM2-integrated transgenic nematodes according to the embodiments of the present invention;

[0101] Figure 3E Representative images from an example of the present invention show PDE neurons of wild-type and htrpm2-expressing nematodes at day 7 of adulthood. Scale bar = 20 μm.

[0102] Figure 3F Quantification of PDE neurons in WT and htrpm2 expressing nematodes according to the examples of the present invention. P values ​​were tested using the Mann-Whitney test.

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

[0104] Figure 3H ADPR (1 mM) and Ca2+ (10 μM) induce TRPM2 currents in ADE and CEP neurons of WT and htrpm2-expressing nematodes (adult day 2) according to the examples of the present invention. (A) Statistical analysis of currents in panel h. Data are presented as mean ± sem. n = 4-7. Two-way ANOVA was performed using the Holm-Sidak multiple comparison test. Fig. 3I Schematic diagram of the changes in PA values ​​of ADE and CEP neurons in WT and htrpm2-expressing nematodes according to an embodiment of the present invention;

[0105] Figure 3J Quantification of ADE and CEP neurons in WT and TRPM2 worms treated with 2 mM MPP for 7 days according to an example of the present invention. P values ​​are from two-way ANOVA with Holm-Sidak multiple comparison test.

[0106] Figure 4A Representative confocal images of ADE and CEP dopaminergic neurons in WT and hTRPM2-expressing nematodes at day 1, day 4, and day 7 of adulthood according to an embodiment of the present invention. Scale bar = 20 μm;

[0107] Figure 4B is the quantification of ADE and CEP dopaminergic neurons in WT and hTRPM2 animals according to the examples of the present invention. Error bars represent mean ± sem. P values ​​are from two-way ANOVA with Holm-Sidak multiple comparison test;

[0108] Figure 4C Schematic diagram of the hTRPM2 channel structure according to an embodiment of the present invention. The inactivating E960Q mutation is located in the pore region between the transmembrane (TM) segments S5 and S6. The inactivating mutation R1433A is located in the C-terminal ADPR binding domain;

[0109] Figure 4D are representative confocal images of ADE and CEP DA neurons in mutant hTRPM2-expressing nematodes according to an embodiment of the present invention;

[0110] Figure 4E Quantification of ADE dopaminergic neurons in TRPM2, TRPM2(E960Q) and TRPM2(R1433A) animals according to the embodiments of the present invention. Error bars represent mean ± sem. P values ​​are from Dunnett's multiple comparison test after one-way ANOVA;

[0111] Figure 5A 1 is a bar graph of the cytosolic Ca2+ of different transgenic strains measured using CaMP3 and superprobe according to the embodiment of the present invention. The P value is from the one-way ANOVA with Dunnett's multiple comparison test.

[0112] Figure 5B 1 is a bar graph of the determination of cytosolic ROS in different transgenic strains using CaMP3 and superprobe according to an embodiment of the present invention. The P value is from one-way ANOVA with Dunnett's multiple comparison test.

[0113] Figure 5C It is a typical confocal image according to an embodiment of the present invention, showing the effect of 100 μM BAPTA-AM, 1 mM NAC and 1 mM CsA on htrpm2-expressing animals. Scale bar = 20 μm.

[0114] Figure 5D Quantification of ADE neurons in the experiment of panel A according to the embodiment of the present invention. One-way ANOVA was performed on the P value using Kruskal-Wallis test.

[0115] Figure 5E Representative confocal images of the effects of egl-1(n1086n3082) and cyn-1(tm4171) mutations on htrpm2-expressing animals according to the examples of the present invention. Scale bar = 20 μm.

[0116] Fig. 5F It is the quantification of ADE neurons in the experiment of group C according to the embodiment of the present invention. The P value was subjected to one-way ANOVA using Kruskal-Wallis test.

[0117] Fig. 6A Figure 1 is the dopaminergic neurons of WT and TRPM2 knockout mice after MPTP or solvent treatment according to the embodiments of the present invention. Confocal images show the expression of TRPM2 in TH-positive DA neurons in the SNc and VTA regions. Scale bar = 50 μm; Scale bar for magnified views = 20 μm;

[0118] Figure 6B is the quantification of TH-positive neurons in the SNc region according to the embodiment of the present invention. Scale bar = 50 μm or 200 μm. Two-way ANOVA was performed using the Holm-Sidak multiple comparison test. Data are expressed as mean ± sem n = 6-9;

[0119] Figure 6C is a schematic diagram of a rotenone-treated PD mouse model and an experimental design according to an embodiment of the present invention;

[0120] Fig.6D TRPM2 knockout reduces apo-induced rotational behavior at week 1 and week 2 after Rot treatment according to the present invention. Data are presented as mean ± sem, n = 9-13. Two-way ANOVA was performed using the Holm-Sidak multiple comparison test;

[0121] Fig. 6E The right side is the result of the rotarod test of PD mice treated with rot according to the embodiment of the present invention. The data are expressed as mean ± sem. n = 9-13. Two-way ANOVA was performed using the Holm-Sidak multiple comparison test;

[0122] Fig. 6F Representative confocal images of TH-immunoreactive (TH+) fibers in the striatum of four groups after Rot treatment according to the present invention. The magnified area is 20 μm. The scale bar of the central image = 1000 μm;

[0123] Figure 6G It is the quantification 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 ± sem n = 5,7. Two-way ANOVA was performed using the Holm-Sidak multiple comparison test;

[0124] Fig. 7A It is the key regulatory gene for apoptosis of Caenorhabditis elegans according to the embodiment of the present invention, and the red one is the human homologous gene;

[0125] Figure 7B 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 the embodiments of the present invention. Scale bar = 20 μm;

[0126] Figure 7C It is the quantification of ADE neurons in the experiment of group B according to the embodiment of the present invention. One-way ANOVA and Dunnett's multiple comparison test;

[0127] Fig.7D The following are representative confocal images, scanning electron microscope images, and scanning electron microscope 3D reconstruction images of mitochondria of ADE and CEP neurons of wild-type and hTRPM2-expressing animals according to the embodiments of the present invention. Confocal images: scale bar = 2 μm. SEM: scale bar = 500 nm. 3D scanning electron microscope: scale bar = 1 μm;

[0128] Fig. 7E It is a statistical analysis of the mitochondrial major axis / minor axis ratio based on confocal image data according to an embodiment of the present invention. One-way ANOVA and Dunnett's multiple comparison test;

[0129] Figure 7F The confocal images according to the embodiments of the present invention show the effect of the ced-9 inactivation mutation on the mitochondrial morphology of ADE and CEP neurons of wild-type and hTRPM2 nematodes. Scale bar = 2 μm;

[0130] Figure 7G The confocal images show the effects of drp-1 RNAi, fzo-1 RNAi, DRP-1 (280-502) overexpression and FZO-1 (346-595) overexpression on WT and hTRPM2 strains according to the examples of the present invention. Scale bar = 20 μm;

[0131] Figure 7H is the quantification of ADE neurons in the experiment of panel G in the embodiment of the present invention. Two-way ANOVA was performed using the Holm-Sidak multiple comparison test;

[0132] Fig.7I is the positive PLA signal (red spots) showing the interaction between Bcl-2 and Mfn2 in WT and TRPM2-knockout mice after MPTP or solvent treatment according to the examples of the present invention. Scale bar = 5 μm;

[0133] Figure 7J is the quantification of the number of PLA spots according to the embodiments of the present invention, which indicate the strength of the interaction of Mfn2 / Bcl-2 in the SNc and VTA regions. Data are expressed as mean ± semn = 4,5. Two-way ANOVA was performed using the Holm-Sidak multiple comparison test;

[0134] Fig. 8A Schematic diagram of key enzymes involved in the synthesis and metabolism of ADPR according to an embodiment of the present invention. TRPM2 channel is activated by co-agonist, ADPR and Ca2+;

[0135] Figure 8B 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 the examples of the present invention. Scale bar = 20 μm;

[0136] Figure 8C It is the quantification of ADE and CEP neurons in the experiment of group B according to the embodiment of the present invention. Two-way ANOVA was performed using the Holm-Sidak multiple comparison test;

[0137] Fig.8D is the quantification of ADE neurons in Experiment B (PJ-34 and PDD treatment) according to the embodiment of the present invention. One-way ANOVA and Dunnett's multiple comparison test;

[0138] Fig. 9A It is the immunofluorescence of iPSC markers OCT-4, Nanog, Tra-1-60 and Sox-2 according to the example of the present invention. Scale bar = 50 μm.

[0139] Fig. 9B This is a micrograph of DA neurons differentiated from iPSCs of healthy controls and SPD patients according to an embodiment of the present invention. 15 days after the in vitro cultured DA neurons matured, the differentiated DA neurons were all TH / MAP2 / Girk2 positive. Scale bar = 20 μm.

[0140] Fig. 9C Quantification of differentiated TH+ neurons relative to DAPI-stained cells of healthy controls and SPD patients according to an embodiment of the present invention. Points represent individual values ​​from independent experiments.

[0141] Fig.9D is a representative trace of 4 s suprathreshold current-evoked action potential discharges recorded from iPSC-derived DA neurons according to an example of the present invention.

[0142] Fig.9E is a voltage clamp recording of sodium current in iPSC-derived DA neurons according to an example of the present invention.

[0143] Fig. 10A The effect of ACA (0.5 μM) and A10 (0.6 μM) treatment on the viability of ipsc-derived DA neurons from SPD patients was detected by CTG according to the embodiment of the present invention. Data are expressed as mean ± sem; P value is from one-way ANOVA with Holm-Sidak multiple comparison test;

[0144] Fig. 10B The effect of PJ-34 (2 μM) treatment on the viability of ipsc-derived DA neurons from SPD patients was detected by CTG according to an embodiment of the present invention. The data are expressed as mean ± sem, and the P value is from the Student's t test;

[0145] Fig. 10C The effect of PDD (1 μM) on the viability of ipsc-derived DA neurons from SPD patients was determined by CTG experiment according to the examples of the present invention. Data are presented as mean ± sem, and P values ​​are from Student's t test;

[0146] Fig. 10D Statistical analysis of the relative length of mitochondrial branches according to the embodiment of the present invention. Data are expressed as mean ± sem; P values ​​are from one-way ANOVA with Dunnett's multiple comparison test;

[0147] Fig.10E Representative confocal images according to the examples of the present invention show the mitochondrial network of PD patient iPSC-derived DA neurons treated with PJ-34, PDD, A10, ACA or PEP. Scale bar = 20 μm;

[0148] Fig.10F The PLA positive signals (red spots) show that the interaction between Bcl-2 and Mfn2 in human iPSC-derived DA neurons was determined by PLA experiments according to the examples of the present invention. Scale bar = 5 μm;

[0149] Figure 10GQuantification of the number of PLA spots in human iPSC-derived DA neurons according to the present invention, which indicates the strength of Mfn2 / Bcl-2 interaction. Data are presented as mean ± sem. One-way ANOVA with Holm-Sidak multiple comparison test;

[0150] Fig.11A The representative Western blot method according to the embodiment of the present invention shows the multi-(ADPR) in the ipsc-derived dopamine neurons of healthy control (WT) and SPD patients, and the effect of PJ-34 (2μM) or PDD (1μM) treating the ipsc-derived dopamine neurons of SPD patients.

[0151] Fig. 11B The Ca2+ levels of iPSC-derived DA neurons from healthy controls and SPD patients were analyzed by flow cytometry according to an example of the present invention.

[0152] Fig. 11C is the normalized intensity of the calcium ion level measured in b according to the quantitative analysis of the embodiment of the present invention. The data are expressed as mean ± sem Student's t test.

[0153] Fig.11D According to the embodiments of the present invention, flow cytometry was used to detect the Ca2+ level of DA neurons derived from ipsc of SPD patients after treatment with PJ-34 (2 μM), PDD (0.5 μM), A10 (0.6 μM), ACA (0.5 μM) or synthetic peptide (PEP, 2 μM).

[0154] Fig.11E is the normalized intensity of calcium ion level measured according to the quantitative analysis d in the embodiment of the present invention. Data are expressed as mean ± sem. One-way ANOVA with Dunnett's multiple comparison test.

[0155] Fig. 12A is a localization map showing the coiled colon domains of nematode FZO-1 and human Mfn2 according to an embodiment of the present invention;

[0156] Fig. 12B Representative confocal images according to the embodiments of the present invention show that the synthetic peptide PEP (10 μM) inhibits the interaction between Bcl-2 and Mfn2 in SH-SY5Y cells after MPP+ or solvent treatment. The red fluorescent dot-like area indicates that PLA detects the interaction between Mfn2 and Bcl-2. Scale bar = 5 μm;

[0157] Fig. 12CIt is the quantification of the red points in Figure b according to the embodiment of the present invention. The data are expressed as mean ± semn = 6-9. Two-way ANOVA was performed using the Holm-Sidak multiple comparison test;

[0158] Fig.12D The viability of iPSC-derived DA neurons from SPD patients and PEP-treated DA neurons was detected by CTG according to the embodiments of the present invention. Data are expressed as mean ± semP values ​​are from one-way ANOVA with Holm-Sidak multiple comparison test;

[0159] Fig.13 Schematic diagram of an experiment in which TRPM2 mediates the vulnerability of DA neurons by sensing ROS and calcium homeostasis disorders according to an embodiment of the present invention. In different models, TRPM2 channels are preferentially activated in vulnerable DA neurons, and PARP-1 / PARG / ADPR activates TRPM2 in 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 DESCRIPTION

[0160] Studies have reported that certain dopaminergic (DA) neuron populations are more vulnerable to stress-induced damage than other subpopulations, and that DA neuron subpopulations have different degrees of susceptibility that is conserved across species, but the molecular mechanisms that mediate the differential susceptibility of DA neuron subpopulations are largely unknown. The inventors have conducted extensive and in-depth studies and found that transient receptor potential M2 (TRPM2) plays a conservative role in mediating DA neuron susceptibility, and for the first time clarified the mechanism by which TRPM2 channels are preferentially activated in ADE neurons through the PARP-1 / PARG / ADPR axis, driving FZO-1 / CED-9 complex-dependent mitochondrial superfusion, leading to ADE neuron death, and found that TRPM2 channels also mediate the susceptibility of SNc DA neurons in MPTP-treated mice through the homologs of nematode FZO-1 / CED-9, Mfn2 / Bcl-2. Since the loss of susceptible SNc DA neurons is a major symptom of PD, new drugs for the treatment of PD can be developed by targeting multiple potential targets in the above-mentioned mechanism.

[0161] Peptides

[0162] The present invention also relates to an isolated polypeptide, which can specifically block the interaction between human Mfn2 and Bcl-2.

[0163] In some preferred embodiments, the polypeptide has the coiled-coil domain (aa391-434) of Mfn2.

[0164] In a preferred embodiment of the present invention, the polypeptide comprises a sequence selected from the group consisting of: (i) an amino acid sequence as shown in SEQ ID NO: 23;

[0165] (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 (activity of binding to the coiled-coil domain of human Mfn2) as SEQ ID NO: 23;

[0166] (iii) having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 23, but retaining the same biological activity as SEQ ID NO: 23 (blocking the interaction between human Mfn2 and Bcl-2).

[0167] In a preferred embodiment of the present invention, the length of the polypeptide is 20-100 bp, preferably 22-63 bp.

[0168] In a preferred embodiment of the present invention, the amino acid sequence of the polypeptide is selected from any one of the following:

[0169] (i) the amino acid sequence shown in SEQ ID NO: 1-33;

[0170] (ii) a sequence formed by substitution, deletion or addition of one or more amino acids in SEQ ID NOs: 1-33;

[0171] (iii) has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NOs: 1-33.

[0172] In a preferred embodiment of the present invention, the amino acid sequence of the polypeptide is selected from any one of the following:

[0173] (i) the amino acid sequence shown in SEQ ID NO: 1; or

[0174] (ii) a sequence formed by substitution, deletion or addition of one or more amino acids in SEQ ID NO: 1;

[0175] (iii) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0176] In the present invention, a sequence formed by substitution, deletion or addition of one or more amino acids in the target sequence and a sequence 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.

[0177] Peptide derivatives

[0178] The present invention also relates to derivatives of the above-mentioned polypeptides, and the polypeptide derivatives include the above-mentioned polypeptides and functional peptides connected to the above-mentioned polypeptides, such as cell-penetrating peptides, and the functional peptides are connected to the N-terminus and / or C-terminus of the above-mentioned polypeptides. In the present invention, the cell-penetrating peptide is a cell-penetrating peptide conventionally described in the art, as long as it can assist in delivering the polypeptide into cells to exert its effect. Generally speaking, the cell-penetrating peptide is a short peptide molecule composed of 10-30 amino acids. In a preferred embodiment of the present invention, the cell-penetrating peptide is a pH-responsive cell-penetrating peptide, such as octaarginine. In a preferred embodiment of the present invention, the functional peptide can also include a tag peptide, and the tag peptide is a Biotin or a derivative peptide thereof. For example, the polypeptide is shown in SEQ ID NO:34:

[0179] SEQ ID NO:34:

[0180] Biotin-EEMREERQDRLKFIDKQLELLAQDYKLRIKQITEEVERQVSTAM-RRRRRRR (PEP polypeptide).

[0181] 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 modification or function. As used herein, the term "polypeptide" refers to a protein that occurs naturally or is produced or altered by recombinant chemical or other means, and which is essentially a three-dimensional structure of a protein that is processed post-translationally in the same manner as the native protein.

[0182] The polypeptides of the present invention can be prepared by conventional methods in the art, for example, they can be synthesized by chemical synthesis based on the known polypeptide sequence.

[0183] Pharmaceutical composition

[0184] The present invention also relates to a pharmaceutical composition, which comprises the polypeptide or polypeptide-derived peptide of the present invention and a pharmaceutically acceptable carrier.

[0185] In the present 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 a pharmaceutically acceptable excipient, filler or diluent, etc. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the above-mentioned polypeptide or a derivative of the polypeptide, and 0.01 to 99.99% of a pharmaceutical carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0186] Use of polypeptide or its derivative peptide

[0187] The present invention also relates to the use of polypeptides for: (i) inhibiting DA neuron death or enhancing DA neuron activity; (ii) reducing DA neuron susceptibility; (iii) inhibiting the formation of 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 drugs for preventing and / or treating Parkinson's disease; and / or (vii) inhibiting TRPM2 activation. The polypeptides of the present invention can be used alone or prepared into a pharmaceutical composition. Among the aforementioned uses, (i)-(iv) and (vii) can all be in vitro non-therapeutic uses, such as use as scientific research reagents. The polypeptides of the present invention, when used as drugs for preventing and / or treating Parkinson's disease, can alleviate DA neuron degeneration / death in patients with Parkinson's disease, or prevent or reduce nerve damage, or slow down the progression of Parkinson's disease, or alleviate Parkinson's disease symptoms.

[0188] Treatment

[0189] The present invention also relates to a method for preventing and / or treating Parkinson's disease, comprising the step of administering a therapeutically effective amount of the polypeptide or polypeptide-derived peptide of the present invention to a subject.

[0190] As used herein, the term "subject" is defined herein to include animals, such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In a specific embodiment, the subject is a human.

[0191] As used herein, "therapeutically effective amount" refers to the amount of a drug sufficient to provide a therapeutic effect in the treatment or management of a disease or disorder, or sufficient to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of a drug refers to the amount of a therapeutic agent that can provide a therapeutic effect in the treatment or management of a disease or disorder when used alone or in combination with other therapies. The term "therapeutically effective amount" can include an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent.

[0192] In the present invention, the administration route of the drug or pharmaceutical composition is a conventional polypeptide drug administration route, preferably injection or oral administration. The injection preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is a conventional dosage form in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules, injections or infusions, etc.

[0193] In addition to the polypeptides or polypeptide-derived peptides provided by the present invention, the present invention also provides other drugs for treating diseases associated with DA neuron death (such as Parkinson's disease), such as other drugs capable of inhibiting the formation of Mfn2 and Bcl-2 complexes, RARP-1 inhibitors or PARG inhibitors.

[0194] The present invention also relates to the use of a drug capable of inhibiting the formation of a complex of Mfn2 and Bcl-2, for:

[0195] (i) inhibiting DA neuron death or enhancing DA neuron activity;

[0196] (ii) reducing the susceptibility of DA neurons (preferably reducing the susceptibility of DA neurons under stress);

[0197] (iii) Inhibition of Ca2+ in DE neurons 2+ and increased ROS levels.

[0198] (iv) inhibiting excessive mitochondrial fusion in DA neurons;

[0199] (v) prevention and / or treatment of Parkinson's disease; and / or

[0200] (vi) preparing a medicament for preventing and / or treating Parkinson's disease.

[0201] As used in the present invention, the term "susceptibility" refers to the sensitivity of neurons to stress stimuli such as aging, oxidative stress, calcium homeostasis imbalance and other cell physiological homeostasis imbalances, which 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 as a large number of deaths in Parkinson's patients.

[0202] In the present invention, the degree of mitochondrial fusion can be determined by the following method: labeling mitochondria with mitochondrial-specific dyes, imaging with a fluorescent confocal microscope, processing the images with Image J software, and analyzing the length of mitochondria. If the length of mitochondria in dopaminergic neurons differentiated from pluripotent stem cells derived from Parkinson's patients is significantly increased relative to that of differentiated neurons derived from normal people, it can be considered that mitochondria are excessively fused.

[0203] The present invention also relates to the use of PARP-1 inhibitors and / or PARG inhibitors for:

[0204] (i) inhibiting DA neuron death or enhancing DA neuron activity;

[0205] (ii) reducing the susceptibility of DA neurons (preferably reducing the susceptibility of DA neurons under stress);

[0206] (iii) Inhibition of Ca2+ in DE neurons 2+ and increased ROS levels.

[0207] (iv) inhibiting excessive mitochondrial fusion in DA neurons;

[0208] (v) prevention and / or treatment of Parkinson's disease; and / or

[0209] (vi) preparing a medicament for preventing and / or treating Parkinson's disease.

[0210] In a preferred embodiment of the present invention, the PARP-1 inhibitor is represented by the following formula I:

[0211]

[0212] In a preferred embodiment of the present invention, the PARG inhibitor is represented by the following formula II:

[0213]

[0214] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0215] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0216] Unless otherwise indicated, the term "or" means and is used interchangeably with the term "and / or".

[0217] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0218] Materials and methods

[0219] Caenorhabditis elegans

[0220] C. elegans husbandry: C. elegans were cultured under standard conditions on nematode growth medium (NGM) plates at 20°C and fed OP50 as described previously (Corsi et al., 2015). The strains used in this study are listed in the Reagents and Tools table.

[0221] Passaging and construction of Caenorhabditis elegans strains

[0222] The coding sequence of human TRPM2 was cloned into the plasmid Pdat-1::sl2::DsRed2b under the control of the Pdat-1 promoter, and the sl2 sequence and DsRed2b were retained as positive markers for hTRPM2 expression. hTRPM2 and WT (DsRed2b is specifically expressed only in DA neurons) transgenic Caenorhabditis elegans were constructed, and 50 ng / μL of plasmid and 10 ng / μL of co-labeled Punc-122-GFP were injected into the gonads of young adult nematodes. Progenies expressing DsRed2b were isolated for integrated strain construction. The array was integrated into the genome by trimethylpsoralen / ultraviolet (TMP / UV) mutagenesis. Extrachromosomal chip transgenic animals were prepared by gonadal injection of 40-50 ng / μL of the constructed plasmid and 10 ng / μL of the co-injected marker (Plin-44-mCherry).

[0223] Neurodegeneration test

[0224] 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, and the nematode head was observed and captured using a 546 nm excitation laser to better image the ADE and CEP neurons, and the Z layer was processed using FV10-ASW 4.2Viewer. ADE and CEP neurons with visible soma and continuous processes were counted as surviving neurons, and each nematode was examined by 2 independent researchers.

[0225] Quantification of neuronal calcium and reactive oxygen species levels

[0226] The coding sequence of the probe was cloned into a plasmid under the regulation of the Pdat-1 promoter, and the calcium and ROS levels were detected by genetic GCaMP3 and Hyper fluorescent probes, respectively. The probe plasmid and linker (Plin-44-mCherry) were then injected into the gonads of young adults. We acquired images under a confocal microscope with 488nm and 546nm excitation using a 60x objective (Olympus, FV1000). The calcium and ROS levels were quantified by measuring the mean fluorescence of the neuronal cell bodies (F488). The average fluorescence of the epidermis of the head was taken to obtain the baseline fluorescence f488 background. The normalized calcium and ROS levels were calculated as (F488-F488-background) / (F546-F546-background), respectively.

[0227] Electrophysiology

[0228] Day 2 adult worms were glued to the surface of sylgard-coated coverslips using cyanoacrylate-based glue. An incision was made dorsolaterally using a sharp glass pipette to expose the dopamine neurons located in the head. Whole-cell current recordings were performed on a fluorescence microscope (Olympus, BX51W1) using an EPC-10 amplifier and Patchmaster software (HEKA). The extracellular solution (ECS) included 145 mM NaCl, 2.5 mM KCl, 5 mM CaCl2, 1 mM MgCl2, 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. 1 mM ADPR and 10 μM CaCl2 were added to ICS to detect Ca2+-coactivated TRPM2, and 20 μM CA was added to ECS. The data were processed using Igor software (WaveMetrics).

[0229] Neuronal RNAi

[0230] Since neurons of C. elegans are not susceptible to systemic feeding RNAi (Calixto et al., 2010; Firnhaber and Hammarlund, 2013), we injected RNAi of mixed gene fragments in sense and antisense directions under the Phsp-16.2 promoter to obtain its time-specific priming 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 were injected, and 10 ng / μL of the marker Plin-44-mCherry was co-injected. Young L4 worms were heat-shocked at 35°C for 30 min every day, and then incubated at 20°C until the 7th day of adulthood for neuron counting.

[0231] Drug treatment

[0232] Detailed information on antibodies, drugs, and kits used in this study is listed in the Reagents and Tools Table. 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 make 10 mM stock solutions. Drugs were added to the bacterial lawns inoculated on NGM plates, and the drug NGM plates were allowed to dry for several hours. L4 worms were transferred to drug plates for drug treatment. Considering the decline in drug effects, the worms were transferred to another fresh drug dish every 2 days.

[0233] Scanning electron microscopy and 3D reconstruction

[0234] The SEM preparation method of nematodes was as described previously (Shu et al., 2011) with some modifications. To better determine the location of ADE and CEP neurons, each nematode was first observed with a confocal microscope (Olympus FV1000) to determine the distance from the front to the target neuron cell body based on DsRed2b fluorescence. The nematodes were then cleared into PCR tubes for SEM sample preparation. The nematodes were fixed with 2% PFA and 1% glutaraldehyde in 0.1M sodium bicarbonate (CAS) buffer at pH 7.4 for 2 hours, and then the cuticle was sharply cut to allow the 4% glutaraldehyde fixation buffer to diffuse for better fixation overnight. The nematodes were washed three times with CAS buffer, and the worm bodies were removed and placed in diaminobenzidine (DAB) buffer and co-oxidized with bubble oxygen under strong light at 480nm for 5min. The worm bodies were washed three times with CAS buffer, fixed in 2% diammonium tetroxide 0.1M CAS buffer on ice for 30min, and washed three times with ddH2O. The samples were stained with 1% uranyl acetate aqueous solution at 4°C for 1 h and then washed again with ddH2O for 3 times. Dehydrated in a graded ethanol series (30%, 50%, 70%, 90%, 100%) for 30 min each step. The worms were washed twice with 1:1 acetone and methanol and pure acetone. The mixed solutions of 3:7, 7:3, and 1:0 resin and acetone were soaked for 12 h, 24 h, and 48 h, respectively. Finally, the samples were placed in a vacuum oven at 65°C for 48 hr. The position of the worm body was adjusted parallel to the sample hole to locate the neurons. According to the previously recorded distance, the resin sample was cut into the desired area at the micrometer level, and the image of each thin section (10 nm) was collected on a Nova Nano 450 (Thermo, FEI). The serial images were aligned and reconstructed using Amira software (ThermoFisher Scientific). Finally, the mitochondrial boundaries marked with photooxidized dab staining were manually segmented for 3D reconstruction.

[0235] mouse

[0236] TRPM2- / - male mice (8-10 weeks old, 22-25 g) were introduced from the University of Leeds and then raised at Zhejiang University. Transgenic mice were generated at the University of Leeds (Zou et al., 2013). They were collectively housed in a controlled environment (21 ± 2 °C, 12 h light / dark cycle) with free access to food and water. The experiments followed the ethical standards set by the Animal Care and Use Committee of Zhejiang University. Efforts were made to minimize the number of animals used and their discomfort.

[0237] MPTP-induced PD model in mice

[0238] Mice were randomly divided into a solvent group and an MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) group. The MPTP group was intraperitoneally injected with MPTP 25 mg / kg / d for 5 consecutive days (Jackson-Lewis and Przedborski, 2007; Wei et al., 2020), while the control group mice were injected with an equal volume of standard suspension vehicle (0.9% NaCl, w / v) (Jackson-Lewis and Przedborski, 2007; Tatton and Kish, 1997). Six days after the last injection, the mice were euthanized after completing the rotarod test. 20 μM thick coronal cryosections were prepared by fusion with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for immunofluorescence staining.

[0239] Stereotaxic brain surgery (Rotenone-induced PD mouse model)

[0240] We used the animal surgery 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 instrument (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; a microinfusion set was used at a flow rate of 1 μl / min. Sham-operated mice received an equal amount of solvent only.

[0241] Immunofluorescence staining of tyrosine hydroxylase (TH)

[0242] The midbrain SNc and striatum were sectioned coronally at a height of 20 μm on a freezing sliding microtome (Leica, Wetzlar, Germany). Sections were collected for TH immunofluorescence staining. Briefly, brain slices were blocked in 3% BSA and 1% TBST for 2 h at room temperature. After the blocking step, anti-tyrosine hydroxylase antibody (clone LNC1; MAB318, Sigma) (1:500) was used in 0.3% PBST supplemented with 1% BSA at 4°C. The next day, brain slices were rinsed in PBS three times for 5 min each time and then incubated in fluorescently labeled goat anti-mouse IgG-H&L antibody for 2 h. We analyzed multiple slices (1 for every 4 slices of the entire SNc from -2.7 mm to -4.04 mm) for TH-stained neuron counting. Fluorescence images were taken under 10x and 20X objectives using an Olympus VS120 microscope. Images were subsequently quantified and analyzed in CellSens Dimension.

[0243] Rotarod Test

[0244] The motor coordination of mice was assessed using a rotarod apparatus (Bieri et al., 2019). The mice were trained at a constant speed of 5 rpm / min for 300 s for 3 consecutive days to adapt to the rotarod apparatus. The rotarod test was performed under a constant acceleration (5-30 r / min) for 300 s, and the falling latency of 3 trials was recorded. The rotarod and chamber were wiped with 70% ethanol between two trials.

[0245] Apomorphine-induced rotation

[0246] After the animals were habituated to the environment for 30 min, mice were given apomorphine (0.5 mg / kg; on days 7 and 14). Consistent with our previous standards (Zhou et al, 2019), rotations (360° in one cycle) were counted. The video recording lasted for 60 min.

[0247] iPSC

[0248] Dopamine neuron differentiation

[0249] We performed floor plate (FP) cell-based DA neuron induction as described previously (Fedele et al., 2017; Ke et al., 2020). Briefly, human iPSCs were first digested with accutase and seeded on Matrigel-coated plates, and then cultured with FP cell induction medium N1 (containing SB431542 (10 μM) and LDN193189 (100 nM), SHH-C24 (100 ng / mL), FGF8 (100 ng / mL), and purmorphamine (2 μM)) from day 0 to day 5. The culture medium was replaced with N2 medium at a ratio of 75% (N1) : 25% (N2), 50% (N1) : 50% (N2) on days 5, 6, 7, and 8, 25% (N1) : 75% (N2) on days 9 and 10, and 100% (N2) on day 11. N1 medium (50 ml) contains 41 ml of knockout DMEM, 7.5 ml of knockout serum replacement, 0.5 ml of GlutaMAX, 0.5 ml of NEAAs, and 0.5 ml of penicillin / streptomycin. N2 medium (50 ml): 48.5 ml of DMEM / F12 containing HEPES buffer / Neural basal, 0.5 ml of N2 supplement, 0.5 ml of GlutaMAX, and 0.5 ml of penicillin / streptomycin. On day 11, FP cells were passaged, expanded, and cryopreserved. After 6 generations of expansion, FP cells were dissociated with accutase and seeded onto fresh Matrigel-coated plates, and the medium was replaced with DA induction 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). Then, the cells were dissociated with accutase and diluted to 5 × 10 5 / mL, and then the cells were re-seeded into culture dishes pre-coated with poly-d-lysine hydrobromide (PDL) and laminin until the desired maturation stage.

[0250] Drug treatment of iPSC-derived dopamine neurons

[0251] All drugs (PJ-34, PDD, ACA, A10 and synthetic peptides (PEP, dendritic bacteria)) were dissolved at 10 mM as stock solutions. DA neurons were treated with drugs at corresponding concentrations on the 15th day after maturation, and the drug-containing culture medium was replaced every 4 days.

[0252] Western blot experiments

[0253] Briefly, cultured neurons were collected and lysed with 50 μL of pre-cooled RIPA buffer containing 1% PMSF and 1% cocktail (ThermoFisher). The cells were resuspended and sonicated, and then centrifuged at 12500g for 20 min at 4°C. The supernatant was collected, the concentration of each sample was determined using a BCA assay kit (Beyotime Biotechnology), mixed with 5x loading buffer, and sample preparation and electrophoresis were performed. Band intensity was analyzed using Image Studio Lite software.

[0254] PLA proximity linking technology

[0255] To detect the direct interaction between Bcl-2 and Mfn2, PLA assays were performed according to the standard protocol of DuolinkTM in situ detection reagent (SigmaAldrich). Day 18 differentiated DA neurons, SH-SY5Y cells and mouse brain sections were first fixed with 4% PFA. Briefly, samples were incubated in blocking solution at 37°C for 60 min and washed twice. Anti-Bcl-2 (Santa Cruz, sc-7382) and anti-Mfn2 (Abcam, ab124773) antibodies were diluted with Duolinkantibody diluent and added to the samples and incubated overnight at 4°C. Next, samples were washed three times with wash buffer A and PLUS MINUS PLA probes diluted 1:5 in antibody diluent were applied and incubated at 37°C for 1 hr. The probes were washed with wash buffer A, and the connection solution was added and incubated at 37°C for 30 min. After washing, the amplification solution was added and incubated at 37°C for 100 min. Samples were washed with wash buffer B and Duolink In Situ Mounting Medium containing DAPI was added. Confocal imaging was performed using Zeiss, LSM 880 and Airyscan.

[0256] Ca 2+ Flow cytometry

[0257] The calcium ion probe Fluo-4 (Invitrogen, Fluo-4, AM, F14201) was dissolved in the extracellular working solution (containing 130mM NaCl, 4.6mM KCl, 2mM MgCl2, 10mM HEPES, 5mM D-Glucose, 100μM EGTA). The probe solution was preheated at 37°C and added to the wells for incubation for 60min. The probe solution was discarded and the cells were washed twice with ECS. Another 300μL-ECS was taken and incubated for 30min. ECS was discarded again and digested with preheated Accutase at 37°C for 5min. ECS was removed and resuspended in ECS for flow cytometry (Beckman, DxFLEX) experiments. FlowJo 10 software was used to process the data.

[0258] Mitochondrial imaging

[0259] Mitochondrial morphology was observed using MitoTracker staining. Briefly, differentiated DA neurons on day 18 were stained with 500 nM MitoTracker Red (Thermo, M22425) in culture medium at 37 °C for 30 min. The cells were then washed with DPBS. To ensure that the desired dopamine neurons were observed, Girk2 antibody (Proteintech, 21647-1-1AP) was used to label fragile DA neurons, which were stained with Girk2 antibody for 12 h and with secondary antibody for 12 h after PFA fixation. The samples were washed three times and 50% glycerol was added for fluorescence protection. Neuronal staining was observed using a laser confocal microscope (Zeiss, LSM880+Airy scanning, NA 100 nm, 63x oil). Mitochondrial branch length was analyzed using Image J software.

[0260] Immunofluorescence staining

[0261] In brief, cells were attached to culture dishes, fixed with 4% paraformaldehyde for 20 minutes at room temperature, then washed with PBS, and blocked with 3% BSA and 0.5% TBST (PBS containing 0.5% Triton X-100) for 1 hour at room temperature. After the blocking step, cells were incubated 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) at 4°C overnight. The next day, the cells were washed 3 times with PBS, 5 min each time, and then incubated with secondary antibodies [goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 488 (A32723, Invitrogen, 1:100) / goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 546 (A11010, Invitrogen, 1:100)]. Finally, the cells were washed 3 times with PBS and DAPI-containing adhesion medium was added for imaging. Fluorescence images were taken using Zeiss LSM 880 and Airyscan, 20X objective lens.

[0262] Statistical analysis

[0263] GraphPad Prism 8 software was used to process the data, which were expressed as mean ± sem. The distribution of quantitative data was analyzed by Shapiro-Wilk test. One-way analysis of variance or two-way analysis of variance and appropriate post hoc tests were used for comparison among multiple groups. Two-tailed t-test was used for statistically significant individual differences between the two groups, and Mann-Whitney test was used for those that did not conform to normal distribution. Significance was defined as P less than 0.05.

[0264] Data and code availability

[0265] The datasets used or analyzed in the current study are available from the corresponding author on reasonable request.

[0266] Example 1: TRPM2 is enriched in vulnerable DA neuron clusters

[0267] In this example, the inventors performed single-cell transcriptome sequencing on DA neurons differentiated from induced pluripotent stem cells (iPSCs), analyzed the data of 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 1B), indicating that it is more vulnerable. It is reported that the G protein-gated inwardly rectifying 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). We used the above-mentioned marker neurons and the results showed that SNc DA neurons, rather than VTA DA neurons, were mainly expressed in the vulnerable DAn1 cluster ( Figure 1C and 1D ), indicating that iPSC-derived DAn1 neuron clusters are consistent with the vulnerability of vulnerable SNcDA neurons in the human brain. In addition, we also 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 vulnerable DAn1 clusters ( Figure 1E ). To further verify this finding, we also analyzed the expression of TRPM2 based on three other public databases. The midbrain TRPM2 expression profile obtained from the Human Protein Atlas dataset (www.proteinatlas.org) showed that TRPM2 expression in the SNc region was higher than that in the VTA region, regardless of gender ( Figure 2A -C). We also analyzed the expression of TRPM2 in SNc neurons in the SNc single-cell transcriptome profile of PD patients. The results showed that TRPM2 expression was positively correlated with aging in PD patients ( Figure 1F We also found that Girk2-positive neurons and TRPM2 but not Calbindin neurons were more abundant in this fragile DAn1 neuron cluster ( Fig. 1I -K). To verify these RNA-seq-based data, we also detected the expression of TRPM2 in the mouse midbrain by RNA-scope. The results showed that the mRNA level of TRPM2 in the ventral layer of SNc DA neurons in aged mice was indeed significantly higher than that in VTA ( Figure 1L This is consistent with the above RNA-seq results. Therefore, TRPM2 may be involved in mediating the susceptibility of DA neurons.

[0268] Example 2: Ectopic expression of hTRPM2 in nematodes induces selective dopaminergic neuron degeneration

[0269] There is no homologue of human TRPM2 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). In order to explore whether human TRPM2 does confer susceptibility to DA neurons in nematodes, in this example, the inventors constructed a TRPM2 C. elegans strain that expresses human TRPM2 and DsRed fluorescent protein independently and specifically in DA neurons under the control of the Pdat-1 promoter ( Figure 3A -D). Interestingly, ADE, PDE neurons and CEP neurons showed completely different phenotypes in terms of TRPM2 expression. Both ADE and PDE neurons in TRPM2-expressing nematodes showed neurodegeneration, characterized by a gradual loss of neurons within 7 days from the first day of adulthood ( Figure 4A -B and Figure 3E and F). In contrast, CEP neurons from the same human TRPM2-expressing worms remained unaffected even at 7 and 10 adult days ( Figure 4A and B and Figure 3G elegans is susceptible to TRPM2 expression. Since the age-related increase in intestinal autofluorescence severely confounds the quantification of PDE neurons, we focused on ADE and CEP neurons in subsequent studies.

[0270] To determine whether the selective susceptibility of DA neurons caused by the expression of hTRPM2 in C. 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 saturating concentrations of ADPR and Ca2+ as previously reported (for detailed methods, see Singatulina et al., 2019; Vyas et al., 2014). ADE and CEP neurons of transgenic worms expressing hTRPM2 responded to ADPR / Ca2+ with similar current amplitudes ( Figure 3H and I), indicating that TRPM2 is expressed at similar levels in ADE and CEP neurons. Compared with the expression of WT-hTRPM2, both the integrated hTRPM2 mutants carrying the E960Q mutation in the pore region and the R1433A mutation in the C-terminal ADPR binding pocket significantly reduced the death of ADE neurons ( Figure 4C -E), and the expression of TRPM2 in these two mutants was similar, with an intensity comparable to that of DsRed, indicating that ectopic expression of TRPM2 selectively induced degeneration of ADE DA neurons dependent on its channel activity.

[0271] We also treated nematodes with 2 mM MPP+. MPTP can inhibit complex I of the electron transport system to impair mitochondrial function. The method is described in Nicklas et al., 1987. The results showed that compared with WT, MPP+ treatment led to aggravated ADE death in TRPM2 nematodes ( Figure 3J ), while CEP neurons showed similar toxic effects after MPP+ treatment ( Figure 3J ). These results suggest that it is ADE rather than CEP that is responsible for neuronal susceptibility.

[0272] Since Ca2+ and reactive oxygen species (ROS) are the main ligands for TRPM2 activation, in order to verify the role of Ca2+ in the hTRPM2-induced ADE neuron 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 nematodes, the cytoplasmic Ca2+ levels of ADE neurons were much higher than those of CEP neurons ( Figure 5A The application of the Ca2+ chelator BAPTA-AM significantly inhibited the ADE neuronal death of hTRPM2 nematodes ( Figure 5C and D), further indicating the role of Ca2+ overload in hTRPM2-induced ADE neuron degeneration. Next, we examined ROS levels, which not only induce TRPM2 activation but also lead to neuronal damage during the pathogenesis. By using a ROS sensor, we observed that ROS levels in hTRPM2 C. elegans ADE neurons were much higher than those in CEP neurons ( Figure 5B ). In addition, treatment with the ROS scavenger n-acetylcysteine ​​(NAC) slightly suppressed the death of ADE neurons in hTRPM2 nematodes ( Figure 5C and D). Taken together, this suggests that the selective susceptibility of ADE neurons to hTRPM2 expression is due in part to increased Ca2+ and ROS levels, which activate hTRPM2 to drive neuronal death.

[0273] Example 3: TRPM2 is involved in the susceptibility of mouse SNc DA neurons to stress

[0274] To further verify the role of TRPM2 in the susceptibility of mammalian dopamine neurons, in this example, the inventors used a mouse PD model induced by MPTP and rotenone (Rot). Our results showed that MPTP treatment induced a selective loss of SNc rather than VTA DA neurons in WT mice ( Fig. 6ASimilar to our C. elegans data, TRPM2 deficiency (TRPM2 knockout mice, TRPM2-KO) significantly suppressed MPTP-induced SNc DA neuron loss in mice ( Fig. 6A -B), confirming that TRPM2 is essential in mediating DA neuron susceptibility. In addition, we found that stereotaxic injection of rotenone into one side of the SNc resulted in apomorphine-induced rotations and reduced rotarod time in mice ( Figure 6C -E), suggesting that TRPM2 alleviates PD-like behaviors and has a neuroprotective effect. In addition, in the WT group, progressive degeneration of dopaminergic neurons was observed in the striatum one month after injection ( Figure 6C , F and G). Consistently, these motor deficits and pathological changes were significantly alleviated in TRPM2 ko mice ( Fig.6D -G), further supporting that TRPM2 mediates SNc DA neuron death. These results confirm that TRPM2-mediated SNcDA neuron susceptibility is conserved in different species.

[0275] Example 4: TRPM2 induces ADE death through FZO-1 / ced-9-mediated mitochondrial hyperfusion

[0276] In this example, the inventors investigated whether hTRPM2 induces ADE neuronal death through the core apoptotic mechanism, including EGL-1, CED-9 (anti-apoptotic), CED-4 and CED-3 ( Fig. 7A )(Ellis and Horvitz, 1986). The researchers crossed hTRPM2 worms with mutants with loss-of-function ced-4(n1162) / apoptotic protease activating factor-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 loss-of-function ced-4 in hTRPM2 worms significantly alleviated the death of ADE neurons ( Figure 7B and C and Figure 5E and F). Surprisingly, the loss-of-function ced-9(n2812) mutant significantly suppressed ADE neuron degeneration in hTRPM2 worms, whereas the gain-of-function ced-9 mutant failed to protect ADE neurons ( Figure 7B and C). These results suggest that hTRPM2 induces ADE neuron death via ced-4 and ced-9 but not EGL-1 and CED-3.

[0277] Mitochondrial dysfunction is an important initiator of apoptosis and is of great significance in stress-induced dopamine neurodegeneration. It has been reported that Bcl-2 family proteins located in mitochondria 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 studying its contribution to mitochondrial dysfunction in ADE neurons of hTRPM2 nematodes. Using confocal microscopy, we found that mitochondrial length in ADE neurons was increased compared with CEP neurons of TRPM2 nematodes and wild-type nematodes ( Fig.7D This finding was further confirmed by three-dimensional reconstruction analysis of mitochondrial morphology based on scanning electron microscopy (SEM) results ( Fig.7D ), indicating that mitochondrial hyperfusion or fission defects exist in ADE neurons expressing hTRPM2. In addition, the loss-of-function ced-9(n2812) mutation restored normal mitochondrial morphology in hTRPM2 nematodes ( Fig. 7E and F ), confirming that ced-9 is involved in hTRPM2-induced mitochondrial fusion / fission defects in ADE neurons.

[0278] Previous studies have reported that in Caenorhabditis elegans, CED-9 promotes mitochondrial fission by interacting with EGL-1 and DRP-1, or mediates mitochondrial fusion by interacting with CED-4 and FZO-1, but this remains controversial (Lu et al., 2011; Rowland 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 showed that there was no protective effect when EGL-1 mutants were crossed with hTRPM2 nematodes ( Figure 5E and F), indicating that EGL-1 / CED-9-mediated mitochondrial fission is not involved in ADE death. In contrast, knockdown of fzo-1 but not drp-1 alleviated ADE neuronal degeneration in hTRPM2 transgenic worms ( Figure 7G and H). Consistently, expression of the ced-9 binding domain of fzo-1 (aa346-595, containing a coiled-coil domain) also alleviated ADE neuronal degeneration in hTRPM2 worms. This may be due to competitive blocking of FZO-1 / CED-9-mediated mitochondrial fusion ( Figure 7G and H). Therefore, our data suggest that mitochondrial hyperfusion mediated by the FZO-1 / CED-9 complex plays a crucial role in hTRPM2-induced degeneration of ADE neurons.

[0279] It has been reported that excessive mitochondrial fusion leads to mitochondrial permeability transition (MPT) (Papanicolaou et al., 2011), which can lead to cell death. Therefore, we also investigated whether MPT is involved in TRPM2-mediated neuronal death. In C. elegans, deletion of the cyn-1 gene encoding the regulatory components of MPT reduced the death of ADE neurons in TRPM2 worms ( Figure 5E and F). Blocking MPT pore opening with cyclosporine A (CsA) also improved the viability of ADE neurons ( Figure 5C and D). These results suggest that FZO-1 / CED-9 complex formation driven by TRPM2 activation further induces mitochondrial hyperfusion and that MPT contributes to the death of ADE neurons in hTRPM2 worms.

[0280] To further determine whether the role of the FZO-1 / CED-9 complex in mediating dopaminergic neuron death is conserved in mammals, we analyzed the interaction of Mfn2 / Bcl-2 (the mouse homologue of FZO-1 / CED-9) in a mouse model of MPTP treatment. Using the proximity ligation assay (PLA), we found that TRPM2 deficiency significantly attenuated MPTP-induced Mfn2 / Bcl-2 interaction in SNc dopaminergic neurons, but not in VTA DA neurons ( Fig.7I and J), which supports the idea that TRPM2-induced Mfn2 / Bcl-2 interaction is essential for selective SNc DA neuron death. These results further confirm that TRPM2 activation-mediated Mfn2 / Bcl-2 interaction indeed conservedly mediates the susceptibility of mouse SNc DA neurons during stress.

[0281] Example 5: TRPM2 activation mediated by PARP-1 / PARG-1 / ADPR axis induces ADE selective death

[0282] The results of the above examples show that human TRPM2 plays a new role in dopamine neuron susceptibility. Therefore, in this example, the inventors further used TRPM2 transgenic nematodes to study how TRPM2 selectively mediates ADE neuron death. ADPR is the main endogenous ligand of TRPM2 channels, which are mainly synthesized by poly-ADPR polymerase-1 (PARP-1) and poly-ADPR glycohydrolase (PARG) in response to DNA damage ( Fig. 8A)(Qi et al., 2019; Vyas et al., 2014). To determine whether preferential activation of TRPM2 is responsible for the death of ADE, we first investigated the potential role of PARP-1 and PARG in hTRPM2-induced selective ADE neuron death. Overexpression of C. elegans PARP-1 in DA neurons caused ADE cell death in WT animals and exacerbated ADE neuron death in hTRPM2 animals ( Figure 8B -C). Notably, it also caused significant CEP neuron death ( Figure 8B -C). Similarly, knockdown of parp-1 or parg-1 significantly suppressed the death of ADE neurons in hTRPM2 animals ( Figure 8B -C). In addition, treatment with the PARP-1 inhibitor PJ-34 (Szabo et al., 2002) or the PARG-1 inhibitor PDD (Raghunatha et al., 2020) reduced the death of ADE neurons in hTRPM2 animals ( Figure 8B These results support that TRPM2 activation is essential for DA neuron susceptibility. To explore whether ADPR production plays a role in TRPM2-mediated ADE death, we overexpressed nematode NDX-6 (a homolog of human NUDT9), which hydrolyzes ADPR into adenosine monophosphate (AMP) and 5'-ribose phosphate ( Fig. 8A ). The results showed that NDX-6 overexpression reduced ADE death in hTRPM2 nematodes ( Figure 8B and C), suggesting the importance of ADPR generation in TRPM2-mediated death of ADE neurons. Taken together, these results support that the PARP-1 / PARG-1 / ADPR axis is important for the vulnerability of ADE neurons expressing hTRPM2 in C. elegans.

[0283] Example 6: Death of dopamine neurons derived from ipscs in SPD patients is also mediated by TRPM2

[0284] 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 example, the inventors used iPSCs-differentiated dopamine neurons, a well-established cell model for Parkinson's disease research. Using a floor 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% ( Fig. 9A -C). These differentiated iPSC-DA neurons also expressed Girk2, while calbindin was barely detectable ( Fig. 9BBy whole-cell recording, evoked action potentials and voltage-dependent sodium channel currents were detected in differentiated DA neurons ( Fig.9D and E).

[0285] Importantly, treatment with the TRPM2 inhibitors ACA or A10 improved the viability of SPD iPSC-derived DA neurons ( Fig. 10A ), suggesting that TRPM2 function promotes DA neuron death in our iPSC model. We then used a panel of inhibitors to determine the role of the PARP-1 / PARG / ADPR axis in the viability of SPD iPSC-derived DA neurons. As expected, inhibition of PARP-1 by PJ-34 treatment reduced poly(ADPR) levels, whereas inhibition of PARG by PDD increased poly(ADPR) levels ( Fig.11A Both PJ-34 (PARP-1 inhibitor) and PDD (PARG inhibitor) significantly improved the viability of sPDipsc-derived DA neurons ( Fig. 10B -C). Consistent with the results from our C. elegans PD model, we observed Ca2+ overload in DA neurons derived from SPD patient-specific iPSCs ( Fig.11A and C), treatment with inhibitors of the PARP-1 / PARG / TRPM2 signaling axis (including PJ-34, PDD, ACA, or A10) partially alleviated this condition ( Fig.11D and E). We also observed deficient mitochondrial fusion in iPSC-derived DA neurons from SPD patients but not in healthy controls. Again, the above inhibitors may be useful in treating PD via a mitochondrial mechanism ( Fig. 10D These results suggest a role for the PARP-1 / PARG / ADPR axis in triggering TRPM2 and its downstream Ca2+ toxicity and mitochondrial hyperfusion to mediate DA neuron death in a cellular model of human SPD.

[0286] We further investigated whether the TRPM2 / FZO-1 / CED-9 axis is also conserved in iPSC-derived DA neurons from SPD patients and is involved in mitochondrial hyperfusion. CED-9 has been reported to interact with the coiled-coil domain of FZO-1 (Lu et al., 2011; Roland et al., 2009). We aligned the C. elegans FZO-1 sequence with human Mfn2 and synthesized peptides corresponding to the coiled-coil domain of human Mfn2 (aa391-434) as shown in Table 1 below ( Fig. 12A Using PLA assays, we found that PEP treatment strongly inhibited the interaction between Mfn2 and Bcl-2 in SH-SY5Y cells in the basal state ( Fig. 12Band C). MPP+ treated cells showed enhanced interaction of Bcl-2 and Mfn2, as indicated by an increase in the number of puncta, which was inhibited by PEP treatment ( Fig. 12B This is consistent with our observation that PEP rescues neuronal viability in iPSC-derived DA neurons from SPD patients ( Fig.12D ), excessive fusion mitochondrial morphology ( Fig. 10D -E), downregulation of Mfn2 / Bcl-2 interaction ( Fig.10F -G) and the effect of intracellular Ca2+ ( Fig.11D and E). In ipsc-derived DA neurons from SPD patients, either PARP-1 or PARG activity significantly inhibited the Mfn2 / Bcl-2 interaction compared to healthy controls ( Fig.10F Therefore, our results from SPD patient iPSC-derived DA neurons are consistent with those from C. elegans and mouse models, further supporting a role for Mfn2 / Bcl-2-mediated mitochondrial hyperfusion in DA neuron death across species.

[0287] Table 1

[0288]

[0289]

[0290] discuss

[0291] In this study, we provide clear evidence that TRPM2 plays an important role in mediating DA neuron susceptibility through comprehensive cross-model analyses. We have demonstrated that TRPM2 and its novel associated signaling pathways contribute to ROS- and calcium-dependent mitochondrial superfusion, which in turn leads to selective DA neuron degeneration in C. elegans and mouse models, as well as dysfunction in iPSC-derived DA neurons from PD patients. We also reveal that inhibition of either TRPM2 directly or its associated signaling pathways can conservatively improve mitochondrial function, thereby contributing to the survival of susceptible DA neurons under stress challenges ( Fig.13 These data suggest that TRPM2 acts as a key executive in susceptible DA neurons to tightly control DA neuron fate by sensing ROS and calcium levels.

[0292] In a PD mouse model, our data further demonstrated that TRPM2 deficiency protected susceptible SNc DA neurons from early death. Moreover, treatment with a TRPM2 inhibitor significantly improved the survival of iPSC-derived DA neurons from SPD patients. Therefore, TRPM2 is likely to be an oxidative stress sensor, and its selective activation through calcium-permeable channels may be a key factor in causing the manifestation of 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.

[0293] In our study, we found that both cyn-1 inactivating mutations 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, and its mechanisms include excitotoxicity, neurotoxicity, apoptosis, and necrosis (Bonora et al., 2022; Perez-Pinzon et al., 2012).

[0294] Therefore, our findings suggest that TRPM2 is selectively activated by ROS and calcium in ADE, which further prompts CED-9 / CED-4 to trigger MPT, ultimately leading to ADE death in TRPM2 worms.

[0295] In the TRPM2-related pathway, loss of function of CED-4, but not CED-3 or EGL-1, partially alleviated the death of ADE neurons in TRPM2 nematodes. Loss of function of CED-9, but not gain of function, greatly suppressed the death of ADE neurons, suggesting that CED-4 and CED-9 mediate a cell death pathway. Our findings show for the first time a non-classical apoptotic pathway mediated by CED-4 / CED-9 under specific stress conditions.

[0296] Example 7: TRPM2 switch to CED-9 (Bcl-2) / FZO-1 (Mfn-2) complex enhances mitochondrial hyperfusion and is responsible for the susceptibility of DA neurons

[0297] By using fluorescent probes and 3D electron microscopy, we found that superfused mitochondria increased in TRPM2 worms, but not in wild-type worms. In addition, FZO-1 knockout, but not DRP-1, significantly reduced the death of ADE neurons in TRPM2 worms, confirming that TRPM2 promotes FZO-1-mediated superfusion. We observed that mitochondrial superfusion in TRPM2 worms can also be crossed with TRPM2 worms that inactivate CED-9 or express the CED-9 binding domain in their DA neurons, thereby reducing the interaction of Bcl-2 / Mfn2, supporting the role of CED-9 as a switch to promote mitochondrial fusion. This is contrary to the results 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).

[0298] We also observed that iPSC-derived DA neurons from SPD patients presented mitochondrial hyperfusion compared with healthy controls, and PARP-1 / PARG / ADPR antagonists could reverse mitochondrial hyperfusion in iPSC-derived DA neurons from SPD patients. More importantly, by intervening in Mfn2 / Bcl-2 interacting peptides to disrupt mitochondrial hyperfusion in PD patient-specific iPSC-derived DA neurons, we also found that the interaction between Bcl-2 and Mfn2 was increased in the SNc region of PD mice, but not in controls, in the MPTP mouse model. In addition, TRPM2 deficiency alleviated the interaction between Bcl-2 and Mfn2 in susceptible SNc DA neurons in PD mice.

[0299] These data suggest that antagonizing the PARP-1 / PARG / ADPR pathway, Mfn2, and Bcl-2 interactions could be a promising approach for treating PD.

[0300] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. An isolated polypeptide, characterized in that The polypeptide can specifically block the interaction between Mfn2 and Bcl-2.

2. The polypeptide according to claim 1, characterized in that The polypeptide comprises a sequence selected from the group consisting of: (i) the amino acid sequence shown in SEQ ID NO: 23; (ii) a sequence formed by substitution, deletion or addition of one or more amino acids in SEQ ID NO: 23; and / or (iii) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:

23.

3. The polypeptide according to claim 1, characterized in that The length of the polypeptide is 20-100 bp.

4. The polypeptide according to claim 1, characterized in that The amino acid sequence of the polypeptide is selected from any one of the following: (i) the amino acid sequence shown in SEQ ID NO: 1-33; (ii) a sequence formed by substitution, deletion or addition of one or more amino acids in SEQ ID NOs: 1-33; (iii) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NOs: 1-33.

5. A derivative peptide of the polypeptide according to any one of claims 1 to 4, characterized in that: The derivative peptide of the polypeptide comprises the polypeptide according to any one of claims 1 to 4 and a cell-penetrating peptide connected to the polypeptide, wherein the cell-penetrating peptide is connected to the N-terminus and / or C-terminus of the polypeptide.

6. The derivative peptide of the polypeptide according to claim 5, characterized in that In the derivative peptide of the polypeptide, the N-terminus of the polypeptide is connected to Biotin or its derivative peptide; and the C-terminus of the derivative peptide of the polypeptide is connected to a pH-responsive cell-penetrating peptide.

7. The derivative peptide of the polypeptide according to claim 5, characterized in that The amino acid sequence of the derivative peptide of the polypeptide is shown in SEQ ID NO:

34.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide according to any one of claims 1 to 4 or a derivative peptide of the polypeptide according to any one of claims 5 to 7; and a pharmaceutically acceptable carrier.

9. Use of the polypeptide according to any one of claims 1 to 4 or a derivative peptide of the polypeptide according to any one of claims 5 to 7 for: (i) inhibiting DA neuron death or enhancing DA neuron activity; (ii) reduce the susceptibility of DA neurons; (iii) inhibiting the formation of the Mfn2 / Bcl-2 complex or downregulating the interaction between Mfn2 and Bcl-2 or acting as an Mfn2 / Bcl-2 inhibitor; (iv) inhibiting excessive mitochondrial fusion in DA neurons; (v) prevention and / or treatment of Parkinson's disease; (vi) preparing a medicament for preventing and / or treating Parkinson's disease; and / or (vii) Inhibition of TRPM2 activation.

10. A method for preventing and / or treating Parkinson's disease, characterized in that: The method comprises the step of administering to a subject a therapeutically effective amount of the polypeptide according to any one of claims 1 to 4, or a peptide derived from the polypeptide according to any one of claims 5 to 7, or a pharmaceutical composition according to claim 8.

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