Use of fisetin in the prevention / treatment of kif1a-related neurological diseases

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to the use of fisetin in preventing / treating KIF1A related neurological diseases. The inventor of the present application first discovers and confirms that treating Caenorhabditis elegans with R11Q mutation with plant flavonol fisetin can improve the movement of the animals. The discovery of the present application makes it possible to rescue R11Q deficiency at the biochemical and animal levels, and provides a new clue for the treatment of KIF1A related neurological diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, and particularly relates to the use of non-seco in preventing / treating KIF1A-related neurological diseases. BACKGROUND

[0002] Microtubule (MT)-based axonal transport is essential for neural development and physiology. Transport of axonal cargo is bidirectional, with kinesin superfamily proteins (KIFs) and cytoplasmic dynein-1 driving anterograde (from minus to plus end of microtubule) and retrograde (from plus to minus end of microtubule) transport, respectively. Synaptic vesicles transmit neuronal information, and their components are synthesized in the cell body and transported to axon terminals by kinesin-3 family to synaptic vesicle protein (SVP). The founding member of kinesin-3, UNC-104, was discovered by genetic screening for uncoordinated movement (UNC) phenotype in C. elegans. In unc-104 mutants, SVPs fail to localize to synapses and abnormally accumulate in cell bodies and dendrites. UNC-104 contains an N-terminal motor domain that hydrolyzes ATP to power movement along microtubules, a neck coil domain, a coiled-coil domain, a forkhead associated (FHA) domain, and a C-terminal pleckstrin homology (PH) domain.

[0003] The human homolog of UNC-104 is KIF1A. A large body of genetic evidence indicates that KIF1A mutations cause neurodegenerative diseases and a spectrum of neurological-related diseases, collectively known as KIF1A-associated neurodegenerative diseases (KAND). KKIF1A-associated neurological disorder). KAND symptoms include from congenital neuropathy, hereditary spastic paraplegia, autism, hyperactivity, optic atrophy, etc. C. elegans has become a valuable genetic model to study KAND. Introduction of the human homolog KIF1A into C. elegans can repair the functional defects observed in unc-104 mutants, indicating the evolutionary conservation of the function of this kinesin family. Recent studies used genome editing tools to introduce KAND mutations into the nematode genome and systematically analyzed defects in axonal transport and animal behavior. Combined with biochemical and single-molecule assays of KIF1A in vitro, it was revealed that some de novo KAND mutations upregulate KIF1A motor activity, suggesting that overactivation of the motor rather than loss of function can be the cause of the dominant nature of motor neuron disease (Chiba, K. et al. Disease-associated mutations hyperactivate KIF1A motility and anterograde axonal transport of synaptic vesicle precursors. Proc Natl Acad Sci U S A 116, 18429-18434, doi:10.1073 / pnas.1905690116 (2019).). (Anazawa, Y., Kita, T., Iguchi, R., Hayashi, K. & Niwa, S. Denovo mutations in KIF1A-associated neuronal disorder (KAND) dominant-negatively inhibit motor activity and axonal transport of synaptic vesicle precursors. Proc Natl Acad Sci U S A 119, e2113795119, doi:10.1073 / pnas.2113795119 (2022)).

[0004] In addition to modeling KAND mutations in vivo, C. elegans has the power of robust genetic suppressor screens that can provide new insights into KAND intervention. A pioneering genetic screen for suppressors of the partial loss-of-function mutant unc-104(e1265) identified an intergenic suppressor: mutation in the kinesin cargo adaptor protein UNC-16 (homolog of mouse JIP3 and Drosophila Sunday Driver), and an intragenic suppressor: an amino acid substitution mutation within the motor stalk region of UNC-104 / KIF1A (Byrd, D. T. et al. UNC-16, a JNK-signaling scaffold protein, regulates vesicle transport in C. elegans. Neuron 32, 787-800, doi:10.1016 / s0896-6273(01)00532-3 (2001)) (Byrd, D. T., Pearlman, J. M. & Jin, Y. Intragenic suppressors of unc-104(e1265) identify potential roles of the conserved stalk region. MicroPubl Biol 2022, doi:10.17912 / micropub.biology.000539 (2022)). Additionally, by unbiasedly screening for suppressors that restore presynaptic patterning and axonal transport in the ARL-8 (Arf-like small GTPase) loss-of-function mutant, gain-of-function mutations in unc-104 were found to restore activity (Wu, Y. E., Huo, L., Maeder, C. I., Feng, W. & Shen, K. The balance between capture and dissociation of presynaptic proteins controls the spatial distribution of synapses. Neuron 78, 994-1011, doi:10.1016 / j.neuron.2013.04.035 (2013).). A recent genetic suppressor screen found a mutation that restores activity in a clinical partial loss-of-motor activity KIF1A mutant.It is possible to screen for genetic suppressors of loss-of-function or gain-of-function mutations in unc-104 (Anazawa, Y., Kita, T., Iguchi, R., Hayashi, K. & Niwa, S. De novo mutations in KIF1A-associated neuronal disorder (KAND) dominant-negatively inhibit motor activity and axonal transport of synaptic vesicle precursors. Proc Natl Acad Sci U S A 119, e2113795119, doi:10.1073 / pnas.2113795119 (2022)), and the genetic suppressors of unc-104 mutations provide new ideas for the prevention / treatment of KAND. At present, more than 100 mutations have been identified from KAND patients, however, there are few methods for treating or intervening KAND, and there is currently a lack of effective treatment for this disease.

[0005] Therefore, it is urgent to develop an effective drug for preventing / treating KIF1A-related neurological diseases. SUMMARY

[0006] Fisetin is a plant flavonol that naturally occurs in food, especially fruits and vegetables, and can improve the behavior and physiological functions of old mice, protect cognitive function in Alzheimer's disease models, and promote the survival of C. elegans under heat stress. The inventors of the present application first discovered and confirmed that treating unc-104 (R11Q) mutant C. elegans with plant flavonol fisetin can improve animal movement.

[0007] The present application is directed to the R11Q mutation in the KIF1A motor domain, which comes from a spastic paraplegia (SPG) patient with autism spectrum syndrome and attention deficit hyperactivity disorder. Since R11 is located at the end of the beta1 fold of the nucleotide binding pocket, it is expected that the R11Q mutation will disrupt ATP / ADP binding. It is a mystery whether and how this deleterious mutation around the ATP / ADP binding pocket can be restored.

[0008] The present application aims to at least partially solve one of the technical problems in the related art.

[0009] To this end, the present application provides, in a first aspect, use of fisetin in the preparation of a medicament for preventing or treating a KIF1A-related neurological disease.

[0010] In combination with genetic suppressor screening and biochemical and single molecule analysis, the inventors found that another residue in the mutant nucleotide binding pocket can restore R11Q defects by patching the pocket, and replacing other residues in the motor domain does not restore the motility of R11Q in vitro, but can rescue synaptic and UNC defects in animals. Based on the discovery of genetic suppressors, the inventors of the present application first discovered and confirmed that treating unc-104(R11Q) mutant C. elegans with fisetin, a plant flavonol, can improve animal movement. The discovery of the present application makes it possible to rescue R11Q defects at the biochemical and animal levels, and provides new clues for the treatment of KAND.

[0011] According to an embodiment of the present application, the KIF1A-related neurological disease includes a neurodegenerative disease caused by a lysine at position 11 in the KIF1A motor domain being changed to glutamine.

[0012] According to an embodiment of the present application, the medicament further contains a pharmaceutically acceptable excipient or carrier.

[0013] According to an embodiment of the present application, the excipient includes at least one selected from a binding agent, a disintegrating agent, a lubricant, a glidant, a stabilizer, a filler, a diluent, and a sustained release agent.

[0014] According to an embodiment of the present application, the carrier includes at least one selected from a sugar, a starch, a cellulose and a derivative thereof, a calcium phosphate, an alkaline earth metal salt of stearic acid, a vegetable oil, a surfactant, a fatty alcohol, and a cereal hydrolyzed solid.

[0015] According to an embodiment of the present application, the dosage form of the medicament includes at least one selected from an injection, a powder, a granule, and an emulsion.

[0016] According to an embodiment of the present application, the administration method of the medicament includes at least one selected from injection administration, oral administration, and soaking administration.

[0017] According to an embodiment of the present application, the injection administration includes at least one selected from intravascular injection, injection around a blood vessel through skin or natural body cavity puncture, intravascular wall injection, intramuscular injection, and subcutaneous injection.

[0018] According to an embodiment of the present application, the fisetin has an effective concentration of 1 nmol / L to 200 μmol / L.

[0019] According to an embodiment of the present application, the effective concentration of the non-serotonin is 1 nmol / L-100 μmol / L.

[0020] According to an embodiment of the present application, the effective concentration of the non-serotonin is 10 nmol / L.

[0021] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0022] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the accompanying drawings, wherein:

[0023] Figure 1 A schematic diagram of UNC-104 (R9Q) genome suppressor screening in Example 1 of the present application is shown, wherein, Figure 1 Figure A in the above-mentioned diagram is a schematic diagram of the UNC-104 molecular motor protein domain, NC is a neck coiled coil, CC is a coiled coil region, FHA is a forkhead associated region, PH is a pleckstrin homology domain, the R9Q mutation corresponds to the KIF1A associated nervous system disease (KAND) pathogenic mutation R11Q, which is introduced into the UNC-104 protein coding region by CRISPR-Cas9 genome editing method,

[0024] Figure B on the left side is the position of the R9Q suppressor mutation amino acid site in the UNC-104 motor domain, the number of independent suppressors carrying each type of mutation site is given in the brackets, and the right side pie chart shows the relative proportion of each type of suppressor population in all independent suppressors,

[0025] Figure C is a sequence alignment between the human KIF1A and the nematode UNC-104 protein motor power domain, wherein the conserved identical residues are highlighted in purple, and the mutation site residues identical or similar to KIF1A in the R9Q screening-derived suppressor position of UNC-104 are marked by asterisks or solid circles,

[0026] Figure D is a representative bright field image of wild type, unc-104 (R9Q) mutant and different homozygous suppressor animals identified in the L4 larval stage, wherein the body length measurement data (len) and the calculated curl index value (curl) of the animals in the figure are labeled at the bottom of each image, the data shown in the R9Q image is obtained from the framed animal, and the scale bar is 0.5 millimeter,

[0027] E Figure E shows quantification of the number of body bends in water droplets for different genotypes of nematodes within one minute, where the vertical axis indicates the UNC-104 amino acid change confirmed to be carried by the tested animals, error bars represent SEM, n = 30 animals, statistical significance was calculated using independent sample t-test: ***p < 0.001 compared to animals carrying UNC-104(R9Q) mutation; ###p < 0.001 compared to wild type animals,

[0028] F and G Figures F and G show quantification of the partial rescue of unc-104(R9Q) animal morphological phenotype on body length (Figure F) and on the curling index (Figure G) for five representative suppressor animals, ***p < 0.001 compared to unc-104(R9Q) mutant animals, independent sample t-test; ##p < 0.01, ###p < 0.001 compared to wild type animals, independent sample t-test;

[0029] Figure 2 Figure shows the structure of KIF1A and its mutants, ATPase activity assay and single molecule experiment results in Example 2 of the present application, wherein, Figure 2 A of Figure A shows the potential impact of the amino acid mutations shown on the nucleotide binding pocket structure in the motor domain of KIF1A,

[0030] B of Figure B shows the ATPase activity of KIF1A 1-613 wild type and various mutant proteins under microtubule activation, wherein the data is normalized to 100% ATPase activity of microtubule-activated kinesin-1 heavy chain (KHC). Each experiment was repeated independently three times, the column chart represents the mean ± SD, ND indicates that the data was not detected,

[0031] C of Figure C shows representative kymographs showing the movement of KIF1A 1-613 wild type and various mutants along microtubules, horizontal space scale 5 μm, vertical time scale 10 seconds,

[0032] D of Figure D shows quantification of the speed (top) and run length (bottom) of each molecular motor variant, wherein the histograms show the distribution of movement speed and duration and Gaussian fitting, respectively, the number of tested molecules is represented by N in each graph, the speed and movement length are represented by the mean ± SD,

[0033] E of Figure E shows statistical analysis of the speed (top) and run length (bottom) of each molecular motor variant, wherein the statistical significance was calculated using independent sample t-test compared to wild type (n = 3) (n.s., not significant; **p < 0.01; ****p < 0.0001);

[0034] Figure 3The dynamic of UNC-104::GFP in wild type and mutants observed in FRAP experiment in Example 3 of the present application is shown, wherein, Figure 3 Figure A in the above is a representative fluorescence image of UNC-104::GFP carried by the mutants, wherein, before the R9Q mutation was introduced by genome editing, GFP was knocked into the C-terminal of UNC-104, the mutation site carried by unc-104 is marked in each figure accordingly, the fluorescence at the nerve ring is significantly weakened in unc-104(R9Q) mutant, and this weakening is differentially restored in suppressors, scale bar: 100 pm,

[0035] Figure B is a two-minute recording of the recovery after UNC-104::GFP in the axon is photobleached in the boxed area in the schematic diagram in the upper left corner, showing the images before and after photobleaching at the specified time points in order (0 seconds), scale bar: 5 pm, the recovery process of FRAP signal conforms to a single exponential equation,

[0036] Figures C and D are FRAP experiment tests on UNC-104::GFP in vivo from different variants, the results show that the suppressors rescue the movement characteristics of UNC-104(R9Q)::GFP protein in axons (Figure C) and the proportion of active part in protein population (Figure D), wherein the error bars represent SEM, n = 8 to 15 independent animals, statistical significance is calculated by independent sample t test: ### p < 0.001 compared with wild type animals;

[0037] Figure 4 The distribution and morphology of presynaptic vesicles in axons in wild type and unc-104 mutant animals in Example 3 of the present application are shown, wherein, Figure 4 Figure A in the above is a schematic diagram of DA9 neuron, the blue dots on the axon represent synaptic vesicles, the dorsal axon region framed with a dashed line is the imaging area in each genotype for synaptic vesicle analysis, and the proximal axon is used for velocity analysis. Representative images show the distribution of synaptic vesicles labeled by Pmig-13::mScarlet::RAB-3 in the proximal axon of DA9 neuron in wild type, unc-104(R9Q) mutant and rescue suppressor animals, scale bar: 10 pm,

[0038] B-Figures show quantification of synaptic vesicle distribution characteristics in the dorsal axon of wild type, unc-104(R9Q) mutant and suppressor, where length of synaptic region is B, number of vesicle aggregation morphologies is C, size of vesicle aggregation morphologies is D, fluorescence intensity of vesicle aggregation morphologies relative to non-synaptic axon region is E, and frequency of vesicle aggregation morphologies relative to non-synaptic axon region is F, violin plots with raw data points are shown, n = 39 to 60 independent animals, statistical significance is calculated by independent sample t-test: ***p < 0.001 compared to unc-104(R9Q) mutant; #p < 0.5, ##p < 0.01, ###p < 0.001 compared to wild type animals;

[0039] Figure 5 A-Figures show quantification of synaptic vesicle distribution characteristics in the dorsal axon of wild type, unc-104(R9Q) mutant and suppressor, where length of synaptic region is B, number of vesicle aggregation morphologies is C, size of vesicle aggregation morphologies is D, fluorescence intensity of vesicle aggregation morphologies relative to non-synaptic axon region is E, and frequency of vesicle aggregation morphologies relative to non-synaptic axon region is F, violin plots with raw data points are shown, n = 39 to 60 independent animals, statistical significance is calculated by independent sample t-test: ***p < 0.001 compared to unc-104(R9Q) mutant; #p < 0.5, ##p < 0.01, ###p < 0.001 compared to wild type animals; Figure 5 A-Figures show quantification of synaptic vesicle distribution characteristics in the dorsal axon of wild type, unc-104(R9Q) mutant and suppressor, where length of synaptic region is B, number of vesicle aggregation morphologies is C, size of vesicle aggregation morphologies is D, fluorescence intensity of vesicle aggregation morphologies relative to non-synaptic axon region is E, and frequency of vesicle aggregation morphologies relative to non-synaptic axon region is F, violin plots with raw data points are shown, n = 39 to 60 independent animals, statistical significance is calculated by independent sample t-test: ***p < 0.001 compared to unc-104(R9Q) mutant; #p < 0.5, ##p < 0.01, ###p < 0.001 compared to wild type animals;

[0040] B-Figures show quantification of synaptic vesicle distribution characteristics in the dorsal axon of wild type, unc-104(R9Q) mutant and suppressor, where length of synaptic region is B, number of vesicle aggregation morphologies is C, size of vesicle aggregation morphologies is D, fluorescence intensity of vesicle aggregation morphologies relative to non-synaptic axon region is E, and frequency of vesicle aggregation morphologies relative to non-synaptic axon region is F, violin plots with raw data points are shown, n = 39 to 60 independent animals, statistical significance is calculated by independent sample t-test: ***p < 0.001 compared to unc-104(R9Q) mutant; #p < 0.5, ##p < 0.01, ###p < 0.001 compared to wild type animals;

[0041] D-Figures show quantification of synaptic vesicle distribution characteristics in the dorsal axon of wild type, unc-104(R9Q) mutant and suppressor, where length of synaptic region is B, number of vesicle aggregation morphologies is C, size of vesicle aggregation morphologies is D, fluorescence intensity of vesicle aggregation morphologies relative to non-synaptic axon region is E, and frequency of vesicle aggregation morphologies relative to non-synaptic axon region is F, violin plots with raw data points are shown, n = 39 to 60 independent animals, statistical significance is calculated by independent sample t-test: ***p < 0.001 compared to unc-104(R9Q) mutant; #p < 0.5, ##p < 0.01, ###p < 0.001 compared to wild type animals;

[0042] E-Figures show quantification of synaptic vesicle distribution characteristics in the dorsal axon of wild type, unc-104(R9Q) mutant and suppressor, where length of synaptic region is B, number of vesicle aggregation morphologies is C, size of vesicle aggregation morphologies is D, fluorescence intensity of vesicle aggregation morphologies relative to non-synaptic axon region is E, and frequency of vesicle aggregation morphologies relative to non-synaptic axon region is F, violin plots with raw data points are shown, n = 39 to 60 independent animals, statistical significance is calculated by independent sample t-test: ***p < 0.001 compared to unc-104(R9Q) mutant; #p < 0.5, ##p < 0.01, ###p < 0.001 compared to wild type animals;

[0043] G-Figures show quantification of synaptic vesicle distribution characteristics in the dorsal axon of wild type, unc-104(R9Q) mutant and suppressor, where length of synaptic region is B, number of vesicle aggregation morphologies is C, size of vesicle aggregation morphologies is D, fluorescence intensity of vesicle aggregation morphologies relative to non-synaptic axon region is E, and frequency of vesicle aggregation morphologies relative to non-synaptic axon region is F, violin plots with raw data points are shown, n = 39 to 60 independent animals, statistical significance is calculated by independent sample t-test: ***p < 0.001 compared to unc-104(R9Q) mutant; #p < 0.5, ##p < 0.01, ###p < 0.001 compared to wild type animals;

[0044] Figure 6FIG. 1 shows the schematic diagram of partial restoration of unc-104(R9Q) animal locomotion ability and body morphology by Fisetin in Example 4 of the present application, wherein, Figure 6 FIG. 1A is a representative brightfield image of unc-104(R9Q) animals after treatment with different concentrations of Fisetin,

[0045] FIG. 1B-1D include violin plots of raw data points showing quantification of morphological phenotypes of unc-104(R9Q) mutant animals in swimming behavior (FIG. 1B, n = 25-30 animals), body length (FIG. 1C, n = 45-85 animals), and coiling index (FIG. 1D, n = 230-360 animals) after treatment with different concentrations of Fisetin. Green bars identify the median and quartile positions. Statistical significance was calculated by independent sample t-test: *p < 0.05, ***p < 0.001 compared to unc-104(R9Q) animals cultured on control NGM plates,

[0046] FIG. 1E representative images showing partial rescue of synaptic vesicle distribution defects in DA9 neurons of unc-104(R9Q) mutant animals after treatment with different concentrations of Fisetin. Pmig-13::mScarlet::RAB-3 labels synaptic vesicles. Scale bar: 10 pm,

[0047] FIG. 1F and FIG. 1G are bar graphs showing partial restoration of the length (FIG. 1F) and number of enriched morphologies (FIG. 1G) of the dorsal axon synaptic zone of unc-104(R9Q) mutants after treatment with different concentrations of Fisetin. n = 40-50 independent animals. Statistical significance was calculated by independent sample t-test: **p < 0.01, ***p < 0.001;

[0048] Figure 7 FIG. 2 shows the restoration of KIF1A(R11Q) protein motility activity by Fisetin in in vitro experiments in Example 5 of the present application, wherein, Figure 7 FIG. 2A is a structural model of Fisetin mimetic docking into the KIF1A(R11Q) mutant pocket, the right panel in FIG. 2A shows a structural clash between the guanidino group of wild-type Arg11 and Fisetin,

[0049] FIG. 2B is quantification of microtubule-activated ATPase activity of human and murine KIF1A 1-613 carrying R11Q mutation with or without 10 nM Fisetin, data were normalized to microtubule-activated kinesin-1 heavy chain (KHC) ATPase activity as 100%, data presented in bar graphs represent mean ± SD from three independent repeats of each experiment,

[0050] C. Representative kymographs showing the movement of murine KIF1A(R11Q)1-613 motor protein along microtubules in the presence or absence of 10 nM Fisetin, horizontal spatial scale 2 pm; vertical time scale 5 s,

[0051] D. Quantification of the velocity (upper part) and length (lower part) of the movement of murine KIF1A(R11Q) motor protein after treatment with 10 nM Fisetin, histograms show the distribution of velocity and duration length and Gaussian fits, respectively, and the mean values of three replicates are plotted as a bar graph on the right, the number of test molecules is indicated as N in each graph, velocity and movement length are indicated as mean ± SD;

[0052] Figure 8 The definition of the curling index and more representative images are shown in one embodiment of the present application, wherein, Figure 8 A. Swimming ability test results of animals with UNC-104 amino acid mutation sites obtained by CRISPR-Cas9 gene editing (blue) or EMS chemical mutagenesis suppressor screen (red) in the present application,

[0053] B. Workflow diagram of individual nematode body length measurement and curling index calculation, wherein, tightly coiled animals are excluded from length measurement,

[0054] C. Representative bright field images of two homozygous suppressor animal L4 stage larvae, wherein, the body length measurement (len) and curling index calculation (curl) of the animals in the figure are shown at the bottom, the scale bar is 0.5 mm;

[0055] Figure 9 The expression profile of unc-104 gene in different genetic backgrounds and photobleaching recovery diagram of experimental animals carrying different mutation types are shown in one embodiment of the present application, wherein, Figure 9 A. Standardized RNA expression profile of unc-104 gene in different genetic backgrounds in the present application,

[0056] B-C. After photobleaching the axons of UNC-104(R9QT102I)::GFP (B) or UNC-104(R9QG265R)::GFP (C) suppressor animals in the boxed area of the upper left corner of the diagram and recording the recovery for 2 minutes, representative images before and after bleaching (0 seconds) are shown in order of time points, scale bar: 5 pm;

[0057] Figure 10Distribution and Gaussian fitting of anterograde (top) and retrograde (bottom) synaptic vesicle transport velocity in wild type, unc-104(R9Q) mutant and indicated suppressor animals in one embodiment of the present application.

[0058] Figure 11 Figure 1 shows the schematic diagram of the amino acid sequence alignment of human and mouse KIF1A proteins and the effect of Fisetin on ATPase enzymatic activity and motor domain of KIF1A(R11Q) in one embodiment of the present application, wherein, Figure 11 Figure 1A is the amino acid sequence alignment of human and mouse KIF1A proteins, with the conserved identical amino acids marked with asterisk,

[0059] Figure 1B is the histogram of microtubule gliding event velocity distribution of mouse mKIF1A 1-613 (upper) and mKIF1A(R11Q) 1-613 (lower) motor proteins in microtubule gliding motility assay after pre-treatment with 10 nM Fisetin (Fisetin) and Gaussian fitting,

[0060] Figure 1C is the microtubule-activated ATPase enzymatic activity assay of mouse and human wild type KIF1A 1-613 proteins with or without 10 nM Fisetin (Fisetin), wherein the data is normalized to microtubule-activated kinesin-1 heavy chain (KHC) ATPase activity as 100%, each experiment was repeated independently three times, and the data in the bar graph is shown as mean ± SD;

[0061] Figure 12 Figure 2 shows the example of TIRF imaging of GFP-tagged mKIF1A 1-613 (green) gliding along TAMRA-tagged taxol-stabilized microtubules (red) immobilized on glass coverslips in one embodiment of the present application, with one frame taken every 0.1 second, and the play rate is 10 frames per second;

[0062] Figure 13 Figure 3 shows the example of TIRF imaging of GFP-tagged mKIF1A(R11Q) 1-613 (green) treated with 10 nM Fisetin (Fisetin) gliding along TAMRA-tagged taxol-stabilized microtubules (red) immobilized on glass coverslips in one embodiment of the present application, with one frame taken every 0.1 second, and the play rate is 10 frames per second;

[0063] Figure 14TIRF example imaging combination showing TAMRA-labeled paclitaxel-stabilized microtubules sliding along pre-coated wild type (left panel), R11Q mutant (middle panel) and 10 nM Fisetin pre-treated R11Q mutant (right panel) GFP::mKIF1A 1-613 protein on glass coverslips in one embodiment of the present application. One frame was taken every 1 second. The play rate was 10 frames per second;

[0064] Figure 15 Figure 6 shows the effect of different concentrations of Fisetin on ATPase enzyme activity of KIF1A (R11Q) in one embodiment of the present application, wherein "KHC" represents the standard for detecting ATPase enzyme activity, used as a positive control, "WT" is the ATPase enzyme activity in wild type nematode, "R11Q" represents the ATPase enzyme activity of R11Q mutant, "R11Q-100pm" represents the ATPase enzyme activity of R11Q mutant with 100 pmol / L of Fisetin added, "R11Q-100nm" represents the ATPase enzyme activity of R11Q mutant with 100 nmol / L of Fisetin added. DETAILED DESCRIPTION

[0065] Embodiments of the present application are described in detail below. The embodiments described below are examples for explaining the present application and should not be understood as limiting the present application.

[0066] It should be noted that the terms "first", "second" are used only for descriptive purposes and should not be construed as indicating or implying relative importance or an indicated number of the technical features indicated. Thus, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0067] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges or values should be construed to be approximations that allow for significant variation. Within each range, many

[0068] In order that the present application can be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in this document concur in meaning with the general use of those terms by those skilled in the art to which the present application pertains.

[0069] In the present text, the terms "comprising" or "comprise" are to be construed as open terms, i.e. meaning including, but not excluding, further aspects.

[0070] In the present text, the terms "optionally", "optional" or "option" generally mean that the subsequently described event or circumstance can, but need not, occur, and this description encompasses situations where the event or circumstance occurs and situations where it does not.

[0071] In the present text, the term "excipient" is an additional substance to the active drug in a pharmaceutical formulation, also known as an auxiliary material. For example, the binding agent, the filler, the disintegrant, the lubricant in tablets; the wine, vinegar, medicinal juice, etc. in traditional Chinese medicine pills; the base part in ointment, cream of semi-solid preparation; the preservative, antioxidant, flavoring agent, aromatic agent, cosolvent, emulsifier, solubilizer, osmotic pressure regulator, coloring agent, etc. in liquid preparation can be called excipient.

[0072] According to a specific embodiment of the present application, the non-serotonin can be used as the only active ingredient, or as one of multiple active ingredients.

[0073] According to a specific embodiment of the present application, the present application proposes the use of non-serotonin in the preparation of a medicament for preventing or treating KIF1A-related neurological diseases, wherein the medicament comprises a pharmaceutically acceptable excipient or carrier.

[0074] According to a specific embodiment of the present application, the excipient can enhance the handling properties of the pharmaceutical formulation, i.e. make the formulation more suitable for direct compression by increasing the flowability and / or cohesiveness. Examples of typical "pharmaceutically acceptable carriers" suitable for the above-mentioned formulations are: sugars, such as lactose, sucrose, mannitol and sorbitol; starches, such as corn starch, tapioca starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and methyl cellulose; calcium phosphates, such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinylpyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates, such as magnesium stearate and calcium stearate; stearic acid; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil and corn oil; non-ionic, cationic and anionic surfactants; ethylene glycol polymers; fatty alcohols; and grain hydrolysis solids and other non-toxic, compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants and the like auxiliary materials commonly used in pharmaceutical formulations.

[0075] According to a specific embodiment of the present application, the non-serotonin is encapsulated in any carrier known in the art capable of transporting the active chemical ingredient, such as a liposome.

[0076] The present application provides a use of a nonsteroidal anti-inflammatory drug in the preparation of a medicament for preventing or treating a KIF1A-related neurological disease.

[0077] According to the detailed description of the present application, the KIF1A-related neurological disease includes a neurodegenerative disease caused by a lysine at the 11th position in the KIF1A motor domain being changed to glutamine.

[0078] According to the detailed description of the present application, the medicament further contains a pharmaceutically acceptable excipient or carrier.

[0079] According to the detailed description of the present application, the excipient includes at least one selected from a binding agent, a disintegrating agent, a lubricant, a glidant, a stabilizer, a filler, a diluent, and a sustained-release agent.

[0080] According to the detailed description of the present application, the carrier includes at least one selected from a saccharide, a starch, a cellulose and its derivative, a calcium phosphate, an alkaline earth metal stearate, a vegetable oil, a surfactant, a fatty alcohol, and a cereal hydrolysis solid.

[0081] According to the detailed description of the present application, the dosage form of the medicament includes at least one selected from an injection, a powder, a granule, and an emulsion.

[0082] According to the detailed description of the present application, the administration method of the medicament includes at least one selected from injection administration, oral administration, and soaking administration.

[0083] According to the detailed description of the present application, the injection administration includes at least one selected from intravascular injection, perivascular injection through skin or natural body cavity puncture, intravascular wall injection, intramuscular injection, and subcutaneous injection.

[0084] According to the detailed description of the present application, the effective concentration of the nonsteroidal anti-inflammatory drug is 1 nmol / L-200 μmol / L, further preferably 1 nmol / L-100 μmol / L, and still further preferably 10 nmol / L, which can effectively and specifically restore a neurodegenerative disease caused by KIF1A(R11Q) mutation.

[0085] According to the detailed description of the present application, the human KIF1A(R11Q) mutant protein corresponds to a KIF1A(R9Q) mutant protein in Caenorhabditis elegans.

[0086] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0087] All nematode strains used in the present invention were cultured at 20°C on nematode growth medium (NGM) plates and fed with inoculated Escherichia coli OP50. In the experiment of supplementing with fisetin (purchased from Sigma, cat. no. 528-48-3), the drug was added to the medium at the desired concentration before pouring the NGM plates, and then inoculated with OP50 culture bacteria in the conventional manner. The genetically modified nematode strains in this study were all based on the Bristol N2 nematode strain. All nematode strains used are summarized in Table 1. The PHX4787 (UNC-104::GFP) strain (referred to as wild type in this study) and other CRISPR / Cas9-mediated genome editing strains were constructed by Suny Biotech (Fuzhou, China). All animal experiments were conducted in accordance with the regulations of regulatory authorities at all levels.

[0088] Table 1

[0089]

[0090]

[0091] Example 1: Genetic Screening in C. elegans Identifies Genetic Suppressors of the KIF1A(R11Q) Mutant

[0092] To understand the molecular determinants of cellular defects caused by the pathogenic mutation KIF1A(R11Q), the inventors used a CRISPR-Cas9-based genome editing method to introduce the corresponding unc-104(R9Q) mutation into the UNC-104::GFP knock-in nematode strain to establish a C. elegans disease model. As shown in Table 1, PHX4787[unc-104::gfp KI] is the wild type, and PHX5261[unc-104(R9Q)::GFP] is the unc-104(R9Q) mutant model.

[0093] Depend on Figure 1 The unc-104(R9Q) mutant nematodes are severely coiled, short, and exhibit significant uncoordinated movement disorders with 100% penetrance ( Figure 1Figure D in the Examples section, n > 200), the phenotype is consistent with other loss-of-function unc-104 mutant phenotypes reported earlier (Anazawa, Y., Kita, T., Iguchi, R., Hayashi, K. & Niwa, S. De novo mutations in KIF1A-associated neuronal disorder (KAND) dominant-negatively inhibit motor activity and axonal transport of synaptic vesicle precursors. Proc Natl Acad Sci U S A 119, e2113795119, doi: 10.1073 / pnas.2113795119 (2022)).

[0094] The 100% penetrance of the animal defect facilitates large-scale and high-throughput genetic suppressor screening. However, unc-104(R9Q) mutant animals are poor movers and have small brood sizes (56.2 ± 23.8, mean ± SD, n = 18, about one fourth of wild-type N2 animal brood size), making it difficult to obtain sufficient synchronized offspring for chemical mutagenesis. To enrich L1 larvae, the inventors designed a 15- pm-diameter filtration device to sort nematodes at different developmental stages based on their size.

[0095] The procedure is as follows: Nematodes PHX5261 [unc-104(R9Q)::GFP] are grown on NGM plates containing E. coli OP50 until the population is just starved. Then mixed developmental stage nematodes are collected from 10-20 plates and L1 stage larvae are sorted using a 15- pm-diameter metal filter. The sorted L1 larvae are grown to late L4 and collected for treatment with ethyl methanesulfonate (EMS). As shown in Table 1, the treated nematodes contain mutations at different loci, and the nematodes are washed and aliquoted onto 200-400 NGM plates seeded with OP50, with 5-10 nematodes dropped onto each plate near the edge of the plate. When most of the F2 progeny develop to late L4, the plates are inspected for nematodes that can move to the opposite edge of the plate, which are likely to carry genetic suppressors. Through multiple rounds of screening, the inventors examine about 100,000 haploid genomes, and genetic suppressors from different plates are considered independent. After two generations of phenotype confirmation, the nematodes carrying genetic suppressors are whole-genome sequenced, followed by comprehensive bioinformatic analysis and genetic manipulation to identify and validate genomic mutations responsible for rescuing the unc-104(R9Q) phenotype.

[0096] The inventors isolated 103 independent unc-104(R9Q) suppressors from approximately 100,000 mutagenized haploid genomes. Using whole-genome sequencing and bioinformatics, the inventors found 20 missense mutations within the motor domain, and most of the residues altered by the suppressor mutations are highly conserved between C. elegans and humans (FIG. B-C of Figure 1 The inventors also isolated one intergenic suppressor: the unc-16 E76K mutation, although it is less efficient in rescuing than the intragenic suppressors (FIG. D-E of Figure 1 UNC-16 encodes a conserved JNK signaling scaffold protein (FIG. B of Figure 1 UNC-16, a JNK-signaling scaffold protein, regulates vesicle transport in C. elegans. Neuron 32, 787-800, doi:10.1016 / s0896-6273(01)00532-3 (2001)).

[0097] The inventors obtained multiple alleles carrying the same intragenic mutations from independent genetic screens that have nearly identical effects in rescuing the UNC-104(R9Q) phenotype (FIG. B of Figure 1 UNC-104(R9Q) suppressors. To experimentally validate the recurrent mutations, the inventors introduced the unc-104(T102I) or unc-104(G265R) mutations into wild-type (WT) animals using genome editing tools, and neither of them exhibited any obvious movement or morphological defects (FIG. E of Figure 1 UNC-104(R9Q) (FIG. A of Figure 8 UNC-104(R9Q) (FIG. A of

[0098] Next, the animal swimming assay was used to quantify the recovery of the worm motility (Chiba, K. et al. Disease-associated mutations hyperactivate KIF1A motility and anterograde axonal transport of synaptic vesicle precursors. Proc Natl Acad Sci U S A 116, 18429-18434, doi:10.1073 / pnas.1905690116 (2019)). The inventors mounted a cMOS camera (IMX236, Sony Semiconductor Solutions Corporation) onto a compound microscope to obtain brightfield images. The swimming test was performed using young adults at day 1. All suppressors significantly improved the swimming ability of the worms compared to paralyzed unc-104(R9Q) animals, although the level of recovery varied greatly (Fig. E in Figure 1 The inventors selected five suppressors Y53F, T102I, A344V, A319T and G265R to evaluate their effects on the body length and morphology of unc-104(R9Q) (Figs. F-G in Figure 1 and B-C in Figure 8 ). Synchronized late L4 stage worms were imaged on NGM plates, and body length and body curvature analysis were performed. Fig. S1B describes how to perform image processing, measurement and index calculation with ImageJ (http: / / rsbweb.nih.gov / ij / ), and tightly coiled worms were not included in the body length analysis. The body length of adult unc-104(R9Q) mutant animals was only 58% of that of adult WT animals, and the five suppressors restored the body length of unc-104(R9Q) to 72-94% of the WT level. T102I and Y53F had the highest rescue effect, while G265R had the lowest rescue effect (Figs. D and F in Figure 1 and G in Figure 1 ).

[0099] We defined a coiling index to quantify the body curvature (Figs. B in Figure 8 and Methods), and the coiling index of WT animals was 0.08 ± 0.03 (mean ± standard deviation), while the average coiling index of unc-104(R9Q) mutants was 0.36 ± 0.19. The inventors found that the five suppressors significantly reduced the coiling index from 0.08 ± 0.05 (Y53F) to 0.10 ± 0.06 (G265R) (Figure 1 Graphs D and G in , and Figure 8 These data demonstrate that genetic suppressor mutations identified within the UNC-104 motor domain can rescue unc-104(R9Q) defects and that their rescue effects on animal motility, body length, and morphology are highly correlated.

[0100] Example 2 Biochemical effects of inhibitory factors on the KIF1A nucleotide binding pocket

[0101] To investigate the effects of internal inhibitors on the KAND R11Q mutant protein itself, the inventors tested the motility of purified mouse KIF1A protein (Cong, D. et al. Motor domain-mediated autoinhibition dictates axonal transport by the kinesin UNC-104 / KIF1A. PLoS Genet 17, e1009940, doi: 10.1371 / journal.pgen.1009940(2021)) with reference to previous studies. The specific experiments for measuring ATPase activity and processive motility of single KIF1A motor domain molecules are as follows:

[0102] 1. Protein Expression and Purification

[0103] The DNA fragments encoding mouse KIF1A gene include MD-NC-CC1-FHA tandem (residues 1-613), each KIF1A mutant gene fragment was cloned into a modified version of pET32a vector. All mutations in KIF1A fragments were generated using standard PCR-based mutagenesis methods and confirmed by DNA sequencing. Recombinant proteins were expressed in E. coli BL21 cells at 16 °C. After 16 hours of cultivation, cells were harvested and suspended in a buffer containing 50 mM Tris-HCl, pH 7.5, 500 mM (NH4)2SO4, 2 mM MgCl2, 1 mM EGTA, 1 mM PMSF. Proteins were purified by Ni2+-Sepharose 6 Fast Flow (GE healthcare) affinity chromatography, washing buffer contains 50 mM Tris-HCl, pH 7.5, 500 mM (NH4)2SO4, 2 mM MgCl2, 1 mM EGTA, 40 mM imidazole, elution buffer contains 50 mM Tris-HCl, pH 7.5, 500 mM (NH4)2SO4, 2 mM MgCl2, 1 mM EGTA, 500 mM imidazole. Proteins were further purified by size-exclusion chromatography (Superdex-200 26 / 60, GE healthcare), buffer contains 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 1 mM EGTA, 1 mM DTT.

[0104] 2. Microtubule-stimulated ATPase assay

[0105] Microtubule-stimulated ATPase activity of KIF1A MD-NC-CC1-FHA and various mutants of KIF1A was measured using HTS Kinesin ATPase Endpoint Assay Biochem Kit (Cytoskeleton, Inc., BK053). All measurements were based on the Malachite Green Phosphate Assay to detect inorganic phosphate produced during the reaction. A standard curve of phosphate was made to estimate the amount of phosphate produced. Each protein sample had three replicates, each measurement was repeated independently at least three times. Kinesin-1 heavy chain (KHC) provided in the kit was used as a control. All data were analyzed using GraphPad prism 8 program.

[0106] 3. Purification and labeling of tubulin

[0107] Crude microtubules were obtained from pig brain by a double cycle of polymerization and depolymerization. Microtubulin was further purified using a TOG-based affinity column. Microtubulin was labeled with TAMRA (Thermo Fisher Scientific) following standard protocols (Hyman, A. et al. Preparation of modified tubulins. Methods Enzymol 196, 478-485, doi:10.1016 / 0076-6879(91)96041-o (1991)).

[0108] 4. Polymerized taxol-stabilized microtubules

[0109] A 32 mM pig brain tubulin mix containing 5% tamra-labeled microtubulin was incubated with 1 mM GTP, 4 mM MgCl2 and 4% DMSO to prepare short microtubule seeds. After 5 min incubation on ice, the mix was polymerized overnight in the dark at 37°C in a water bath. The reaction was then stopped by adding 400 μΐ of warm BRB80 buffer (80 mM PIPES / KOH, pH 6.9, 1 mM MgCl2, 1 mM EGTA) containing 20 μΜ taxol. The sample was centrifuged at 15000 x g for 20 min at 25°C and the microtubule seeds were collected in 200 μΐ of pre-warmed taxol-BRB80 buffer.

[0110] 5. Total internal reflection fluorescence microscopy (TIRF) for single molecule experiments

[0111] Imaging was performed using a total internal reflection fluorescence microscope (Olympus) equipped with an Andor 897 Ultra EMCCD camera (Andor, Belfast, UK) under a 100x TIRF objective (NA 1.49, Olympus). Polymerized microtubules were diluted in taxol-brb80 buffer and then flowed into the flow cell, which was incubated at room temperature for 10 min to adsorb on the surface of the coverslip coated with anti-tubulin antibody (Sigma). Subsequently, the polymerized microtubules were protected from laser illumination by alkaline reaction mix buffer (0.08 mg / ml glucose oxidase, 0.032 mg / ml catalase, 0.16 mg / ml casein, 1% β-Mercaptoethanol, 0.001% tween, 1 mM MgCl2, 20 mM taxol, 80 mM D-Glucose). Finally, 50 μΐ of the final reaction mixture consisting of motor molecules, RM and 2 mM ATP was added into the flow chamber, and time-lapse movies were recorded. One image was taken every 0.1 s with an exposure time of 0.05 s and at low laser power to avoid photobleaching when the motor molecules were running continuously.

[0112] 6. Data analysis of single molecule experiments

[0113] In single molecule motility assays, the positions of fluorescently labeled motor molecules were manually tracked using ImageJ software (Fiji, NIH). In kymographs, the vertical distance represents time and the horizontal distance represents the distance of movement. The ratio of the length of movement and time is the velocity. The number of events for each protein sample was plotted as a histogram of velocity and run length and fitted as a Gaussian distribution. All statistical analyses were performed using GraphPad Prism (GraphPad Software) software. All data are presented as mean ± SD, and P values were calculated using a two-tailed unpaired Student t test.

[0114] As shown in Figure A of Figure 2 In the KIF1A motor domain, R11Q is located in the nucleotide binding pocket that captures ATP / ADP. In the WT motor domain, the side chain of R11 forms a hydrogen bond with the side chain of T106 at the bottom of the nucleotide binding pocket, and these two residues encircle the adenine ring of ATP / ADP, making the binding of the nucleotide more stable. Meanwhile, Y56 is adjacent to R11, and the side chain of Y56 forms an additional hydrogen bond with the side chain of D76, which can stabilize the nucleotide binding pocket.

[0115] In the R11Q mutant, the replacement of the long side chain arginine (R) with a short side chain glutamine (Q) may disrupt the hydrogen bond interaction with T106, reducing the spatial fit with the ATP / ADP adenine ring, thereby potentially destabilizing nucleotide binding and inactivating the kinesin motor. This structural analysis is consistent with earlier studies (Anazawa, Y., Kita, T., Iguchi, R., Hayashi, K. & Niwa, S. De novo mutations in KIF1A-associated neuronal disorder (KAND) dominant-negatively inhibit motor activity and axonal transport of synaptic vesicle precursors. Proc Natl Acad Sci USA 119, e2113795119, doi: 10.1073 / pnas.2113795119 (2022)). Figure 2 Panels B and C show that the R11Q mutation disrupts ATPase activity and processive motility of the KIF1A motor domain.

[0116] In the functionally restored mutants, Y56F and T106I substitutions in the nucleotide binding pocket can restore the function of R11Q. In the R11Q and Y56F double mutant KIF1A, the hydroxyl group is removed from Y56 to become F, which is unable to form a hydrogen bond with D76. The bulky aromatic side chain of Y56 initially faces the outside of the pocket. After mutating to F, the aromatic side chain tends to flip inward, occupying the gap left by the R11Q mutation in the nucleotide binding pocket of KIF1A, and making up for the defect caused by the R11Q mutation. In the R11Q and T106I double mutant KIF1A, the short side chain threonine (T) in the nucleotide binding pocket is replaced by the long side chain isoleucine (I), which can also occupy the gap caused by the R11Q mutation and form additional contacts with the adenine ring of ATP / ADP to compensate for the loss of nucleotide binding ( Figure 2 Thus, both the Y56F and T106I substitutions in KIF1A can structurally counteract the defect in the nucleotide binding pocket induced by the R11Q mutation.

[0117] It is noteworthy that the other three suppressor mutations, A319T, A344V, and G265R, which are not located around the nucleotide binding pocket, did not significantly restore KIF1A(R11A) activity in vitro ( Figure 2 D and E in Figure ), thus this group of mutations may rescue animal UNC defects through a mechanism different from that of Y56F and T106I.

[0118] Example 3 Suppressor mutations restore synaptic vesicle distribution and motility in KIF1A (R11A)

[0119] The inventors observed UNC-104::GFP fluorescence throughout the nervous system of WT animals, which is consistent with previously reported results and CeNGEN single-cell RNA-seq datasets (Taylor, SR et al. Molecular topography of an entire nervous system. Cell 184, 4329-4347e4323, doi: 10.1016 / j.cell.2021.06.023 (2021)). The inventors found that although the expression pattern of UNC-104 (R9Q) did not change significantly ( Figure 9 Figure A), but the fluorescence intensity was significantly reduced, especially in the nerve ring area. Figure 3 As shown in Figure A, the intragenic suppressor essentially restored the GFP intensity.

[0120] To measure UNC-104 dynamics, the inventors performed fluorescence recovery after photobleaching FRAP experiments on the dorsal nerve cord ( Figure 3 (B, red outline). The inventors performed FRAP experiments on L4 C. elegans expressing UNC-104::GFP using a laser scanning confocal microscope (LSM900, Zeiss) equipped with a 63x / 1.4NA Plan-Apochromat oil-immersion objective. Photobleaching, image acquisition, and raw fluorescence measurement were performed using the ZEN system, with identical experimental parameters. Dorsal nerve cord neurites without puncta and GFP signal were photobleached using a 488 nm laser within a rectangular region (65 × 30 pixels, 0.085 μm / pixel) for 40 consecutive excitations, with a laser power transmission setting of 10% and a pinhole diameter of 200 μm. Images were collected at 1% 488 nm laser power at 8-bit intensity resolution, using a pixel dwell time of 0.96 μs, over 250 × 60 pixels. Before photobleaching, two frames were collected, and fluorescence recovery was recorded for 120 s, with 2-s intervals to ensure that no further recovery was detected. Fluorescence recovery was calculated after subtracting background fluorescence intensity. The inventors found that UNC-104(R9Q)::GFP was barely detectable in the dorsal nerve cord, but a genetic inhibitor restored GFP fluorescence in this region ( Figure 9 (Figure BC).

[0121] Two of these inhibitors exhibited mobile ratios comparable to WT motor protein as determined by FRAP experiments: wild-type UNC-104 was 0.68 ± 0.10, mean ± SD, UNC-104(R9Q Y53F) was 0.72 ± 0.08, and UNC-104(R9Q T102I) was 0.70 ± 0.10 (Fig. 3, panels B-D, and Fig. 4, panel B). In contrast, consistent with partial rescue of animal motility, UNC-104(R9Q A344V) and UNC-104(R9Q G265R) partially rescued UNC-104::GFP bleaching recovery rates (Fig. 3, panels C-D, and Fig. 4, panel C). Figure 9 Figure 3 Fig. 4, panel D). UNC-104(R9Q T102I) and UNC-104(R9Q Y53F) showed intermediate effects (Fig. 3, panel D, and Fig. 4, panel D). Figure 9

[0122] Next, the inventors assessed the impact of genetic inhibitors on synapse distribution. In the dorsal nerve cord (DNC) of C. elegans, axons emanating from motor neuron cell bodies form en-passant synapses with muscles and other neurons. WT DA9 cholinergic motor neurons typically contain approximately 25 axonal anterograde synapses (Fig. 5, panel A) within the proximal axon segment of the DNC. Figure 4 To visualize these synapses, the inventors constructed transgenic worms expressing mScarlet::RAB-3, a marker for synaptic vesicle precursors (SVPs), driven by the DA9 promoter Pmig-13, and utilized live-cell imaging techniques for Kymograph extraction, image processing, and measurement of axonal synapse morphology and movement velocity in ImageJ, as follows:

[0123] ​​Synchronized late-L4 nematodes were anesthetized with M9 buffer containing 0.1 mmol / L levamisole and immobilized on 2% (weight / volume) agarose pads for live-cell imaging. Our imaging system consisted of an IX83 inverted microscope (Olympus Lifescience, Inc.) equipped with an EM CCD camera (Andor iXon+DU-897D-C00-#BV-500) and a spinning disk confocal scanner (Yokogawa CSU-X1 Spinning Disk Unit) equipped with a Sapphire CW CDRH USB laser system with 488 nm and 561 nm lines. UNC-104::GFP signals were imaged in whole animals using a 10X / 0.3NA UPlan semi-apochromatic WD objective. Static images of mScarlet::RAB-3 fluorescent spots along the axons of DA9 neurons were captured using a 60X, 1.30NA UPlanSAPo silicone oil-immersion objective with an exposure time of 200 ms, and time-lapse images showing mScarlet::RAB-3 trafficking were acquired with 200 frames without interval using a 100X, 1.49NA Apochromat oil-immersion objective with an exposure time of 200 ms.

[0124] In unc-104(R9Q) animals, DA9 neurons almost completely lacked midway synapses. The length of the synaptic region in DA9 neurons was reduced from 115.10 ± 12.69 μm in WT animals to 20.48 ± 26.09 μm in unc-104(R9Q) animals. Figure 4 AC in Figures 1A and 1B). The number of mScarlet::RAB-3 puncta decreased from 28.36 ± 5.77 in WT to 2.08 ± 2.04 in unc-104(R9Q) animals ( Figure 4 AC in Figures 1A and 1B). In addition, the size and intensity of mScarlet::RAB-3 in individual axonal synaptic puncta were reduced in unc-104(R9Q) ( Figure 4 Notably, the intragenic inhibitor significantly restored the distribution area, number, and size of synapses ( Figure 4 Figures A and E in the figure).

[0125] To investigate whether the axonal synaptic defects in unc-104(R9Q) mutants were caused by vesicle trafficking, we examined the movement of mScarlet::RAB-3 puncta in the proximal region of DA9 axons ( Figure 4 Kymography analysis revealed that RAB-3 was frequently transported axonally in both anterograde and retrograde directions in WT animals ( Figure 5In contrast, the unc-104(R9Q) mutation disrupts anterograde trafficking of RAB-3 and significantly reduces retrograde trafficking events ( Figure 5 AF panels in Figure 3), indicating that UNC-104 movement along axons is impaired. Intragenic inhibitors restored the frequency of anterograde and retrograde axonal transport, as well as the distribution and movement of synaptic vesicles in unc-104(R9Q) mutant animals ( Figure 5 Figures A and E and E in the figure explain the mechanism of recovery of the UNC phenotype in animals. The A344V and G265R inhibitors that rescued the motility of animals did not restore the activity of the UNC-104 (R9Q) motor in vitro, but partially restored the velocity and run length of synaptic vesicles ( Figure 5 AG diagram in, Figure 10 ), further indicating that A344V or G265R adopts a different rescue mechanism from Y56F or T106I.

[0126] Example 4 Fisetin alleviates UNC-104(R9Q) animal movement and morphological defects

[0127] Having demonstrated that UNC-104(R9Q) abnormalities can be restored through various pathways in vivo, the inventors next aimed to investigate whether treatment with small molecule chemicals could restore animal behavior. To test the potential of fisetin in alleviating the symptoms of KAND-related diseases, the inventors added fisetin to the nematode growth medium and found that fisetin improved locomotion and alleviated the developmental and behavioral defects of unc-104(R9Q) mutant animals. Figure 6 These effects were dose-related, with higher doses showing greater improvements in locomotor capacity, body length, and posture ( Figure 6 In addition, the inventors observed that the distribution and number of synapses were partially restored after fisetin treatment ( Figure 6 (Figure EF in Figure 5). This suggests that the improvement in behavioral abnormalities in the animals may be attributable to the restoration of synaptic function. These results highlight the potential of fisetin supplementation as a promising therapeutic strategy for alleviating symptoms associated with KAND.

[0128] Example 5 Fisetin restores the ATPase activity and processive motility of the motor domain of KIF1A(R11Q)

[0129] The inventors further evaluated the potential direct effect of fisetin on the KIF1A protein. To evaluate this, the inventors first performed docking analysis between fisetin and the KIF1A motor with the R11Q mutation: using PyMOL (version 2.5, LLC) introduced the R11Q mutation into the KIF1A structure (PDB ID: 2ZFI). The protein was expressed using BioLuminate software (Maestro BioLuminate 4.4, LLC, New York, NY, 2021) After minimization of the prefabricated KIF1A(R11Q) and fisetin structures, induced fit docking was performed between the two molecules, and the side chain conformations of Tyr67 and Gln70 were set to be adjustable.

[0130] Docking results showed that fisetin can effectively insert into the structural gap caused by the R11Q mutation, possibly restoring the structural stability of the nucleotide binding pocket destroyed by the R11Q mutation. These findings provide a valuable structural basis for the observation that fisetin treatment can restore the behavior of R11Q mutant nematodes. In contrast, in the wild-type KIF1A motor domain, the guanidinyl portion of R11 occupies part of the pocket, thereby hindering the binding of emodin ( Figure 7 These results highlight that fisetin specifically recognizes and restores KIF1A-R11Q without affecting the wild-type KIF1A motor.

[0131] Subsequently, the inventors purified mouse and human KIF1A (R11Q) proteins ( Figure 11 (Figure A in the middle), and biochemical analysis was performed to evaluate the effect of fisetin on its ATPase activity. The specific method for measuring ATPase activity is described in Example 2. The inventors observed that the ATPase activity of KIF1A (R11Q) treated with 0.1% DMSO was negligible. However, when fisetin was added, ATPase activity was detected ( Figure 7 B in the figure), the inventors tried to add different concentration gradients of fisetin, such as Figure 15 As shown in the results, it was found that different concentrations of fisetin could enhance ATPase activity. Although in many concentration gradients, 10 nmol / L fisetin could only increase the ATPase activity of KIF1A(R11Q) to about 12% of wild-type KIF1A, while Y57F and T102I inhibitors increased the ATPase activity of KIF1A(R11Q) to 15% and 22% of wild-type levels, respectively. Figure 2 However, both modestly restored the defects induced by KIF1A-R11Q (Figure 2B), suggesting that even modest restoration of ATPase activity may be sufficient to rescue behavioral deficits in animals.

[0132] In addition, the inventors performed microtubule gliding assays to examine whether fisetin treatment could induce force generation in KIF1A(R11Q). KIF1A(R11Q) did not exhibit any microtubule gliding in vitro in the absence of fisetin. However, the addition of 10 nmol / L of fisetin in the gliding assay could make microtubules glide at a speed of 0.49 micrometers / second, comparable to that of wild-type KIF1A Figure 11 FIG. 2B and Figure 14 ). In addition, using single molecule assays, the specific method of measuring the progressive movement of KIF1A motor domain single molecules is described in Example 2. The inventors demonstrated that fisetin treatment enabled KIF1A(R11Q) to perform continuous movement on microtubules, although the speed and distance of movement were lower than wild-type KIF1A Figure 7 FIG. 2C-D, Figure 12 , Figure 13 ). In line with the prediction of molecular docking, the inventors did not observe any effect of fisetin on the ATPase enzymatic activity or continuous movement of wild-type KIF1A Figure 11 FIG. 2C). These findings suggest that fisetin is able to specifically enhance the function of KIF1A(R11Q) mutant protein, while having no effect on wild-type KIF1A.

[0133] The present invention introduced the clinically reported KIF1A(R11Q) mutation into the C. elegans homolog UNC-104, which resulted in uncoordinated (UNC) mutant animals. Through suppressor genetic screening, the inventors isolated 20 different amino acid mutations in the UNC-104 motor domain that rescued the cellular and behavioral defects of KAND mutant animals. In vitro experiments showed that two of these suppressor mutations, by alleviating the structural defect of the KIF1A nucleotide binding pocket caused by R11Q, could partially restore the motility activity of the molecular motor, based on which the inventors found that the supplementation of a plant flavonol fisetin could improve the movement and morphological defects of KIF1A(R11Q) mutant animals. Biochemical and single molecule analysis results showed that fisetin directly acted to restore the ATPase activity and progressive movement of human KIF1A(R11Q) protein, and did not affect wild-type KIF1A. The above findings suggest that small molecule fisetin intervention has the potential function of enhancing KIF1A(R11Q) molecular motor activity to improve the defects of KAND-related diseases.

[0134] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", "some implementations" or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples, without contradiction.

[0135] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. Use of fisetin in the preparation of a medicament for alleviating motor or morphological defects caused by the conversion of lysine at position 11 in the KIF1A motor domain to glutamine; or Restores the reduced ATPase activity or progressive motor defects of the motor domain caused by the conversion of lysine 11 in the KIF1A motor domain to glutamine.

2. The use according to claim 1, characterized in that The drug further contains a pharmaceutically acceptable excipient or carrier.

3. The use according to claim 2, characterized in that The excipients include at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent, and a sustained-release agent.

4. The use according to claim 2, characterized in that The carrier comprises at least one selected from sugars, starches, cellulose and its derivatives, calcium phosphates, vegetable oils, fatty alcohols, and hydrolyzed cereal solids.

5. The use according to claim 1, characterized in that The dosage form of the drug includes at least one selected from injection, powder, granule, and emulsion.

6. The use according to claim 1, characterized in that The administration method of the drug includes at least one selected from injection administration, oral administration, and immersion administration.

7. The use according to claim 6, characterized in that The injection administration includes at least one selected from intravascular injection, percutaneous or natural body cavity puncture perivascular injection, intravascular wall injection, intramuscular injection, and subcutaneous injection.

8. The use according to claim 1, characterized in that The effective concentration of fisetin is between 1 μmol / L and 200 nmol / L.

9. The use according to claim 8, characterized in that The effective concentration of fisetin is between 1 μmol / L and 100 nmol / L.

10. The use according to claim 8, characterized in that The effective concentration of fisetin is 10 nmol / L.

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