Vesicular neurotransmitter leakage assay

Abstract

Provided herein are methods of measuring vesicular leakage utilizing a fluorescent false neurotransmitter and methods of identifying a compound that modulates vesicular leakage utilizing a fluorescent false neurotransmitter.

Description

VESICULAR NEUROTRANSMITTER LEAKAGE ASSAY

[0001] This International Patent Application claims the benefit of and priority to U.S. Provisional Application No. 63 / 468,701, filed May 24, 2023, entitled “VESICULAR NEUROTRANSMITTER LEAKAGE ASSAY”, the contents of which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grants ES007322, DA052498, and ES023839 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.INCORPORATION BY REFERENCE

[0004] All patents, patent applications and publications, and other literature references cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.TECHNICAL FIELD

[0005] The present invention relates, in general, to a method of measuring vesicular leakage and a method of identifying a compound that modulates vesicular leakage. More particularly, the present invention relates to the use of a fluorescent false neurotransmitter and determination of the rate of fluorescence decay of the fluorescent signal of the fluorescent false neurotransmitter.BACKGROUND OF THE INVENTION

[0006] Neuronal circuits depend on chemical communication between neurons through the vesicular transport of neurotransmitters. Quantitative analysis of vesicular neurotransmitter sequestration and retention is essential for the study of neuronal activity and neurological disorders. Described herein is an assay using false neurotransmitters to quantify vesicular neurotransmitter sequestration dynamics.SUMMARY OF THE INVENTION

[0007] In certain aspects, described herein is a method of measuring vesicular leakage, the method comprising: (a) contacting a cell culture with a false neurotransmitter, (b) measuring a signal of the false neurotransmitter in real-time, and (c) determining a rate of fluorescence decay.

[0008] In certain aspects, described herein is a method of identifying a compound that modulates vesicular leakage, the method comprising: (a) contacting a cell culture with a fluorescent false neurotransmitter, (b) measuring a fluorescence signal of the false neurotransmitter, (c) contacting the cell with a test compound, (d) measuring a fluorescence signal of the fluorescent false neurotransmitter in real-time, (e) determining a rate of fluorescence decay of the fluorescence signal, and (f) identifying the test compound as a compound that modulates vesicular leakage if: the rate of fluorescence decay determined in step e) is higher or lower than a rate of fluorescence decay of a cell culture under a control condition.

[0009] In certain aspects, described herein is a method of identifying a compound that modulates vesicular leakage, the method comprising: (a) contacting a cell culture with a test compound (b) contacting the cell with a fluorescent false neurotransmitter, (c) measuring a fluorescent signal of the fluorescent false neurotransmitter in real-time, (c) determining a rate of fluorescence decay of the fluorescence signal, and (d) determining a rate of fluorescence decay of the fluorescence signal, and (e) identifying the test compound as a compound that modulates vascular leakage if the rate of fluorescence decay determined in step c) is higher or lower than a rate of fluorescence decay of a cell culture under a control condition.

[0010] In certain aspects, described herein is a method of treating a neurological disorder in a subject in need thereof, comprising: (a) contacting a cell culture with a fluorescence falseneurotransmitter, (b) measuring a fluorescence signal of the fluorescent false neurotransmitter, (c) contacting the cell culture with a test compound, (d) measuring a fluorescence signal of the fluorescent false neurotransmitter in real-time, and (e) determining a rate of fluorescence decay, and (f) administering the test compound to the subject if the rate of fluorescence decay determined in step e) is higher than a rate of fluorescence decay of a cell culture under a control condition.

[0011] In some embodiments, the cells of the cell culture are genetically modified cells. In some embodiments, the cells of the cell culture have reduced synaptic vesicle glycoprotein 2C (SV2C) expression compared to a cell without genetic modification. In some embodiments, the cells of the cell culture have reduced expression of synaptic vesicle localized proteins compared to a cell without genetic modification. In some embodiments, the cells of the cell culture have reduced expression of plasma membrane localized proteins compared to a cell without genetic modification. In some embodiments, the cells of the cell culture have reduced expression of organelle localized proteins compared to a cell without genetic modification.

[0012] In some embodiments, the false neurotransmitter is a substrate for vesicular neurotransmitter transporter proteins. In some embodiments, the false neurotransmitter mimics catecholamines. In some embodiments, the false neurotransmitter mimics monoamines. In some embodiments, the false neurotransmitter mimics dopamine. In some embodiments, the false neurotransmitter is a fluorescent false neurotransmitter. In some embodiments, the fluorescent false neurotransmitter comprises false fluorescent neurotransmitter 206 (FFN206). In some embodiments, the fluorescent false neurotransmitter comprises a dopamine analogue.

[0013] In some embodiments, the rate of fluorescence decay of the fluorescence signal is determined by i) for each fluorescence signal value of the real-time measurement, subtracting a background fluorescence signal and calculating a percent fluorescence signal change relative to the fluorescence signal measured in step b), and ii) plotting the percentage of fluorescence signal changes versus time; and iii) calculating the slope of the percentage of fluorescence signal change versus time.

[0014] In some embodiments, the rate of fluorescence decay of the fluorescence signal is determined by i) for each fluorescence signal value of the real-time measurement, subtractinga background fluorescence signal, and ii) plotting the fluorescence signal value versus time; and iii) calculating the slope of the fluorescence signal value.

[0015] In some embodiments, a cell culture under a control condition comprises a cell culture cultured under standard cell culturing conditions for such a cell culture. In some embodiments, a cell culture under a control condition comprises a cell culture contacted with a control compound. In some embodiments, the control compound is a solvent. In some embodiments, the control compound is sterile ultrapure water. In some embodiments, the control compound is dimethyl sulfoxide (DMSO). In some embodiments, the test compound is a pharmacological regulator. In some embodiments, the test compound comprises tetrabenazine (TBZ). In some embodiments, the test compound is an environmental or toxicological regulator. In some embodiments, the test compound comprises l-methyl-4- phenyl- 1 ,2,3 ,6-tetrahydropyri dine (MPTP).BRIEF DESCRIPTION OF THE FIGURES

[0016] The patent or application file contains at least one drawing executed in color. To conform to the requirements for PCT patent applications, many of the figures presented herein are black and white representations of images originally created in color.

[0017] Figure 1 shows a schematic showing that synaptic vesicle glycoprotein 2C (SV2C) promotes retention of dopamine and dopamine analogues (FFN206, MPP+) within synaptic vesicles.

[0018] Figures 2A-2C show western blot and immunocytochemistry demonstrating SV2C expression in HEK-VMAT2-SV2C cells. Figure 2A shows western blot performed on whole cell lysates to identify SV2C protein expression HEK-VMAT2-SV2C cells compared to HEK-VMAT2 cells lacking SV2C. Figure 2B shows representative 40x immunocytochemistry images visualizing VMAT2 (green) and SV2C (red) protein expression in HEK-VMAT2 cells and HEK-VMAT2-SV2C cells (transmitted light with scale bar). Figure 2C shows co-localization of SV2C with VMAT2 in HEK-VMAT2-SV2C cells demonstrated by overlap of enlarged inset.

[0019] Figures 3A-3C show that SV2C enhances vesicular uptake of the fluorescent dopamine analogue FFN206. Figure 3 A shows representative lOx images of FFN206 uptake in HEK-VMAT2 (top) and HEK-VMAT2-SV2C (bottom) cells. Figure 3B shows FFN206uptake measured in HEK-VMAT2 and HEK-VMAT2-SV2C cells seeded on 96-well plates following control (DMSO) or tetrabenazine (IpM) pretreatment. Values were transformed into percent control of the average HEK-VMAT2 DMSO value following background subtraction. For each experiment there were 12 technical replicates for each cell and treatment condition which were averaged to a single value for comparison across seven experimental replicates. Two-way ANOVA with Sidak's multiple comparisons test, n = 7 experimental replicates, a:b ****p <0.0001, b:c ****p <0.0001, a:c **p <0.005. Figure 3C shows IC50 calculation for tetrabenazine in HEK-VMAT2 vs HEK-VMAT2-SV2C cells. FFN206 uptake was measured in HEK-VMAT2 and HEK-VMAT2-SV2C cells seeded on 96-well plates following control (DMSO) or tetrabenazine (0.00 IpM - lOOpM) pretreatment. Values were transformed into percent control of the average HEK-VMAT2 DMSO value. For each experiment there were 4-6 technical replicates for each cell and treatment condition which were averaged to a single value for comparison across 4-8 experimental replicates. Non-linear regression to calculate IC50 values for each cell line demonstrated HEK-VMAT2 IC50 = 0.1348pM and HEK-VMAT2-SV2C IC50 = 0.2749pM. Comparison of non-linear regressions determined the curves of best fit were significantly different for each cell line p <0.05.

[0020] Figures 4A-4B show that SV2C enhances retention of the dopamine analogue FFN206. Figures 4A and 4B show FFN206 retention measured in HEK-VMAT2 and HEK- VMAT2-SV2C cells seeded on 96-well plates. Following a scan for baseline fluorescence, control (DMSO) or tetrabenazine (IpM) was added and the plate was scanned for 45m in 2.5m intervals. Each well was normalized to its own baseline fluorescent value and values are plotted as percent of baseline. Each experiment had 12 experimental replicates per cell and treatment combination, with 10 DMSO experimental replicates and 4 tetrabenazine experimental replicates. DMSO was analyzed using linear regression and TBZ was analyzed by performing non-linear regression single-phase decay. Regressions were compared and determined that line of best fit was significantly different for each cell line in the DMSO (p < 0.01) and TBZ condition (p < 0.0001).

[0021] Figures 5A-5B show that SV2C increases vesicular [3H]-dopamine uptake and retention in isolated vesicles. Figure 5A shows vesicles isolated from HEK-VMAT2 and HEK-VMAT2-SV2C cells before undergoing radiolabeled dopamine uptake assay. Vesicles isolated from HEK-VMAT2 cells averaged 2951.19fmol / ug of tritiated dopamine uptakecompared to HEK-VMAT2-SV2C cells which averaged 4369.47fmol / ug of tritiated dopamine uptake. Unpaired t-test, n = 11-12, *p < 0.05. B. SV2C slows vesicular leakage of [3H]-dopamine from isolated vesicles. Figure 5B shows values transformed into percent control of the average time value within cell line. Non-linear regression performed using one- phase decay to calculate half-life values for each cell line. HEK-VMAT2 tl / 2 = 8.4m; n = 11- 12 for each time-point. HEK-VMAT2-SV2C tl / 2 = 12.7m; n = 11-12 for each time-point. Comparison of non-linear regressions determined the curves of best fit were significantly different for each cell line ****p < 0.0001.

[0022] Figure 6 shows that genetic ablation of SV2C increases vesicular leakage of [3H]- dopamine in brain-derived vesicles. Values were transformed into percent control of the average timeo value within mouse line. Non-linear regression performed using one-phase decay to calculate half-life values for each mouse line. WT ti / 2 = 3.3m; n = 3-4 for each timepoint. SV2C-KOti / 2 = 2.0m; n = 3-4 for each time-point. Comparison of non-linear regressions determined the curves of best fit were significantly different for each mouse line ***p < 0.005.

[0023] Figures 7A-7B show that SV2C increases vesicular uptake and retention of [3H]- MPP+. Figure 7A shows vesicles isolated from HEK-VMAT2 and HEK-VMAT2-SV2C cells before undergoing uptake assay. Vesicles isolated from HEK-VMAT2 cells averaged1390.3 Ifmol / ug of tritiated MPP+uptake compared to HEK-VMAT2-SV2C cells which averaged 2010.14fmol / ug of tritiated MPP+uptake. Unpaired t-test, n = 12, *p < 0.05. Figure 7B shows that SV2C increases vesicular retention of [3H]-MPP+. Values were transformed into percent control of the average timeo value within cell line. Non-linear regression performed using one-phase decay to calculate half-life values for each cell line. HEK- VMAT2 ti / 2 = 3.5m; n = 10-12 for each time-point. HEK-VMAT2-SV2C ti / 2 = 4.3m; n = 10- 12 for each time-point. Comparison of non-linear regressions determined the curves of best fit were significantly different for each cell line ****p < 0.0005.

[0024] Figure 8 shows that genetic ablation of SV2C enhances dopamine vulnerability to MPTP. Representative TH immunohistochemistry of the striatum and midbrain of wild-type and SV2C-KO animals treated with saline or MPTP demonstrates that the lesion to the basal ganglia is enhanced in both the dorsal striatum and substantia nigra of SV2C-KO animals (bottom) as compared to wild-type controls (top).

[0025] Figure 9 shows that genetic ablation of SV2C results in increased loss of nigral TH+ neurons following MPTP as compared to wild-type animals. Quantification of intact dopaminergic cells of the SNc using unbiased stereological cell counting confirms a significant loss dopaminergic nigral cells in SV2C-KO, but not WT animals following MPTP. Nigral TH+ neuronal counts: Two-way ANOVA with Tukey’s multiple comparisons test, n = 4-10. WT:saline vs. SV2C-KOMPTP *p<0.05; WT:MPTP vs. SV2C-KO: saline *p<0.05; SV2C-KO: saline vs. SV2C-KO:MPTP **p<0.01. Striatal TH expression: Two-way ANOVA with Tukey’s multiple comparisons test, n = 4-10. WT:saline vs. WT:MPTP ****p <0.0001; WT:saline vs. SV2C-KO:MPTP ****p <0.0001; WT:MPTP vs. SV2C-KO: saline ****p <0.0001; SV2C-KO: saline vs. SV2C-KO:MPTP ****p <0.0001..

[0026] Figure 10 shows wild-type vs. SV2C-KO uptake of tritiated dopamine. Radiolabeled dopamine uptake in isolate vesicles from WT and SV2C-KO animals (n = 7 animals per genotype) across multiple concentrations of dopamine (0.03 pM - lOpM).

[0027] Figure 11 shows that genetic ablation of SV2C results in increased loss of nigral TH+ neurons following MPTP as compared to wild-type animals. Quantification of intact dopaminergic cells of the SNc using unbiased stereological cell counting confirms a significant loss dopaminergic nigral cells and striatal TH expression in SV2C-KO, but not WT animals following MPTP. Nigral TH+ neuronal counts: Two-way ANOVA with Tukey’s multiple comparisons test, n = 2-4. WT:saline vs. SV2C-KOMPTP *p<0.05; WT:MPTP vs. SV2C-KO:MPTP *p<0.05; SV2C-KO: saline vs. SV2C-KO:MPTP *p<0.05. Striatal TH expression: Two-way ANOVA with Tukey’s multiple comparisons test, n = 2-4. SV2C- KO:saline vs. SV2C-KO:MPTP *p <0.05.

[0028] Figure 12 shows the effect that MPP+ has on the vesicular sequestration of the VMAT2 substrate FFN206. To investigate the effect that MPP+ has on the vesicular sequestration of the VMAT2 substrate FFN206, the uptake of FFN206 in HEK-DAT- VMAT2 pre-treated with a dose response of MPP+ cells was measured. Dose-response experiments revealed an IC50 of 79.74nM (non-linear regression [inhibitor] vs. normalized response, n = 2 experimental replicates for each dose).

[0029] Figure 13 shows that fluorescence decays linearly over time in HEK-DAT- VMAT2 cells in the retention assay. The slope of HEK-DAT-VMAT2 cells in a sterile water (i.e. OpM MPP+) condition equals -0.18, representing a 0.18% loss of fluorescence perminute, whereas the slope for HEK-DAT-VMAT2 cells with increasing doses of MPP+ show a slope of -0.22, -0.34, -0.49, -0.56 with O. lpM, IpM, lOpM, and lOOpM MPP+ respectively (linear regression, n = 2 experimental replicates, p <0.0001).

[0030] Figure 14 shows SV2C expression in HEK cells co-expressing VMAT2

[0031] Figure 15 shows that SV2C enhances vesicular uptake of the fluorescent dopamine analogue FFN206.

[0032] Figure 16 shows that SV2C increases vesicular [3H]-dopamine uptake and retention in isolated vesicles.

[0033] Figure 17 shows that SV2C increases vesicular uptake and retention of [3H]- MPP .

[0034] Figures 18A-B show genetic ablation of SV2C results in loss of nigral TH+ neurons following mild MPTP treatment regimen. Figure 18A shows nigral TH+ neurons treated with either saline or MPTP. Figure 18B shows striatal TH expression following saline or MPTP treatment.

[0035] Figure 19 shows that genetic ablation of SV2C does not affect vesicular capacity of [3H]-dopamine uptake in brain-derived vesicles. Radiolabeled dopamine uptake in isolated vesicles from WT and SV2C-KO animals (n = 7 animals per genotype) across multiple concentrations of dopamine (0.03 pM - lOpM). Data points represent mean values ± SEM.

[0036] Figure 20 shows that genetic ablation of SV2C results in increased loss of nigral TH+ neurons following MPTP (Sigma) as compared to wild-type animals. Quantification of intact dopaminergic cells of the SNc using unbiased stereological cell counting confirms a significant loss dopaminergic nigral cells in SV2C-KO, but not WT animals following MPTP. Nigral TH+ neuronal counts: Two-way ANOVA with Tukey’s multiple comparisons test, effect of genotype (F(l, 23) = 0.03027, p=0.8634); effect of treatment (F,(l, 23) = 16.45, p =0.0005; effect of interaction (F(l, 23) = 0.01901, p =0.8915). n = 4-10 animals per genotype:treatment group. WT:saline vs. SV2C-KO:MPTP p =0.0347; WT:MPTP vs. SV2C- KO:saline p =0.0469; SV2C-KO: saline vs. SV2C-KO:MPTP p =0.0071. Striatal TH expression: Two-way ANOVA with Tukey’s multiple comparisons test, effect of genotype (F(l, 23) = 0.1351, p=0.7166); effect of treatment (F,(l, 23) = 106.3, p =<0.0001; effect ofinteraction (F(l, 23) = 0.6757, p =0.4195). n = 4-10 animals per genotype:treatment group.WT:saline vs. WT:MPTP p <0.0001; WT:saline vs. SV2C-KO:MPTP p <0.0001;WT:MPTP vs. SV2C-KO: saline p <0.0001; SV2C-KO: saline vs. SV2C-KO:MPTP p <0.0001.

[0037] Figure 21 shows that genetic ablation of SV2C results in increased loss of nigral TH+ neurons following MPTP (MedChemExpress) as compared to wild-type animals. Quantification of intact dopaminergic cells of the SNc using unbiased stereological cell counting confirms a significant loss dopaminergic nigral cells and striatal TH expression in SV2C-KO, but not WT animals following MPTP. Nigral TH+ neuronal counts: Two-way ANOVA with Tukey’s multiple comparisons test, effect of genotype (F(l, 6) = 14.92, p=0.0652); effect of treatment (F,(l, 6) = 9.214, p =0.0229; effect of interaction (F(l, 6) = 5.075, p =0.0652). n = 2-4 animals per genotype:treatment group. WT:saline vs. SV2C- KO:MPTP p =0.0151; WT:MPTP vs. SV2C-KO:MPTP p = 0.0135; SV2CKO: saline vs. SV2C-KO:MPTP p = 0.480. Striatal TH expression: Two-way ANOVA with Tukey’s multiple comparisons test, effect of genotype (F(l, 10) = 0.01255, p=0.9130); effect of treatment (F,(l, 10) = 12.98, p =0.0048; effect of interaction (F(l, 10) = 2.889, p =0.1200). n = 2-4 animals per genotype:treatment group. SV2C-KO: saline vs. SV2C-KO:MPTP p =0.0167.DETAILED DESCRIPTION

[0038] All patent applications, published patent applications, issued and granted patents, texts, and literature references cited in this specification are hereby incorporated herein by reference in their entirety to more fully describe the state of the art to which the present disclosed subject matter pertains.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0040] Neuronal circuits depend on chemical communication between neurons through the vesicular transport of neurotransmitters. Quantitative analysis of vesicular neurotransmitter sequestration and retention is essential for the study of neuronal activity and neurological disorders. While methods have been developed to quantify the speed and quantity of vesicular transport, assays for neurotransmitter retention or leakage are limited.

[0041] Disruption of vesicular integrity or function results in decreased release of neurotransmitters and increased cytoplasmic neurotransmitters, both toxic for neurons (Alter S. et. al. Curr Neurol Neurosci Rep. 2013 Jul; 13(7): 362). Dysfunction in the vesicular synapse may contribute to the pathogenesis of PD. (Alter S. et. al. Curr Neurol Neurosci Rep. 2013 Jul; 13(7): 362). Cytoplasmic accumulation of dopamine can lead to oxidative stress and metabolic dysfunction, both observed in Parkinson’s disease patients. (Lotharius J. Human Molecular Genetics. 2002 Oct; 11(20): pp. 2395-2407). Vesicle leakage events are less characterized and difficult to discern with current ensemble assays (Kyoung M. Proc Natl Acad Sci USA. 2011 Jul; 108(29): pp. 304-313).

[0042] In certain aspects, described herein is a method of measuring vesicular leakage, the method comprising: a) contacting a cell culture with a fluorescent false neurotransmitter, b) measuring a fluorescence signal of the fluorescent false neurotransmitter in real-time, and c) determining a rate of fluorescence decay of the fluorescence signal.

[0043] In certain aspects, described herein is a method of measuring vesicular retention or leakage, the method comprising: a) contacting a cell culture with a fluorescent false neurotransmitter, b) measuring a fluorescence signal of the fluorescent false neurotransmitter in real-time, and c) determining a rate of fluorescence decay of the fluorescence signal.

[0044] In some embodiments, a fluorescence signal of the fluorescent false neurotransmitter is measured in real-time. In some embodiments, real-time measurement comprises continuous or near-continuous data collection that can be used in any of the methods described herein. For example, in some embodiments, real-time measurement comprises repeated collection of data from the same cell culture (e.g., a well of a tissue culture plate) over the entire time period of the method (e.g., a time-lapse). In some embodiments the real-time measurements are collected over at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments the real-time measurements are collected over 45 minutes.

[0045] In some embodiments, the cell culture comprises cells cultured in a tissue culture plate. In some embodiments, the cell culture comprises cells cultured in a multiwell plate, such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, 96-well plate, a 96- well half area plate, 384-well plate, or a 1536-well plate. In some embodiments, the multiwell plate is a black walled plate. In some embodiments, the multiwell plate is a black walled with clear flat bottom. In some embodiments, the multiwell plate is a black walled with clear flat bottom half-area 96-well plate.

[0046] In some embodiments, cell culture comprises cells that grow in suspension. In one embodiment, cell culture comprises cells that grow as attached cells. In some embodiments, the cell culture is a confluent cell culture before the contacting of step (b). In some embodiments, the cells of the cell culture are incubated in a cell culture medium.

[0047] In some embodiments, the cells of the cell culture are an immortalized mammalian cell line. Examples of immortalized cells lines include but not limited to PC 12, RCSN-3, BE2, SH-SY5Y, and HEK-293 cells. In some embodiments, the cells of the cell culture are induced pluripotent stem cells. In some embodiments, the cells of the cell culture are neuronal cultures differentiated from induced pluripotent stem cells. In some embodiments, the cells of the cell culture are primary neuronal cultures. In some embodiments, the cells of the cell culture are HEK-293 cells.

[0048] In some embodiments, the cells of the cell culture express a protein of interest. In some embodiments, the cells of the cell culture an increased expression level of a protein of interest. In some embodiments, the cells of the cell culture have an increased expression level of a protein of interest compared to normal, wild-type cells of the same cell type. In some embodiments, the cells of the cell culture do not express a protein of interest. In some embodiments, the cells of the cell culture have a reduced expression level of a protein of interest. In some embodiments, the cells of the cell culture have a reduced expression level of a protein of interest compared to normal, wild-type cells of the same cell type.

[0049] In some embodiments, the cells of the cell culture express vesicular monoamine transporter 2 (VMAT2). In some embodiments, the cells of the cell culture are HEK-293 cells that express VMAT2. In some embodiments, the cells of the cell culture express synaptic vesicle glycoprotein 2C (SV2C). In some embodiments, the cells of the cell culture are HEK- 293 cells that express SV2C. In some embodiments, the cells of the cell culture expressVMAT2 and SV2C. In some embodiments, the cells of the cell culture are HEK-293 cells that express VMAT2 and SV2C. In some embodiments, the cells of the cell culture express at least one of VMAT2, dopamine transporter (DAT), or synaptic vesicle glycoprotein 2C (SV2C). In some embodiments, the cells of the cell culture are HEK-293 cells that express at least one of the following proteins: VMAT2, DAT, or SV2C. In some embodiments, the cells of the cell culture are genetically modified cells. In some embodiments, the cells of the cell culture have reduced synaptic vesicle glycoprotein 2C (SV2C) expression level. In some embodiments, the level of SV2C is reduced as compared to normal, wild-type cells of the same cell type. In some embodiments, the cells of the cell culture are genetically modified to knock out SV2C. In some embodiments, the cells of the cell culture have reduced expression of synaptic vesicle localized proteins compared to a cell without genetic modification. In some embodiments, the cells of the cell culture have reduced expression of plasma membrane localized proteins compared to a cell without genetic modification. In some embodiments, the cells of the cell culture have reduced expression of organelle localized proteins compared to a cell without genetic modification.

[0050] In some embodiment, targeted gene expression can be reduced by several genome editing techniques such as RNAi (RNA interference), zinc finger nucleases (ZFNs), a TALE- effector domain nuclease (TALLEN), prime editing and base editing, CRISPR / Cas9 systems which are known in the art. In some embodiment, the CRISPR / Cas9 systems comprise a guide RNA (gRNA) or a single-molecule guide RNA (sgRNA). In some embodiment, the gRNA or sgRNA comprises a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding CASP8. In some embodiment, the gRNA or sgRNA comprises a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding VMAT2. In some embodiment, the gRNA or sgRNA comprises a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding SV2C. In some embodiment, the gRNA or sgRNA comprises a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding a protein of interest. Inhibition of RNA encoding a protein of interest, such as VMAP2 and / or SV2C, can effectively modulate the expression of these proteins. Inhibitors can include shRNAs encoding siRNAs, siRNA; interfering RNA or RNAi; dsRNA; RNA Polymerase III transcribed DNAs; ribozymes; GalNac-siRNA;GalN Ac- Anti sense Oligonucleotide (ASO) and antisense nucleic acids, which can be RNA, DNA, or an artificial nucleic acid.

[0051] Antisense oligonucleotides, including antisense DNA, RNA, and DNA / RNA molecules, act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the DNA sequence encoding a protein of interest, such as SV2C or VMAT2 can be synthesized, e.g., by conventional phosphodiester techniques. Antisense nucleotide sequences include, but are not limited to: morpholinos, 2’- O-methyl polynucleotides, DNA, RNA and the like.

[0052] In some embodiments, the false neurotransmitter mimics various types of neurotransmitters including, but not limited to dopamine, catecholamine, monoamine, adrenalin, serotonin, histamine, norepinephrine, GABA, glutamate, endorphins, oxytocin, adenosine triphosphate, adenosine, carbon monoxide, nitric oxide or acetylcholine. In some embodiments, the false neurotransmitter is a dopamine analogue.

[0053] In some embodiments, the false neurotransmitter is a fluorescent false neurotransmitter. In some embodiments, the false neurotransmitter could be any fluorescent compound that mimics a neurotransmitter. In some embodiments, the fluorescent false neurotransmitter comprises false fluorescent neurotransmitter 206 (FFN206), false fluorescent neurotransmitter 511 (FFN511), false fluorescent neurotransmitter 102 (FFN102), false fluorescent neurotransmitter 200 (FFN200) or false fluorescent neurotransmitter 270 (FFN270). In some embodiments, the fluorescent false neurotransmitter is false fluorescent neurotransmitter 206 (FFN206), false fluorescent neurotransmitter 511 (FFN511), false fluorescent neurotransmitter 102 (FFN102), false fluorescent neurotransmitter 200 (FFN200) or false fluorescent neurotransmitter 270 (FFN270). In some embodiments, the fluorescent false neurotransmitter comprises false fluorescent neurotransmitter 206 (FFN206). In some embodiments, the fluorescent false neurotransmitter consists essentially of false fluorescent neurotransmitter 206 (FFN206). In some embodiments, the fluorescent false neurotransmitter consists of false fluorescent neurotransmitter 206 (FFN206). In some embodiments, the false neurotransmitter is a fluorescent neurotransmitter ligand. In some embodiments, the fluorescent neurotransmitter ligand comprises a Danysl-dopamine. In some embodiments, the fluorescent neurotransmitter ligand is a Danysl-dopamine. In some embodiments, the false neurotransmitter is a radiolabeled neurotransmitter. In some embodiments, the false neurotransmitter comprises radiolabeled dopamine. In some embodiments, the falseneurotransmitter is radiolabeled dopamine. In some embodiments, the false neurotransmitter is monitored using an assay kit.

[0054] In some embodiments, FFN206 comprises

[0056] In some embodiments, FFN102 comprises, or salts thereof. In some embodiments, FFN102 comprises bodiments, FFN200 comprises, or salts thereof. In some embodiments, FFN200 comprises

[0058] In some embodiments, FFN270 comprises

[0059] In some embodiments, Dansyl-dopamine comprises

[0060] In some embodiments, the rate of fluorescence decay is determined by i) for each fluorescence signal measurement of the real-time measurement, subtracting a background fluorescence signal, and calculating a percent fluorescence signal change relative to the fluorescence signal measured in step b), and ii) plotting the percentage of fluorescence signal change versus time; and iii) calculating the slope of the percentage of fluorescence signal change versus time. In some embodiments, the slope is calculated by performing linear or non-linear regression.

[0061] In some embodiments, the rate of fluorescence decay of the fluorescence signal is determined by i) for each fluorescence signal value of the real-time measurement, subtracting a background fluorescence signal, and ii) plotting the fluorescence signal value versus time; and iii) calculating the slope of the fluorescence signal value. In some embodiments, the slope is calculated by performing linear or non-linear regression.

[0062] In some embodiments, the background signal comprises a fluorescence signal value measured for cells of a cell culture that have not been contacted with a fluorescent false neurotransmitter. In some embodiments, the background signal is measured at about the same time point as the measurement of the cell culture that has been contacted with a fluorescent false neurotransmitter. In some embodiments, the background signal is measured at the same time point as the measurement of the cell culture that has been contacted with a fluorescent false neurotransmitter. In some embodiments, the background signal comprises a fluorescence signal value measured for cells of a cell culture that have not been contacted with a fluorescent false neurotransmitter, wherein the cell culture comprises cells cultured in a tissue culture plate. In some embodiments, the background signal comprises a fluorescence signal value measured for cells of a cell culture that have not been contacted with a fluorescent false neurotransmitter, wherein the cell culture comprises cells cultured in that same tissue culture plate as cells of the cell culture that have been contacted with a fluorescent false neurotransmitter.Methods of Identifying Compounds

[0063] In certain aspects, described herein is a method of identifying a compound that modulates vesicular retention or leakage: a) contacting a cell culture with a fluorescent false neurotransmitter, b) measuring a fluorescence signal of the fluorescent false neurotransmitter, c) contacting the cell culture with a test compound, d) measuring a fluorescence signal of the fluorescent false neurotransmitter in real-time, e) determining a rate of fluorescence decay of the fluorescence signal, and f) identifying the test compound as a compound that modulates vesicular leakage if the rate of fluorescence decay determined in step e) is higher or lower than a rate of fluorescence decay of a cell culture under a control condition.

[0064] In certain aspects, described herein is a method of identifying compound that modulates vesicular leakage, the method comprising: a) contacting a cell culture with a test compound, b) contacting the cell with a fluorescent false neurotransmitter, c) measuring a fluorescent signal of the fluorescent false neurotransmitter in real-time, d) determining a rate of fluorescence decay of the fluorescence signal, and e) identifying the test compound as a compound that modulates vascular leakage if the rate of fluorescence decay determined in step c) is higher or lower.

[0065] In some embodiments, the cells of the cell culture are an immortalized mammalian cell line. Examples of immortalized cells lines include but not limited to PC 12, RCSN-3, BE2, SH-SY5Y, and HEK-293 cells. In some embodiments, the cells of the cell culture are induced pluripotent stem cells. In some embodiments, the cells of the cell culture are neuronal cultures differentiated from induced pluripotent stem cells. In some embodiments, the cells of the cell culture are primary neuronal cultures. In some embodiments, the cells of the cell culture are HEK-293 cells.

[0066] In some embodiments, the cells of the cell culture express a protein of interest. In some embodiments, the cells of the cell culture an increased expression level of a protein of interest. In some embodiments, the cells of the cell culture have an increased expression level of a protein of interest compared to normal, wild-type cells of the same cell type. In some embodiments, the cells of the cell culture do not express a protein of interest. In some embodiments, the cells of the cell culture have a reduced expression level of a protein of interest. In some embodiments, the cells of the cell culture have a reduced expression level of a protein of interest compared to normal, wild-type cells of the same cell type.

[0067] In some embodiments, the cells of the cell culture express vesicular monoamine transporter 2 (VMAT2). In some embodiments, the cells of the cell culture are HEK-293 cells that express VMAT2. In some embodiments, the cells of the cell culture express synaptic vesicle glycoprotein 2C (SV2C). In some embodiments, the cells of the cell culture are HEK- 293 cells that express SV2C. In some embodiments, the cells of the cell culture express VMAT2 and SV2C. In some embodiments, the cells of the cell culture are HEK-293 cells that express VMAT2 and SV2C. In some embodiments, the cells of the cell culture express at least one of VMAT2, dopamine transporter (DAT), or synaptic vesicle glycoprotein 2C (SV2C). In some embodiments, the cells of the cell culture are HEK-293 cells that express at least one of the following proteins: VMAT2, DAT, or SV2C. In some embodiments, the cells of the cell culture are genetically modified cells. In some embodiments, the cells of the cell culture have reduced synaptic vesicle glycoprotein 2C (SV2C) expression level. In some embodiments, the level of SV2C is reduced as compared to normal, wild-type cells of the same cell type. In some embodiments, the cells of the cell culture are genetically modified to knock out SV2C. In some embodiments, the cells of the cell culture have reduced expression of synaptic vesicle localized proteins compared to a cell without genetic modification. In some embodiments, the cells of the cell culture have reduced expression of plasma membranelocalized proteins compared to a cell without genetic modification. In some embodiments, the cells of the cell culture have reduced expression of organelle localized proteins compared to a cell without genetic modification.

[0068] In some embodiments, the rate of fluorescence decay is determined by i) for each fluorescence signal measurement of the real-time measurement, subtracting a background fluorescence signal, and calculating a percent fluorescence signal change relative to the fluorescence signal measured in step b), and ii) plotting the percentage of fluorescence signal change versus time; and iii) calculating the slope of the percentage of fluorescence signal change versus time.

[0069] In some embodiments, the rate of fluorescence decay of the fluorescence signal is determined by i) for each fluorescence signal value of the real-time measurement, subtracting a background fluorescence signal, and ii) plotting the fluorescence signal value versus time; and iii) calculating the slope of the fluorescence signal value.

[0070] In some embodiments, the background signal comprises a fluorescence signal value measured for cells of a cell culture that have not been contacted with a fluorescent false neurotransmitter. In some embodiments, the background signal is measured at about the same time point as the measurement of the cell culture that has been contacted with a fluorescent false neurotransmitter. In some embodiments, the background signal is measured at the same time point as the measurement of the cell culture that has been contacted with a fluorescent false neurotransmitter. In some embodiments, the background signal comprises a fluorescence signal value measured for cells of a cell culture that have not been contacted with a fluorescent false neurotransmitter, wherein the cell culture comprises cells cultured in a tissue culture plate. In some embodiments, the background signal comprises a fluorescence signal value measured for cells of a cell culture that have not been contacted with a fluorescent false neurotransmitter, wherein the cell culture comprises cells cultured in that same tissue culture plate as cells of the cell culture that have been contacted with a fluorescent false neurotransmitter.

[0071] In some embodiment, the test compound is a pharmacological regulator. In some embodiment, the test compound is an environmental or toxicological regulator. In some embodiment, the test compound comprises tetrabenazine (TBZ). In some embodiment, the test compound is tetrabenazine (TBZ). In some embodiments, the test compound comprisesl-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). In some embodiments, the test compound is l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP).

[0072] In some embodiments, tetrabenazine comprises, or salts thereof. In some embodiments, tetrabenazine comprises

[0073] In some embodiments, MPTP comprises

[0074] In some embodiments, a cell culture under a control condition comprises a cell culture cultured under standard cell culturing conditions for such a cell culture. In some embodiments, a cell culture under a control condition comprises a cell culture contacted with a control compound. In some embodiments, the control compound is a solvent. In some embodiments, the control compound is sterile ultrapure water. In some embodiments, the control compound is dimethyl sulfoxide (DMSO). DMSO is an organosulfur compound thatcan be used as a vehicle for a test compound. Other known vehicles for test compounds can be used. Accordingly, in some embodiments, a control is contacted with the vehicle used to deliver the test compounds of interest.

[0075] In some embodiments, the cell culture comprises cells cultured in a tissue culture plate. In some embodiments, the cell culture comprises cells cultured in suspension. In some embodiments, the cell culture comprises cells cultured in a multiwell plate, such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, 96-well plate, a 96-well half area plate, 384-well plate, or a 1536-well plate. In some embodiments, the multiwell plate is a black walled plate. In some embodiments, the multiwell plate is a black walled with clear flat bottom. In some embodiments, the multiwell plate is a black walled with clear flat bottom half-area 96-well plate. In some embodiments, cells are cultured in a multi-well plate and each well of the multi-well plate is used to test one or more different test compounds or different concentrations of one or more test compounds. In some embodiments, the control cell is cultured in a well of the same multi-well plate as the cells contacted with a test compound. In some embodiments, the multi -well plate is a 384 well plate. In some embodiments, the multi-well plate is a 6 well plate, a 12 well plate, a 24 well plate, a 48 well plate, a 96 well plate, a 96-well half area plate, a 384 well plate, or a 1536 well plate.

[0076] In some embodiments, the false neurotransmitter mimics various types of neurotransmitters including, but not limited to dopamine, catecholamine, monoamine adrenalin, serotonin, histamine, norepinephrine, GABA, glutamate, endorphins, oxytocin, adenosine triphosphate, adenosine, carbon monoxide, nitric oxide or acetylcholine. In some embodiments, the false neurotransmitter targets dopamine.

[0077] In some embodiments, the false neurotransmitter is a fluorescent false neurotransmitter. In some embodiments, the false neurotransmitter could be any fluorescent compound that mimics a neurotransmitter. In some embodiments, the fluorescent false neurotransmitter comprises false fluorescent neurotransmitter 206 (FFN206), false fluorescent neurotransmitter 511 (FFN511), false fluorescent neurotransmitter 102 (FFN102), false fluorescent neurotransmitter 200 (FFN200) or false fluorescent neurotransmitter 270 (FFN270). In some embodiments, the fluorescent false neurotransmitter is false fluorescent neurotransmitter 206 (FFN206), false fluorescent neurotransmitter 511 (FFN511), false fluorescent neurotransmitter 102 (FFN102), false fluorescent neurotransmitter 200 (FFN200) or false fluorescent neurotransmitter 270 (FFN270). In some embodiments, the fluorescentfalse neurotransmitter comprises false fluorescent neurotransmitter 206 (FFN206). In some embodiments, the fluorescent false neurotransmitter consists essentially of false fluorescent neurotransmitter 206 (FFN206). In some embodiments, the fluorescent false neurotransmitter consists of false fluorescent neurotransmitter 206 (FFN206). In some embodiments, the false neurotransmitter is a fluorescent neurotransmitter ligand. In some embodiments, the fluorescent neurotransmitter ligand comprises a Danysl-dopamine. In some embodiments, the fluorescent neurotransmitter ligand is a Danysl-dopamine. In some embodiments, the false neurotransmitter is a radiolabeled neurotransmitter. In some embodiments, the false neurotransmitter comprises radiolabeled dopamine. In some embodiments, the false neurotransmitter is radiolabeled dopamine. In some embodiments, the false neurotransmitter is monitored using an assay kit.

[0078] In some embodiments, the cells are contacted with the test compound for 1 hour. In some embodiments, the cells are contacted with the test compound for 1 day. In some embodiments, the cells are contacted with the test compound for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours. In some embodiments, the cells are contacted with the test compound for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. In some embodiments, the cells are contacted with the test compound for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or longer.

[0079] In some embodiments, the cells are contacted with the fluorescent false neurotransmitter before the cells are contacted with the test compound. In some embodiments, the cells are contacted with the test compound immediately after being contacted with the fluorescent false neurotransmitter. In some embodiments, additional steps can be performed between contacting the cells with the test compound and the fluorescent false neurotransmitter, such as, but not limited to, measuring a fluorescence signal of the false neurotransmitter. In some embodiments, the cells are contacted with the fluorescent falseneurotransmitter simultaneously with the test compound. In some embodiments, the cells are contacted with the fluorescent false neurotransmitter after the cells are contacted with the test compound. In some embodiments, the cells are contacted with the fluorescent false neurotransmitter immediately after being contacted with the test compound. In some embodiments, additional steps can be performed between contacting the cells with the test compound and the fluorescent false neurotransmitter.

[0080] Stereological cell counting can be performed as described in Lohr K. et. Al. PNAS 111(27) 9977-9982 (2014). Every fourth SNpc section can be counted using the optical fractionator method. Counting frames of 50pm X 50pm on a 120pm X 120pm counting grid can be sampled. Cell counts can be weighted based on manually measured section thickness. Only counts with a Gunderson coefficient of error (m=0) of <0.10 can be included for analysis.

[0081] Test compounds can be screened from large libraries of synthetic or natural compounds, including small molecule drugs, as wells as peptides, proteins, peptidomimetic molecules, saccharides, and nucleic acid-based compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available, or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.

[0082] In some embodiments, the test compound is a small molecule. In some embodiments, the test compound is an antibody. In some embodiments, the test compound is a nanobody. In some embodiments, the test compound is an antisense oligonucleotide.

[0083] Methods for preparing libraries of molecules are well known in the art and many libraries are commercially available. Libraries of interest include peptide libraries, randomized oligonucleotide libraries, synthetic organic combinatorial libraries, and the like. Degenerate peptide libraries can be readily prepared in solution, in immobilized form as bacterial flagella peptide display libraries or as phage display libraries. Peptide ligands can be selected from combinatorial libraries of peptides containing at least one amino acid. Libraries can be synthesized of peptoids and non-peptide synthetic moieties. Such librariescan further be synthesized which contain non-peptide synthetic moieties, which are less subject to enzymatic degradation compared to their naturally-occurring counterparts. Libraries are also meant to include for example but are not limited to peptide-on-plasmid libraries, polysome libraries, aptamer libraries, synthetic peptide libraries, synthetic small molecule libraries, neurotransmitter libraries, and chemical libraries. The libraries can also comprise cyclic carbon or heterocyclic structure and / or aromatic or polyaromatic structures substituted with one or more of the functional groups.

[0084] Small molecule combinatorial libraries can also be generated and screened. A combinatorial library of small organic compounds is a collection of closely related analogs that differ from each other in one or more points of diversity and are synthesized by organic techniques using multi-step processes. Combinatorial libraries include a vast number of small organic compounds. One type of combinatorial library is prepared by means of parallel synthesis methods to produce a compound array. A compound array can be a collection of compounds identifiable by their spatial addresses in Cartesian coordinates and arranged such that each compound has a common molecular core and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, with each compound identified and tracked by its spatial address. Examples of parallel synthesis mixtures and parallel synthesis methods are known in the art. Numerous examples of chemically synthesized libraries are described in the art.

[0085] In some embodiments, the rate of fluorescence decay determined in step (c) is higher than a rate of fluorescence decay of a cell culture under a control condition. In some embodiments, the rate of fluorescence decay may be higher by at least 5%, e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, relative to a rate of fluorescence decay of a cell culture under a control condition.

[0086] In some embodiments, the rate of fluorescence decay determined in step (c) is lower than a rate of fluorescence decay of a cell culture under a control condition. In some embodiments, the rate of fluorescence decay may be higher by at least 5%, e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, relative to a rate of fluorescence decay of a cell culture under a control condition.Methods of Treatment

[0087] The practice of aspects of the present invention can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Molecular Cloning A Laboratory Manual, 3rdEd., ed. By Sambrook (2001), Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In Enzymology (Academic Press, Inc., N.Y.), specifically, Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Caner and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-FV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). All patents, patent applications and references cited herein are incorporated by reference in their entireties.

[0088] In certain aspects, described herein is a method of treating a neurological disorder in a subject in need thereof, comprising: a) contacting a cell culture with a fluorescent false neurotransmitter, b) measuring a fluorescence signal of the fluorescent false neurotransmitter, c) contacting the cell culture with a test compound, d) measuring a fluorescence signal of the fluorescent false neurotransmitter in real-time, and e) determining a rate of fluorescence decay of the fluorescence signal, and f) administering the test compound to the subject if the rate of fluorescence decay determined in step e) is higher than a rate of fluorescence decay of a cell culture under a control condition.

[0089] In certain aspects, described here is a method of treating a neurological disorder in a subject in need thereof, comprising: a) contacting a cell culture with a test compound, b) contacting the cell with a fluorescent false neurotransmitter, c) measuring a fluorescent signal of the fluorescent false neurotransmitter in real-time, d) determining a rate of fluorescencedecay of the fluorescence signal, and e) identifying the test compound as a compound that modulates vascular leakage if the rate of fluorescence decay determined in step c) is higher or lower.

[0090] In some embodiments, the cells of the cell culture are an immortalized mammalian cell line. In some embodiments, the cells of the cell culture are induced pluripotent stem cells. In some embodiments, the cells of the cell culture are neuronal cultures differentiated from induced pluripotent stem cells. In some embodiments, the cells of the cell culture are primary neuronal cultures. In some embodiments, the cells of the cell culture are any immortalized cell lines, primary neuronal cultures, ex vivo brain slices, any induced pluripotent stem cells (iPSCs) and differentiated cultured cells from human iPSCs. Examples of immortalized cells lines include but not limited to PC12, RCSN-3, BE2, SH- SY5Y, and HEK-293 cells. In some embodiments, the cell culture is derived from a sample from the subject.

[0091] In some embodiments, the cells of the cell culture express a protein of interest. In some embodiments, the cells of the cell culture an increased expression level of a protein of interest. In some embodiments, the cells of the cell culture have an increased expression level of a protein of interest compared to normal, wild-type cells of the same cell type. In some embodiments, the cells of the cell culture do not express a protein of interest. In some embodiments, the cells of the cell culture have a reduced expression level of a protein of interest. In some embodiments, the cells of the cell culture have a reduced expression level of a protein of interest compared to normal, wild-type cells of the same cell type.

[0092] In some embodiments, the cells of the cell culture express vesicular monoamine transporter 2 (VMAT2). In some embodiments, the cells of the cell culture are HEK-293 cells that express VMAT2. In some embodiments, the cells of the cell culture express synaptic vesicle glycoprotein 2C (SV2C). In some embodiments, the cells of the cell culture are HEK- 293 cells that express SV2C. In some embodiments, the cells of the cell culture express VMAT2 and SV2C. In some embodiments, the cells of the cell culture are HEK-293 cells that express VMAT2 and SV2C. In some embodiments, the cells of the cell culture express at least one of VMAT2, dopamine transporter (DAT), or synaptic vesicle glycoprotein 2C (SV2C). In some embodiments, the cells of the cell culture are HEK-293 cells that express at least one of the following proteins: VMAT2, DAT, or SV2C.

[0093] In some embodiments, the rate of fluorescence decay is determined by i) for each fluorescence signal measurement of the real-time measurement, subtracting a background fluorescence signal, and calculating a percent fluorescence signal change relative to the fluorescence signal measured in step b), and ii) plotting the percentage of fluorescence signal change versus time; and iii) calculating the slope of the percentage of fluorescence signal change versus time.

[0094] In some embodiments, the rate of fluorescence decay of the fluorescence signal is determined by i) for each fluorescence signal value of the real-time measurement, subtracting a background fluorescence signal, and ii) plotting the fluorescence signal value versus time; and iii) calculating the slope of the fluorescence signal value.

[0095] In some embodiments, the test compound is a pharmacological regulator. In some embodiment, the test compound is an environmental or toxicological regulator. In some embodiment, the test compound is tetrabenazine (TBZ). In some embodiments, the test compound is l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP).

[0096] In some embodiments, a cell culture under a control condition comprises a cell culture cultured under standard cell culturing conditions for such a cell culture. In some embodiments, a cell culture under a control condition comprises a cell culture contacted with a control compound. In some embodiments, the control compound is a solvent. In some embodiments, the control compound is sterile ultrapure water. In some embodiments, the control compound is dimethyl sulfoxide (DMSO). DMSO is an organosulfur compound that can be used as a vehicle for a test compound. Other known vehicles for test compounds can be used. Accordingly, in some embodiments, a control is contacted with the vehicle used to deliver the test compounds of interest.

[0097] In some embodiments, the test compound is a small molecule. In some embodiments, the test compound is an antibody. In some embodiments, the test compound is a nanobody. In some embodiments, the test compound is an antisense oligonucleotide.

[0098] In various embodiments, the method is to treat a neurological disorder including, but not limited to Parkinson’s disease, addiction, attention hyperactivity disorder (ADHD), schizophrenia, depression, bipolar disorder, obsessive-compulsive disorder, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), ataxia, brain tumors, epilepsy and seizures,Guillain-Barre syndrome, meningitis, multiple sclerosis, stroke, muscular dystrophy and neuromuscular diseases. In some embodiment, the method is to treat Parkinson’s disease.

[0099] In some embodiments, the rate of fluorescence decay determined in step (c) is higher than a rate of fluorescence decay of a cell culture under a control condition. In some embodiments, the rate of fluorescence decay may be higher by at least 5%, e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, relative to a rate of fluorescence decay of a cell culture under a control condition.

[0100] In some embodiments, the rate of fluorescence decay determined in step (c) is lower than a rate of fluorescence decay of a cell culture under a control condition. In some embodiments, the rate of fluorescence decay may be lower by at least 5%, e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, relative to a rate of fluorescence decay of a sample from a healthy subject.

[0101] In some embodiments, the expression of SV2C may be reduced by at least about 5% to about 95%, e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, relative to corresponding expression level of SV2C in a subject not suffering from the neurological disorder.

[0102] In some embodiments, a biological sample comprises any immortalized cell lines, primary neuronal cultures, ex vivo brain slices, any induced pluripotent stem cells (iPSCs) and differentiated cultured cells from human iPSCs. Examples of immortalized cells lines include but not limited to PC12, RCSN-3, BE2, SH-SY5Y, and HEK-293 cells.Methods of Administering

[0103] Indications, dosage and methods of administration of the drugs of the present invention are known to one of skill in the art. In some embodiments, a drug of the present invention can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. Choice of the excipient and any accompanying elements of the composition will be adapted in accordance with the route and device used for administration. In some embodiments, a composition comprising a drug of the present invention can also comprise, or be accompanied with, one or more other ingredients that facilitate the delivery or functional mobilization of the drugs of the present invention.

[0104] These methods described herein are by no means all-inclusive, and further methods to suit the specific application is understood by the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

[0105] According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.

[0106] Pharmaceutical compositions for use in accordance with the invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration.Techniques and formulations generally can be found in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa (20thed., 2000), the entire disclosure of which is herein incorporated by reference.

[0107] Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human. In certain embodiments, the therapeutic applications described herein can be applied to a human.

[0108] Administration of a drug of the present invention is not restricted to a single route, but may encompass administration by multiple routes. Multiple administrations may be sequential or concurrent. Other modes of application by multiple routes will be apparent to one of skill in the art.***

[0109] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methodsand materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0110] All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art.EXAMPLESEXAMPLE 1 - Synaptic vesicle glycoprotein 2C enhances vesicular storage of dopamine and counters dopaminergic toxicity

[0111] The dopaminergic neurons of the substantia nigra that degenerate in Parkinson’s disease exist in a state of constant vulnerability resulting from high baseline oxidative stress, high energy demand, and broad unmyelinated axonal arborizations. Impairments in the storage of dopamine compound this stress due to cytosolic reactions that transform the vital neurotransmitter into an endogenous neurotoxicant. To maintain neuronal health, the cytosolic pool of dopamine is minimized through sequestration of dopamine into synaptic vesicles. While vesicular sequestration via vesicular monoamine transporter 2 (VMAT2) has been extensively researched, synaptic vesicles are inherently leaky, and much less is understood about factors regulating the retention of dopamine within synaptic vesicles. Synaptic vesicle glycoprotein 2C (SV2C) is identified as a modifier of vesicular dopamine dynamics, demonstrating that genetic ablation of SV2C in mice results in decreased dopamine content and evoked dopamine release in the striatum. Furthermore, SV2C has been implicated as a modifier of Parkinson’s disease risk. Described herein is data indicating that SV2C mediates the retention of dopamine and dopamine analogues within vesicles. The previously published in vitro assay utilizing false fluorescent neurotransmitter 206 (FFN206) was used to visualize how SV2C regulates vesicular dynamics and identified that SV2C promotes the uptake and retention of FFN206 within vesicles. These findings were confirmed with radiolabeled dopamine in uptake and retention assays performed on isolated vesicles from cells and mouse brain. Finally, data show that SV2C mediates the uptake and retention of MPP+, and genetic ablation of SV2C results in enhanced MPTP -induced neurotoxicity in mice. Together, these findings suggest that SV2C functions to enhance vesicular storage of dopamine and neurotoxicants and helps maintain the integrity of dopaminergic neurons.Introduction

[0112] Deficiency in dopamine neurotransmission, resulting from dysfunction and degeneration of dopaminergic neurons in the substantia nigra, is the key pathologic feature of Parkinson’s disease. Effective dopamine neurotransmission regulates motor function and depends on proper dopamine homeostasis including cytosolic synthesis, vesicular packaging, evoked synaptic release, post-synaptic receptor activation, pre-synaptic reuptake, and enzymatic metabolism. Dysregulation of dopamine homeostasis can cause deficits in dopamine neurotransmission and jeopardize neuronal health. At a baseline, cytosolic dopamine that undergoes enzymatic catabolism by monoamine oxidase generates electrons, which are utilized by the electron transport chain in mitochondria to promote energy production and regulate neuronal activity (Graves S. et. al., Nat. Neuroscience 23, 15- 20(2020)). However, dopamine also has the potential to act as an endogenous neurotoxin when cytosolic dopamine undergoes toxic metabolic processes that generate highly reactive metabolites such as the dopamine quinone and toxic aldehyde, DOPAL, and reactive oxygen species (Bucher M. et. al., NPJ parkinsons Dis. 6: 34 (2020)), all of which contribute to cellular dysfunction through interactions with proteins, lipids, and nucleic acids (Sulzer D. & Zecca L. 1(3): 181-95 (2000); Graves S. et. al., Nat. Neuroscience 23, 15-20(2020)). This can compound the high vulnerability of dopamine neurons to degeneration due to high baseline levels of oxidative stress, high energy demand, and broad unmyelinated axons (Guzman J. et. al., Nature 468, 496-700 (2010); Pissadaki E & Bolam J. 7 (2013); Matsuda W. et. al., JNeurosci 29(2) 444-453 (2009)).

[0113] The vesicular monoamine transporter 2 (VMAT2) minimizes the cytosolic pool of dopamine by sequestering dopamine into synaptic vesicles. However, even with functioning VMAT2, it is known that vesicles are inherently leaky (Floor E. et. al. JNC 64(2) 689-699 (1995)); thus, it is important to evaluate other factors that contribute to the cytosolic pool of dopamine such as factors that regulate the retention of dopamine within vesicles. There is significant evidence that dysregulation of dopamine homeostasis can cause dopaminergic neurons to become vulnerable to dysfunction and degeneration, and replicate features of Parkinson’s disease (Caudle W. et. al., JNeurosci 27(30) 8138-8148 (2007), Bucher M. et. al., NPJ parkinsons Dis. 6: 34 (2020); Taylor T. et. al. Neuropharmacology 76 97-105 (2014); Taylor T. et. al. JNeurosci 29 (25) 8103-8113 (2009)). The vesicular sequestration of dopamine by VMAT2 has been extensively implicated in Parkinson’s disease risk andpathogenesis. There are several documented cases of infantile parkinsonism resorting from variants in SLC18A2 - the gene for VMAT2 - that are believed to result from decreased VMAT2 expression or function (Saida K. et. al., Genetics in Med. 25(1) P90-102 (2023)). Conversely, rare SNPs in SLC18A2, which are believed to be gain-of-function mutations resulting in enhanced VMAT2 expression or function, confer a decrease in the risk of developing Parkinson’s disease (Brighina et. al. Neurobiolo. Aging 34 (6) 1712.e9-1712.el3 (2013); Glatt C. et. al. Hum Mol. Genet. 15(2):299-305 (2006); Yang X. et. al. Parkinsons Dis. (2015)), and two low-activity variants in VMAT2 have been identified that may be associated with an increased risk of developing the disease (Xiong N. et. al., Neurotherapeutics 13, 623-634 (2016)). Furthermore, post-mortem tissue from Parkinson’s disease patients demonstrates decreased VMAT2 immunoreactivity, and analysis of vesicles isolated from post-mortem brain tissue demonstrate decreased VMAT2-mediated dopamine uptake in Parkinson’s disease patients (Miller G. et. al Exp. Neurol. 156(1): 138-48 (1999), Pifl C. et. al. JNeurosci 34 (24) 8210-8218 (2014)). The impairments in VMAT2 are not limited to the brain, as analysis of circulating platelets in Parkinson’s disease patients demonstrated decreased VMAT2 mRNA suggesting a systemic deficiency in VMAT2 (Sala G. et. al. J. Neural Transm. 117(9): 1093-8 (2010)).

[0114] Recent investigation has focused on additional factors that regulate vesicular dynamics of dopamine, both at the basic biological level and for disease relevance. For example, the leakage of neurotransmitters from the vesicle into the cytosol has been hypothesized as a contributor to dopamine neuron vulnerability to degeneration. Thus, in addition to the vesicular sequestration of dopamine, it is important to understand factors that regulate the retention of dopamine within synaptic vesicles. Although it is unclear which vesicular components may contribute to vesicular leak (Alter S. et. al. Curr. Neuro & Neurosci Rep. 13, 362 (2013), Eisenhofer G. et. al. Ann. N. Y. Acad. Sci 1018:224-30 (2004)), several lines of evidence suggest that synaptic vesicle glycoproteins (SV2s) may be capable of facilitating the storage of intravesicular molecules, including neurotransmitters, due to a heavily glycosylated intraluminal loop.

[0115] Synaptic vesicle glycoproteins (SV2s), of which there are three isoforms (SV2A, SV2B, and SV2C), are synaptic vesicle localized glycoproteins in the SLC22B family of solute carriers (Bohnert T. et. al. Drug Metab. Dispos. 44(8): 1399-423 (2016)). The three SV2 isoforms have differential expression throughout the brain, with SV2A and SV2Bhaving more ubiquitous expression, and SV2C having enriched expression in the basal ganglia, particularly in dopaminergic neurons (Dunn A. et. al. Brain Research 1702 (1) 85-95 (2019)). While SV2 proteins are identified as solute carriers, the substrates for SV2B and SV2C remain unidentified. Evidence suggests SV2A is a galactose transporter (Madeo M. et. al. J. Bio. Chem. 289(48):33066-71 (2014)), which corroborates analysis identifying sequence homology of SV2 proteins to bacterial proteins that transport sugars (Bajjalieh S. et. al. 257(5074): 1271-3 (1992)). SV2 proteins also appear to enable calcium-mediated exocytosis of synaptic vesicles (Ciruelas K. et. al. Semin. Cell Dev. Biol. 95:130-141 (2019); Chang W. & Sudhof T. JNeurosci 29(4) 883-897 (2009)), and it has further been hypothesized that SV2C may be a novel neurotransmitter transporter (Feany M. et. al. Cell 70(5) P861-867 (1992)).

[0116] SV2C has been implicated in Parkinson’s disease following an initial identification in a genome-wide association study (GWAS) as a modifier of nicotine’s protective effect against developing Parkinson’s disease (Hill-Bums E. et. al.Pharmacogenomics J. 13(6):530-7 (2013)). Intriguingly, we identified that genetic ablation of SV2C alters the effect of nicotine on dopamine transmission (Dunn A. et. al. PNAS 114(111) E2253-E2262 (2017)), and a 2022 study investigating this relationship demonstrated the necessity of drosophila SV2 orthologs in nicotine-mediated protection against alpha- synuclein induced neurodegeneration (Olsen A. et. al. Movement Disorder 38(2) 244-255 (2022)). Furthermore, subsequent GWAS identified SV2C as a modifier of GBA-associated PD risk (Gan-OR et. al. J. Neurol. 262(11):2443-7 (2015)), Parkinson’s disease patient’s response to L-DOPA (Altmann V. et. al. Pharmacogenomics 17(5) (2016)), and most recently directly as a risk-modifier for Parkinson’s disease (Foo J. et. al. JAMA Neurol. 77(6)746-754 (2020); Grover S. et. al. Movement Disorders 36(7) 1689-1695 (2021)). Additionally, our lab has demonstrated aberrant SV2C staining patterns in post-mortem brain tissue from people with Parkinson’s disease (Dunn A. et. al. PNAS 114(111) E2253-E2262 (2017)), and an interaction between SV2C and alpha-synuclein (Dunn A. et. al. PNAS 114(111) E2253- E2262 (2017)).

[0117] Although the exact function of SV2C is unknown, there is ample evidence to suggest that SV2C plays a role in synaptic transmission. We have previously shown that animals lacking SV2C have decreased dopamine content in the striatum, as well as impaired evoked dopamine release (Dunn A. et. al. PNAS 114(111) E2253-E2262 (2017)). Theseanimals also displayed decreased locomotor activity correlating with the decrease in dopamine transmission (Dunn A. et. al. PNAS 114(111) E2253-E2262 (2017)). To better understand the biological function of SV2C and its role mediating Parkinson’s disease risk, this work uses cellular models, isolated vesicles, and in vivo toxicant experiments to show that SV2C controls vesicular retention of dopamine and ameliorates dopaminergic damage from toxicants.Methods

[0118] Cell lines: Human embryonic kidney (HEK293) cells were stably transfected with human vesicular monoamine transporter 2 (VMAT2; HEK-VMAT2) utilizing zeocin selection. A secondary stable transfection was performed on the HEK-VMAT2 cell line to add human synaptic vesicle glycoprotein 2C (SV2C) expression (HEK-VMAT2-SV2C) utilizing geneticin selection. HEK-VMAT2 cells were maintained in media consisting of Dulbecco’s Modified Eagle Medium (DMEM) with 4.5g / L glucose (Corning), 10% fetal bovine serum (Fisher), 0.5% Penicillin-Streptomycin (Sigma), and lOOmg / ml zeocin (Fisher). HEK-VMAT2-SV2C cells were maintained in media consisting of DMEM with 4.5g / L glucose, 10% fetal bovine serum, 0.5% Penn / Strep, lOOmg / ml zeocin, and 250ug / ml geneticin (Fisher). All cells were maintained in an incubator at 37°C with 5% CO2 on 10cm cell culture dishes coated with poly-D-Lysine (Sigma)..

[0119] Western blot: Western blots were performed as previously described (Dunn A. et. al. PNAS 114(111) E2253-E2262 (2017)). Cell culture samples were collected in RIPA buffer, sonicated, centrifuged and the nuclear fraction discarded, the cytoplasmic fraction was used for subsequent western analysis. Samples were run through SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked with 5% nonfat dry milk and incubated in primary antibody (SV2C, 1 : 1,000 (Sigma, MABN367); VMAT2, 1 :2000 (Invitrogen, MAS- 24939); ACTIN, 1 :5,000 (Sigma, A5060); GAPDH, 1 :5,000 (Invitrogen, PAI-9046)) overnight at 4°C with gentle agitation. Secondary antibodies (Fluorescent channels 700 and 800, 1 :20,000 (Licor, IRDye 800cw and IRDye 680id (Fisher)) were incubated at room temperature for 1 hour. Signal was visualized using the Licor Odyssey system. Unilateral striatal dissections were homogenized and underwent differential centrifugation to achieve a crude synaptosomal protein preparation. 20pg of protein was run through an SDS-PAGE gel and transferred to a PVDF membrane. Nonspecific antibody binding was blocked with a 7.5% nonfat dry milk solution, and the membrane was incubated in primary antibodyovernight at 4°C with gentle agitation. Membranes were then incubated in HRP-conjugated secondary antibody for 1 hour at room temperature. Protein was visualized using chemiluminescence (Thermo) and a BioRad UV imager. Protein was quantified using ImageLab software and normalized to an actin loading control. Rabbit anti-TH (1 : 1,000 (Millipore AB 152)).

[0120] Immunocytochemistry: Cells were seeded onto a poly-D-Lysine (Sigma) coated chamber slide and allowed to attach overnight. To perform immunocytochemistry, media was removed and cells were fixed with 2% paraformaldehyde warmed to 37°C for 5m. After 5m, cells were fixed with 4% paraformaldehyde at room temperature for 20m with gentle agitation. Cells were then rinsed with phosphate buffered saline (PBS (Fisher)) three times for 5m each before treatment with blocking solution (PBS with 10% normal goat serum (Fisher), 0.1% triton-x (Sigma), and 10% bovine serum albumin (Sigma)) at room temperature for Ih with gentle agitation. Following blocking, cells were treated with primary antibody solution (PBS with 10% normal goat serum, 0.1% triton-x, and 10% bovine serum albumin) with Mouse anti-SV2C (1 :500 (Sigma, MABN367)) and Rabbit anti-VMAT2 (1 :500 (Miller lab)) at 1 :500 overnight at 4°C with gentle agitation. After overnight incubation with primary antibodies, cells were washed in PBS with 0.1% triton three times for 5m each before treatment with secondary antibody solution (PBS with 1% normal goat serum, 0.1% triton-x, and 10% bovine serum albumin) containing 1 :400 Goat anti-mouse Alexafluor 594 (ThermoFisher) and 1 :400 Goat anti-rabbit AlexaFluor 488 (ThermoFisher) for Ih at room temperature with gentle agitation. Following secondary incubation, cells were washed with PBS three times for 5m each before coverslipping with Vectashield (Fisher).

[0121] FFN206 uptake assay: FFN206 uptake assay was performed as previously described (Black C. et. al. Chem. Res. Toxicol. 34, 5, 1256-1264 (2020)). Briefly, HEK- VMAT2 and HEK-VMAT2-SV2C cells were seeded in DMEM + 0.5% P / S + 10% FBS in a black walled with clear flat bottom half-area 96-well plate (Corning) at 40,000 cells. 24h later, at confluency, culture media was removed and cells were pre-incubated in standard incubator conditions with DMSO or IpM tetrabenazine for 30m in DMEM with 4.5g / L Glucose, Sodium Pyruvate; Without L-Glutamine, Phenol Red (Corning). After 30m, FFN206 prepared in DMEM with 4.5g / L Glucose, Sodium Pyruvate; Without L-Glutamine, Phenol Red (Coming) was added to a final concentration of 2pM and incubated for Ihr. After the incubation period, media was removed and replaced with DMEM with 4.5g / L Glucose,Sodium Pyruvate; Without L-Glutamine, Phenol Red (Coming) containing 1 :25 trypan blue and plate was scanned on BioTek Synergy Hl multiplate reader at 37°C using endpoint fluorescent intensity measurements with excitation = 369 and excitation = 464. Fluorescent values were corrected by subtracting background fluorescent values obtained from cells on the same plate with same treatments without FFN206. For uptake experiments, each 96-well plate contained 12 technical replicates per cell line: treatment group. An average for the control condition (HEK-VMAT2:DMS0) was calculated and each well value was expressed as a percent of this control. A single averaged value was calculated for each cell line: treatment group to represent an experimental replicate. Two-way ANOVA with Sidak's multiple comparisons test was performed in GraphPad Prism 9 on 7 experimental replicates. Tetrabenazine ICso calculation was performed in GraphPad Prism 9 by non-linear regression ([inhibitor] vs. normalized response) and regressions were compared to determine significant differences. Data points were excluded only in cases of pipetting errors (e.g., well received no FFN solution). Microscopy images were acquired on and EVOSfl microscope.

[0122] FFN206 retention assay: HEK-VMAT2 and HEK-VMAT2-SV2C cells were seeded in DMEM + P / S + FBS in a black walled with clear flat bottom half-area 96-well plate (Coming) at 40,000 cells. 24h later, at confluency, culture media was removed and cells were incubated in standard culture incubator conditions with 2pM FFN206 in DMEM with 4.5g / L Glucose, Sodium Pyruvate; Without L-Glutamine, Phenol Red (Corning) for Ihr. Following this Ihr incubation, media was removed and replaced with DMEM with 4.5g / L Glucose, Sodium Pyruvate; Without L-Glutamine, Phenol Red (Corning) containing 1 :25 trypan blue and plate was scanned on BioTek Synergy Hl multiplate reader at 37°C using endpoint fluorescent intensity measurements with excitation k = 369 and excitation = 464 for a baseline fluorescence value. DMSO (Fisher) or tetrabenazine (Sigma) were added for a final concentration of 0.02% DMSO and IpM tetrabenazine. Fluorescent values were corrected by subtracting background fluorescent values obtained from cells on the same plate with same treatments without FFN206. For retention experiments, each 96-well plate contained 12 technical replicates per cell line: treatment group. Each well underwent background subtraction at each timepoint, and the fluorescent value was expressed as a percent of its own baseline fluorescent value at each timepoint. The percent of fluorescence was graphed over time and linear and non-linear regression (plateau followed by one-phase decay) analysis was performed in GraphPad Prism 9 to determine the rate of fluorescent decay and regressions were compared to determine significant differences. Data points wereexcluded only in cases of pipetting errors (e.g., well received no FFN solution). Data in repository features extended retention data to 60m.

[0123] Vesicle isolation: Cells were grown to confluency in large (500cm2) cell culture dishes. Cells were collected in 20ml room temperature PBS, and pelleted (2 minutes at 1500 x g). After removing the supernatant, cells were re-suspended in 1200pl of ice-cold incomplete assay buffer (ICB) (lOOmM Potassium Tartrate, 25mM HEPES, O. lmM EDTA, 0.05 mM EGTA, pH to 7.4), placed in a 3ml dounce homogenizer and subjected to 30 strokes. Lysates were then run through the width of a 27-gauge needle twice for further homogenization. Lysates were centrifuged (8000 x g, 8 minutes, 4°C) and the supernatant collected as a crude vesicle preparation. Vesicles were further diluted in ice-cold ICB according to the number of samples needed for each experiment. A small aliquot was reserved for later BCA (Pierce) to determine protein concentration. All chemical reagents were from Sigma Aldrich. Brain-derived vesicles were collected from wild-type and SV2C- KO mice as previously described (Lohr K. et. al. PNAS 111(27) 9977-9982 (2014)). Briefly, the cytoplasmic vesicular fraction was prepared from homogenized bilateral striata via differential centrifugation.

[0124] Vesicular leak: Crude vesicle fractions were isolated from HEK-VMAT2 cells and HEK-VMAT2-SV2C cells as described above. Ascorbate (1.7mM) and Mg2+ATP salt (2mM) were added to ICB to make complete assay buffer (CAB). Dopamine hydrochloride (2pM) and 2% [3H]-dopamine (40nM) tracer were added to the CAB. 450pl of the CAB / dopamine solution was added to each glass test tube in the experiment and kept in racks at 30°C in a water bath. Vesicles were isolated from WT and SV2C-KO mice, as described above. Samples were kept on ice and sequentially added to a 30°C water bath. Cell culture vesicle samples were kept on ice and added sequentially in 50pl volumes to the CAB / dopamine tubes to begin the uptake reaction. Uptake proceeded for 10 minutes, at which time the VMAT2 inhibitor reserpine was added to the reactions to yield a final concentration of lOpM. Reserpine effectively blocks the transporter, preventing any further uptake of radiolabeled dopamine. Vesicular leak proceeded for 0, 5, 10, 15, and 20 minutes, with all time points staggered so that all samples concluded at the same time. After the “0” leak time point , cells were immediately harvested through a Brandel cell harvester onto GF / F filter paper coated with 0.5% polyethylenimine in ICB, and washed 3x with ice-cold ICB. Filter papers were cut out, placed in scintillation vials and vortexed. Samples were stored inthe dark and read 12 hours later using a liquid scintillation counter (Packard). A small portion of the crude vesicle lysate was analyzed by BCA (Pierce) analysis to normalize uptake to the amount of vesicles used and expressed as fmol of [3H] dopamine remaining per pg of vesicles. Each time point was replicated 4 times across 3 different experiments. VMAT2 specific uptake was calculated by subtracting the average uptake of vesicles pre-treated with reserpine. Experiments using radiolabeled MPP+were performed in exactly the same way, substituting MPP+(2pM) for dopamine hydrochloride and [3H] MPP+for [3H] dopamine. Figures 4A and 6A represent total uptake without leak, or the “0” time point as an expression of maximal uptake. All chemical reagents were from Sigma Aldrich with the exception of radiolabeled compounds provided by Perkin Elmer. Brain-derived vesicle samples were incubated for five minutes and then [3H] dopamine spiked in to a final concentration of 30nM. Uptake proceeded for 10 minutes, at which time VMAT2 inhibitor tetrabenazine was added to the samples to yield a final concentration of lOpM. This addition effectively froze the transporter, preventing additional uptake of radiolabeled dopamine. Vesicular leak proceeded for 0, 1, 2.5, 5, 7.5, and 10 minutes. The experiment was arranged such that all samples concluded at the same time. Each time point was assessed in three specific and one nonspecific samples per animal. Nonspecific uptake, measured from samples that lacked ATP, was subtracted from total counts to generate specific uptake counts. Counts are shown as percentages of the zero leak time point. Non-linear regression (one-phase decay) was performed in GraphPad Prism 9 to determine the rate of radiolabeled decay and regressions were compared to determine significant differences. Data points were removed due to technical complications during experimental process or due to outlier exclusions based on values greater or less than one standard deviation from the mean.

[0125] Animals: Male mice (5-7 mo) were used for all MPTP experiments. Animals were kept on a 12 / 12 light / dark cycle and given food and water ad libitum. SV2C-KO mice were created as described previously (Dunn A. et. al. PNAS 114(111) E2253-E2262 (2017)). Briefly, animals were generated using the EUCOMM “knockout first allele” construct. These animals contained a / z / cZ / neomycin resistance cassette flanked by FRT sites inserted into the Sv2c gene. Animals were crossed with a line globally expressing Flp-recombinase to excise the cassette, resulting in a line of mice containing a floxed exon 2 of the Sv2c gene. These mice were then crossed with a line containing a nestin-driven Cre-recombinase in order to achieve a pan-neuronal knockout of SV2C.

[0126] MPTP injections'. MPTP (Sigma or MedChemExpress) was administered according to a 5 x 20mg / kg dose paradigm. Mice were weighed prior to MPTP administration and injected (s.c.) with 20mg / kg MPTP or (freebase) or an equivalent volume of saline (control) once per day for five days, with an intrainjection interval of 24hr. The lesion was allowed to stabilize for 21 days following the final injection. Mice were sacrificed by rapid decapitation. Brains were removed, dissected, and flash frozen (for immunoblot) or post fixed in 4% (w / v) paraformaldehyde (for immunohistochemistry). Due to supply issues, data reported in Fig. 9 was acquired from experiments using MPTP sourced from Sigma, and the representative immunohistochemistry images in Fig. 8 was acquired from a pilot experiment using MPTP sourced from MedChemExpress. Quantification of the pilot experiment is included in Fig. 11 and demonstrates comparable neurotoxicity as seen in the experiments using MPTP sourced from Sigma. A three-way ANOVA to assess main effects and interactions of MPTP source, treatment, and SV2C genotype indicated no significant main effect (cell loss: F(1 ,4.17), p=0.05; striatal TH: F(l,0.59), p=0.45) or interaction with other factors (cell loss: Treatment* Source: F(l, 1.08), p=0.31; cell loss:Genotype*Treatment* Source: F(l,2.35), p=0.14; striatal TH: Treatment*Source: F(l,l .24), p=0.27; cell loss: Genotype*Treatment* Source: F(l,3.46), p=0.07) of MPTP source on neurotoxicity, thus stereological counts and striatal TH expression were compiled into analysis in Figures 18A-B.

[0127] Immunohistochemistry: Immunohistochemistry was performed as described previously (Dunn A. et. al. PNAS 114(111) E2253-E2262 (2017)). Briefly, brains were sectioned to 40pm. Sections underwent antigen retrieval (70°C citra buffer (Biogenix) for 1 hour) and endogenous peroxidase was quenched with 10% hydrogen peroxide. Nonspecific antibody binding was blocked with 10% normal horse serum in PBS with 0.2% Triton X-100. Tissue was incubated in primary antibody overnight at 4°C with gentle agitation. Sections were then incubated in biotinylated secondary antibody at room temperature for 1 hour. Signal was enhanced with avidin-biotin complex (Vector) and visualized with 3-3’ - diaminobenzidine (DAB). The DAB reaction was stopped with PBS. Sections were mounted to slides and stained with cresyl violet (0.1% w / v aqueous, Poly Scientific). Sections were destained with 0.1% acetic acid in 95% ethanol, followed by dehydration in ethanol and lipid- clearing in xylenes. Rabbit anti-TH (1 : 1,000 (Millipore AB152)).

[0128] Stereology: Stereological cell counting was performed as described previously using StereoInvestigator software (Lohr K. et. al. PNAS 111(27) 9977-9982 (2014)). Every fourth SNpc section was counted using the optical fractionator method. Counting frames of 50pm X 50pm on a 120pm X 120pm counting grid were sampled. Cell counts were weighted based on manually measured section thickness. Only counts with a Gunderson coefficient of error (m=0) of <0.10 were included for analysis.

[0129] Statistics: Graphical representations and statistical analysis was performed in GraphPad Prism 9.Results

[0130] While the exact function of SV2C is unknown, the previous data demonstrating mice with genetic ablation of SV2C (SV2C-KO) show decreased striatal dopamine content and evoked dopamine release (Dunn A. et. al. PNAS 114(111) E2253-E2262 (2017)) which suggests it plays a key role in mediating the vesicular dynamics of dopamine. To investigate the effect that SV2C has on vesicular dynamics, a double-stable cell line expressing both human VMAT2 and human SV2C was created by introducing human SV2C into the HEK- VMAT2 cells previously utilized in Black et al. 2021 (HEK-VMAT2-SV2C) (Black C. et. al. Chem. Res. Toxicol. 34, 5, 1256-1264 (2020)). The expression of SV2C in HEK-VMAT2- SV2C cells is detectable by Western blot with HEK-VMAT2 cells demonstrating no endogenous expression of SV2C, and HEK-VMAT2-SV2C cells demonstrating robust SV2C expression (Figures 2A, 14). When visualized with immunocytochemistry, SV2C co-localizes with VMAT2 with punctate staining on subcellular compartments (Figures 2B,C).

[0131] The previously published in vitro assay that allows for visualization of vesicular dynamics in HEK293 cells stably expressing human vesicular monoamine transporter 2 (VMAT2; HEK-VMAT2) utilizes the fluorescent dopamine analogue and VMAT2 substrate fluorescent false neurotransmitter 206 (FFN206) (Black C. et. al. Chem. Res. Toxicol. 34, 5, 1256-1264 (2021)). In this assay, HEK-VMAT2 cells are incubated with FFN206, which accumulates within vesicular compartments via VMAT2-mediated transport. The accumulation of FFN206 can be blocked with VMAT2 inhibitors and by dissipating the proton gradient VMAT2 requires to load substrate within subcellular compartments. (Black C. et. al. Chem. Res. Toxicol. 34, 5, 1256-1264 (2021)). When FFN206 accumulates within vesicular compartments, the fluorescent signal is concentrated allowing for visualization andmeasurement of the substrate by microscopy and plate reader analysis. To investigate the effect that SV2C has on the vesicular sequestration of FFN206, the uptake of FFN206 was measured in HEK-VMAT2-SV2C cells and in HEK-VMAT2 cells.

[0132] To investigate the effect that SV2C has on the vesicular sequestration of the VMAT2 substrate FFN206, the uptake of FFN206 in HEK-VMAT2-SV2C cells compared to HEK-VMAT2 cells was measured. The addition of SV2C to HEK-VMAT2 cells results in enhanced uptake of FFN206 as visualized by microscopy (Figure 3 A). Quantification of uptake performed in 96-well plates on a plate reader detected a 33.05% increase in FFN206 fluorescence in HEK-VMAT2-SV2C cells compared to HEK-VMAT2 cells (Two-way ANOVA with Sidak’s multiple comparisons test, n = 7 experimental replicates, p <0.005, Figure 3B; See also Figure 15). The uptake of FFN206 is inhibited in both HEK-VMAT2 and HEK-VMAT2-SV2C cells by pre-treating the cells with IpM of the VMAT2 inhibitor tetrabenazine (TBZ) before FFN206 application. The previously report demonstrated the ICso of TBZ in HEK-VMAT2 cells to be 73.09nM with inhibition plateauing beginning at the IpM dose (Black C. et. al. Chem. Res. Toxicol. 34, 5, 1256-1264 (2020)). HEK-VMAT2- SV2C cells appear to have modest protection against TBZ-based inhibition of uptake at IpM compared to HEK-VMAT2 cells; however, this difference did not reach statistical significance (Two-way ANOVA with Sidak's multiple comparisons test, n = 7 experimental replicates, p = 0.83, Figure 3B; See also Figure 15). As this dose provides maximal inhibition of FFN206 uptake in HEK-VMAT2 cells, a TBZ dose-response was performed to determine whether HEK-VMAT2-SV2C cells are resistant to TBZ-induced inhibition. Dose-response experiments revealed a significant difference in ICso values, with HEK-VMAT2 cells demonstrating an ICso of 134.8nM, and HEK-VMAT2-SV2C cells demonstrating an ICso value of 274.9nM (non-linear regression [inhibitor] vs. normalized response, n = 4-8 experimental replicates for each dose, p < 0.05, Figure 3C).

[0133] To investigate the effect of SV2C on the retention of VMAT2 substrates, a novel method was developed to measure FFN206 fluorescence over time. For the retention assay, cells are incubated with FFN206 to allow for VMAT2-mediated uptake within subcellular compartments. Following incubation, fluorescence can be measured over time to determine the rate at which fluorescence is lost. Removing the FFN206 solution from cells before measuring fluorescence is necessary to be able to measure the fluorescent index of FFN206 sequestered within cells without measuring fluorescence from the solution itself. Byremoving the FFN206 solution, the remaining FFN206 within the cells is sequestered within subcellular compartments, and the cells are depleted of additional FFN206 available to be loaded within vesicles over time. Thus, the fluorescent values measured over time are representative of the retention of FFN206 within subcellular compartments. Using a 96-well plate and plate reader for analysis allows for fluorescence to be quantified at each timepoint for each well expressed as a percent of its baseline fluorescence.

[0134] In the retention assay, fluorescence decays linearly over time in HEK-VMAT2 and HEK-VMAT2-SV2C cells (Figure 4A). While the rate of decay is linear, HEK-VMAT2- SV2C cells demonstrate a modest but significant protection against loss of fluorescence over time based on linear regression. The slope of HEK-VMAT2 cells in a DMSO condition equals -0.41, representing a 0.41% loss of fluorescence per minute, whereas the slope for HEK-VMAT2-SV2C cells in a DMSO condition is -0.25, representing a 0.25% loss of fluorescence per minute (linear regression, n = 10 experimental replicates, p <0.01, Figure 4A). Because the vesicular loading of substrates is a dynamic process requiring active management of vesicular contents by VMAT2, TBZ treatment was treated after baseline fluorescence measurement. Inhibiting VMAT2 with TBZ prevents the reuptake of FFN206 by VMAT2 from the cytosol into the subcellular compartment, thus ensuring that the fluorescent values being measured were representative of the pool of FFN206 being retained within vesicles. To compare the rate of decay of fluorescence in the TBZ condition, nonlinear regression with single-phase decay was performed. HEK-VMAT2 cells demonstrate a rapid loss in fluorescence following TBZ treatment, whereas the HEK-VMAT2-SV2C cells show moderate protection against this loss of fluorescence. Analysis of non-linear regressions determined the decay curves are significantly different for HEK-VMAT2 cells treated with TBZ compared to HEK-VMAT2-SV2C cells treated with TBZ (non-linear regression with one-phase exponential decay, n = 4 experimental replicates, p < 0.0001, Figure 4B).

[0135] Although the fluorescent false neurotransmitters, including FFN206, were designed to be fluorescent substrates for VMAT2 (Hu G. et. al. ACS Chem. Biol. 8, 9, 1947- 1954 (2013)), these findings were replicated with dopamine as the substrate utilizing tritiated dopamine ([3H]-dopamine) in radiolabeled uptake and retention assays in vesicles isolated from HEK-VMAT2 and HEK-VMAT2-SV2C cells. The addition of SV2C to HEK-VMAT2 cells resulted in a 46.7% increase in uptake of [3H]-dopamine compared to HEK-VMAT2 cells lacking SV2C (Unpaired t-test, n = 11-12, p < 0.05, Figure 5A; see also Figure 16). Tomeasure how much [3H]-dopamine was retained within vesicles over time, vesicles were incubated with [3H]-dopamine before the VMAT2 inhibitor reserpine (lOpM) was applied to the vesicles. This effectively froze the transporter and a time course of vesicular leak to uninhibited vesicles was compared. The remaining [3H]-dopamine was measured at five timepoints post-reserpine application (Om, 5m, 10m, 15m, and 20m) to determine the rate of [3H]- dopamine leakage over time. By comparing the half-life calculated from the rate of decay in each cell line, it was determined that HEK-VMAT2-SV2C cells had a slower rate of leakage with a ti / 2 = 12.7m compared to a ti / 2 = 8.4m for HEK-VMAT2 cells (non-linear regression with one-phase exponential decay, n = 11-12 for each time-point, p <0.0001, Figure 5B).

[0136] Because the subcellular compartments on which VMAT2 and SV2C localize in HEK293 cells are not synaptic vesicles per se, the results of the radiolabeled uptake and retention assays were replicated in synaptic vesicles derived from mouse brain tissue. Vesicles were isolated from brain homogenate of wild-type and SV2C-KO animals and radiolabeled dopamine uptake and retention assays were performed. Although SV2C-KO animals showed no difference in [3H]-dopamine uptake compared to wild-type littermate controls as determined by a dose-response measure of vesicular capacity (Figure 19), vesicles derived from SV2C-KO animals had significantly reduced dopamine retention with a ti / 2 = 1.98m compared to vesicles derived from wild-type littermate controls, which had a ti / 2 = 3.32m (non-linear regression with one-phase exponential decay, n = 3-4 for each time-point, p <0.005, Figure 6).

[0137] The expression of VMAT2 is known to mediate dopamine neuron health by sequestering dopamine, thereby preventing dopamine from participating in neurotoxic cytosolic reactions (Bucher M. et. al., NPJ parkinsons Dis. 6: 34 (2020);Caudle W. et. al., JNeurosci 27(30) 8138-8148 (2007)), and through sequestration of the exogenous neurotoxicant and VMAT2 substrate MPP+(Lohr K. et. al. PNAS 111(27) 9977-9982 (2014). Following the results indicating that SV2C mediates the vesicular storage of dopamine and the dopamine analogue FFN206, the role of SV2C in mediating the vesicular storage of MPP+was tested. Radiolabeled uptake and retention assays were performed in vesicles isolated from HEK-VMAT2 and HEK-VMAT2-SV2C cells using radiolabeled MPP+([3H]- MPP+). There was a 44.6% increase in the uptake of [3H]-MPP+in HEK-VMAT2-SV2C cells compared to HEK-VMAT2 cells (Unpaired t-test, n = 12, p < 0.05, Figure 7A; see also Figure 17). In the retention assay, the half-life calculated from the rate of decay in each cellline demonstrated HEK-VMAT2-SV2C cells had a slower rate of leakage with a ti / 2 = 4.3m compared to a ti / 2 = 3.5m for HEK-VMAT2 cells (non-linear regression with one-phase exponential decay, n = 10-12 for each time-point, p <0.0005) (Figure 7B). These data indicate that in addition to regulating the vesicular storage of dopamine, SV2C may confer neuroprotection by mediating vesicular storage of exogenous neurotoxicants.

[0138] To evaluate whether ablation of SV2C results in heightened vulnerability to MPTP, a 5x20mg / kg dose of MPTP (Sigma) consisting of five injections (s.c.) was administered over five days with an inteij ection interval of 24hr in SV2C knock-out (SV2C- KO) mice and wild-type littermate controls. Immunohistochemistry of tyrosine hydroxylase (TH) revealed enhanced loss of immunoreactivity in the striatum and substantia nigra (Figure 7). To determine if there was an exacerbation of dopaminergic cell body loss following MPTP, stereological cell counting of TH-positive cells in the substantia nigra pars compacta (SNc) was performed (Figure 18 A). Baseline dopaminergic cell count was equivalent between SV2C-KO and WT animals (WT: 6221 cells ± 049; SV2C-KO: 5939 cells ± 698, p =0.97). Although there was a trend toward fewer neurons in WT animals following MPTP administration, this did not reach significance; however, SV2C-KO animals demonstrated a significant loss of 37.1% fewer TH-positive neurons in the SNc following MPTP (Fig. 9, Two-way ANOVA with Tukey’s multiple comparisons test, n = 4-10. WT: saline vs.WT:MPTP p = 0.11; SV2C-KO: saline vs. SV2C-KO:MPTP p<0.01). Quantification of TH expression in the striatum performed by Western blot revealed a significant loss of TH expression in both wild-type and SV2C-KO controls exposed to MPTP) (Fig. 9, Two-way ANOVA with Tukey’s multiple comparisons test, n = 4-10. WT:saline vs. WT:MPTP p <0.0001; SV2C-KO: saline vs. SV2C-KO:MPTP p <0.0001; see also Figure 18B, Two-way ANOVA with Tukey’s multiple comparison test, n=6-12. WT:saline vs. WT:MPTP p<0.001; SV2C-KO: saline vs. SV2C-KO:MPTP PO.OOOl).Discussion

[0139] The previous in vivo work in mice with genetically ablated SV2C implicated SV2C as a modifier of vesicular dopamine dynamics. Here, described herein is the first evidence that SV2C mediates the vesicular storage of dopamine and the dopamine analogues FFN206 and MPP+by enhancing total vesicular uptake and promoting vesicular retention. The previous data demonstrated that HEK293 cells expressing human VMAT2 accumulate FFN206 within subcellular components that colocalize with VMAT2 (Black C. et. al. Chem.Res. Toxicol. 34, 5, 1256-1264 (2020)). Described herein is an introduction of HEK293 cells that stably express both human VMAT2 and human SV2C (Figure 2), which show colocalization of VMAT2 and SV2C on subcellular compartments and enhanced accumulation of FFN206 (Figure 3) and radiolabeled dopamine (Figure 5).

[0140] The generation of a double-stable HEK-VMAT2-SV2C cell line revealed SV2C expression as a double band on Western blot appearing at ~95kDa (top band) and ~70kDa (bottom band) (Figure 14). The predicted molecular weight of SV2C is ~82kDa. Due to the highly glycosylated nature of SV2C, it is possible that these two bands correspond to a post- translationally modified and mature form of SV2C (top band), and an “immature” form of SV2C (bottom band) that has yet to undergo post-translational modification. Importantly, VMAT2 expression levels appear to be unchanged in the double-stable cell line (HEK- VMAT2-SV2C) compared to HEK-VMAT2 cells (Figure 14), and SV2C and VMAT2 proteins show a high degree of colocalization by immunocytochemistry (Figure 2B). Thus, the introduction of SV2C does not appear to affect VMAT2 expression in HEK-VMAT2 cell lines

[0141] The use of the fluorescent VMAT2 substrate FFN206 further allows for real-time visualization and monitoring of vesicular dynamics, which was utilized to develop a novel assay to monitor vesicular retention. Because vesicles are inherently leaky, it is possible to measure the retention of substrates within vesicles over time. FFN206 fluorescence is visualizable and measurable only when it is accumulated within subcellular compartments; thus, diffuse and dilute FFN206 is not detected by fluorescent plate reader or on microscopy. This property of FFN206 allows for calculation of vesicular retention by measuring the fluorescence of accumulated FFN206 at baseline and tracking fluorescent values over time. Thus, described herein is a plate reader-based assay using cells seeded on 96-well plates that measures the fluorescence of each well over time. Values can be corrected for background fluorescence by including cells receiving the same treatments (e.g., DMSO and tetrabenazine) that were not incubated with FFN206 each time the assay is run and performing a background subtraction using the average values calculated from these wells. This background subtraction is applied at each time-point that the plate is scanned over the course of the assay and the resulting fluorescent value can be expressed as a percent of its baseline (e.g., time-point 0m) fluorescence. Plotting the percent of baseline fluorescence over time allows for regression analysis to measure the loss of fluorescence over time.

[0142] Measuring vesicular retention of FFN206 identified that HEK293 cells expressing both human VMAT2 and human SV2C retain FFN206 within vesicles better than cells only expressing VMAT2 (Figure 4). Although this is a modest effect in cells treated with DMSO as a control treatment, it is made more apparent by adding the VMAT2 inhibitor TBZ at a saturating concentration (IpM). The vesicular packaging of substrates is a dynamic process involving leakage of vesicular contents requiring reuptake, and active transport of components in and out of the vesicles to maintain vesicular pH and achieve a functional equilibrium. While measuring FFN206 fluorescence over time, in the DMSO condition, it is possible that as FFN206 leaks out of the vesicle it diffuses beyond the local vesicular environment and is unable to be resequestered by VMAT2 or otherwise undergoes degradation. By adding TBZ, the resequestration by VMAT2 of FFN206 that leaks out of the vesicle can be prevented. As an added consequence, the application of TBZ also precludes the active export of FFN206 out of the vesicle by VMAT2. Thus, the observed decay in fluorescence over time is due to FFN206 leaking out of the vesicle in a non-VMAT2 dependent manner and is indicative of how much FFN206 remains sequestered within vesicles.

[0143] Although FFN206 was designed as a dopamine analogue and substrate for VMAT2, the effect of SV2C on vesicular dynamics was confirmed using FFN206 were replicated using dopamine directly. Vesicles isolated from HEK293 cells expressing human VMAT2 or both human VMAT2 and SV2C underwent uptake assays using radiolabeled dopamine ([3H]-dopamine) and demonstrated enhanced uptake and retention of [3H]- dopamine (Figure 4). Retention, or leakage, assays were performed in a similar manner to FFN206 retention assays where vesicles were incubated with [3H]-dopamine before sequestration was blocked with the VMAT2 inhibitors TBZ or reserpine and the amount of [3H]-dopamine remaining within the vesicles was measured at multiple time-points. However, because HEK293 cells are not neurons and do not contain the same factors as neurons, these findings were true in vesicles isolated from mouse brain.

[0144] While uptake of FFN206 and [3H]-dopamine is increased in HEK-VMAT2-SV2C cells and vesicles derived from HEK-VMAT2-SV2C cells respectively, vesicles isolated from mice with genetic ablation of SV2C (SV2C-KO) do not show a difference in total vesicular uptake of [3H]-dopamine (Fig. 10). This may be due to differences in the nature of the compartments within HEK293 cells on which VMAT2 and SV2C localize compared tothe composition of synaptic vesicles. For example, in HEK293 cells, these compartments may have increased capacity for the total amount of substrate uptake based on size or number of copies of VMAT2 expressed on each compartment, or increased uptake due to increased pool of protons within the compartments providing the proton-motive force by which VMAT2 loads substrates. Despite there being no difference in the baseline amount of [3H]- dopamine uptake, synaptic vesicles derived from the brain of SV2C-KO mice displayed enhanced rate of [3H]-dopamine leak compared to those from wild-type animals (Figure 6.).

[0145] HEK-VMAT2-SV2C cells appear to be resistant to the effects of the VMAT2 inhibitor tetrabenazine (TBZ) when measuring FFN206 uptake (Figure 3C). It is possible that the presence of SV2C renders VMAT2 more functional in taking up dopamine or more resistant to the inhibitory effects of TBZ. Furthermore, it is possible that SV2C enhances vesicular dopamine uptake and retention through direct transport of dopamine itself. However, if SV2C could transport dopamine, it is expected to see a significant difference in FFN206 uptake in cells expressing both VMAT2 and SV2C compared to cells only expressing VMAT2 when uptake by VMAT2 is fully inhibited (e.g., with pre-treatment of >lpM TBZ) (Figure 2C). Further experiments will be necessary to determine whether the shift in inhibition is due to an S V2C-mediated decrease of TBZ binding or efficacy at VMAT2. However, the results may also be a result of the initial non-specific accumulation within HEK293 cells observed with FFN206, which can enter HEK293 cells that do not express the plasmalemmal dopamine transporter (DAT). Initial accumulation within the cell is not dependent on dopamine transporters (e.g., DAT and VMAT2); however, once FFN206 has reached diffuse equilibrium throughout the cell, its accumulation within vesicles is dependent upon VMAT2. Thus, SV2C may be acting to retain the FFN206 that has accumulated within vesicles in a non-VMAT2 dependent manner, thereby resisting the effects of TBZ.

[0146] It has been well-established that MPTP toxicity can regulated by dopaminergic synaptic vesicles (Alter S. et. al. Curr. Neuro & Neurosci Rep. 13, 362 (2013), Bernstein A. et. al. 73:89-97 (2014); Gainetdinov R. et. al. J. Neurochem. 70(5) 1973-1978 (2002); Guillot T. & Miller G. Mol. Neurobiol. 39, 149-170 (2009); Przedborski S. et. al. 16(2): 135-142 (2000); Taylor T. et. al. Parkinsons Dis. (2011)). In animal models MPTP administered systemically results in dopaminergic neuron degeneration in the brain due to the toxic metabolite MPP+acting as a substrate for the plasmalemmal dopamine transporter (DAT),allowing for accumulation within dopaminergic neurons. When dopamine vesicles have a greater capacity to store dopamine and sequester toxicants from the cytosol, neurons are resistant to degeneration, and when this process is impaired, neurons are vulnerable to enhanced cellular damage and death. This has been demonstrated repeatedly by our laboratory in models of varying VMAT2 expression. In fact, VMAT2 was originally identified for its role in sequestering the active metabolite of MPTP, MPP+, and protecting against MPTP toxicity (Edwards R., Ann. Neurology 34 (5) 638-645 (1993)).

[0147] The evidence from experiments evaluating the effect of SV2C on FFN206 and [3H]-dopamine uptake and retention suggests that SV2C may similarly interact with the neurotoxicant, and VMAT2 substrate, MPP+. Described herein is the first evidence that another vesicular protein, SV2C, may promote the vesicular retention of the neurotoxicant MPP+and mediate the toxic effects of MPTP. By isolating vesicles from HEK293 cells expressing VMAT2 and SV2C, we were able to directly interrogate the effect SV2C has on uptake and retention of radiolabeled MPP+([3H]-MPP+). Similar to the results from [3H]- dopamine experiments, we found that cells expressing both VMAT2 and SV2C had enhanced uptake and retention of [3H]-MPP+(Figure 7). Interestingly, while the extent of increased uptake in vesicles from cells containing both VMAT2 and SV2C was comparable, with a 48.1% increase in [3H]-dopamine uptake and a 44.6% increase in [3H]-MPP+uptake, the raw values of uptake were much higher for [3H]-dopamine than [3H]-MPP+, with uptake values of 4369.47fmol / pg protein and 2010.14fmol / pg protein respectively. Furthermore, the rate of leakage is different between [3H]-dopamine and [3H]-MPP+, with a ti / 2 equal to 12.7m and 4.3m respectively. Other studies have reported similar affinities of VMAT2 for dopamine and MPP+(Eiden L. & Weihe E. Annal. N. Y. Aca. Sci. 1216 (1) 86-98 (2011)). The design of the experiments conducted in this study does not permit ruling out this difference due to experimental conditions; however, it does provide interesting preliminary data warranting deeper investigation in future studies looking at competition between dopamine and MPP+as substrates.

[0148] Following the data from radiolabeled uptake and retention experiments, the biological implications in vivo was investigated with experiments exposing wild-type and SV2C-KO animals to MPTP. As a result, the present data that showing that SV2C is protective against chemically-induced neurodegeneration. Mice with genetic ablation of SV2C demonstrate a greater effect of MPTP on nigral and striatal tyrosine hydroxylase (TH)immunoreactivity, indicating increased vulnerability (Figure 8). While there is no significant difference in striatal TH expression as determined by Western blot quantitation (Figures 9 and 18B), SV2C-KO mice treated with MPTP have significantly fewer TH-positive neuronal cell bodies in the substantia nigra compared to control treated SV2C-KO mice, whereas wildtype mice treated with MPTP do not show a significant difference in the number of TH- positive neuronal cell bodies in the substantia nigra compared to control treated wild-type mice (Figures 9 and 18 A). Impairment of vesicular function as a result of SV2C-K0 may result in higher cytosolic concentrations of dopamine and / or MPP+. Cytosolic dopamine can act as an endogenous neurotoxicant, and cytosolic MPP+is free to act as a mitochondrial complex I inhibitor, both of which may contribute to neuron vulnerability.

[0149] Although HEK293 cells are non-neuronal and do not contain all the proteins involved in vesicular sequestration that can be found in brain tissue, experiments in these cells that display proton gradient dependent storage of dopamine in a vesicle-like compartment allow for isolation of the factors involved to include only the effect of VMAT2 and SV2C. However, it should be noted that there are limitations due to the derivation of two independent stable cell lines utilized in this manuscript. It is possible that genetic drift occurred during the development of the HEK-VMAT2-SV2C double stable cell line due to the use of geneticin antibiotic resistance, which may have resulted in differences between the HEK-VMAT2 and HEK-VMAT2-SV2C cell lines that may have contributed to the results observed. To evaluate the phenotype of the two cell lines used, a thorough investigation of protein expression and localization was performed to determine whether the HEK-VMAT2- SV2C cells displayed any additional differences such as changes in protein expression or subcellular organelle localization. There were no detectable differences observed in the amount of v-type ATPase protein expression between the two cell lines, or differences in subcellular localization of v-type ATPase and VMAT2 based on colocalization with markers for mitochondria, golgi, endoplasmic reticulum, and lysosomes (data not shown).

[0150] Future experiments are necessary to fully understand the function of SV2C. For instance, SV2 proteins are highly glycosylated and the intraluminal loops of SV2s are thought to comprise the intra-vesicular proteoglycan “gel” matrix, which can be visualized by electron microscopy (Harlow M. et. al. PlosOne 8(7) e69410 (2013)). The proteoglycan matrix within vesicles has been demonstrated as capable of directly adsorbing ATP as visualized by atomic force microscopy and is hypothesized to regulate the release oftransmitter molecules into the synaptic cleft upon endocytosis (de Toledo G. et. al. Nature 363, 554-558 (1993); Harlow M. et. al. PlosOne 8(7) e69410 (2013); Reigada D. et. al. PNAS 100(6) 3485-3490 (2003); Schiffer et. al. Synpase 56(4):243-51 (2006)). In future experiments, the role of glycosylation in SV2C function can be evaluated by mutating out the glycosylation sites on SV2C. Additionally, it is possible that SV2C alters other vesicular kinetics, including but not limited to membrane potential, pH gradient, and successful exocytotic trafficking. (Edward, Neuron 55(6): 835-858 (2007)). Future experiments utilizing tools such as synaptopHluorin or pH-sensitive FFNs can be performed to determine whether SV2C has an effect on pH gradient. Furthermore, synaptopHluorin can also be utilized in vesicular fusion assays to determine whether SV2C influences trafficking and / or exocytosis during evoke release

[0151] It is not well understood how other perturbations to the biological system, such as altered protein expression or toxicant exposure, may affect SV2C expression. For example, Dardou et al. 2013 reported an increase in SV2C mRNA expression following intracranial 6- OHDA (6-hydroxydopamine) lesions and peripheral MPTP treatment. (Dardou D. et. al., Brain Res 1507:61-73 (2013)). This upregulation may be a compensatory response and attempt at preserving neuronal health as a result of neurotoxicant exposure and induction of neurodegeneration. The compensatory upregulation of SV2C mRNA has similarly been reported by Isingrini , which reported the highest increase in mRNA expression for SV2C in mice lacking VMAT2 expression in norepinephrine expressing neurons. (Isingrini E. et. al., Biomolecules 13(3) (2023)). Additional experiments are needed to understand how SV2C expression is altered in response to neuronal insult, particularly to distinguish between mRNA upregulation and protein expression.

[0152] Overall, these data further support the emergence of SV2C as a relevant player in dopamine neuron and vesicle function. The data establish SV2C as a mediator of MPTP toxicity, and suggest that SV2C function and expression is inversely correlated with vulnerability to degeneration. These data point to a role for SV2C in mediating chemically- induced dopaminergic degeneration, and future studies will investigate whether this neuroprotective function of SV2C may extend to additional models of cell death. Additionally, these data may suggest that enhanced SV2C could be neuroprotective, which could have significant implications for the development of Parkinson’s disease therapeutics.References

[0153] Alter, S. P., G. M. Lenzi, A. I. Bernstein and G. W. Miller (2013). "Vesicular integrity in Parkinson's disease." Curr Neurol Neurosci Rep 13(7): 362.

[0154] Altmann, V., A. F. Schumacher-Schuh, M. Rieck, S. M. Callegari-Jacques, C. R. Rieder and M. H. Hutz (2016). "Influence of genetic, biological and pharmacological factors on levodopa dose in Parkinson's disease." Pharmacogenomics 17(5): 481-488.

[0155] Alvarez de Toledo, G., R. Fernandez-Chacon and J. M. Fernandez (1993). "Release of secretory products during transient vesicle fusion." Nature 363(6429): 554-558.

[0156] Bajjalieh, S. M., K. Peterson, R. Shinghal and R. H. Scheller (1992). "SV2, a brain synaptic vesicle protein homologous to bacterial transporters." Science 257(5074): 1271-1273.

[0157] Bernstein, A. I., K. A. Stout and G. W. Miller (2014). "The vesicular monoamine transporter 2: an underexplored pharmacological target." Neurochem Int 73: 89-97.

[0158] Black, C. A., M. L. Bucher, J. M. Bradner, L. Jonas, K. Igarza and G. W. Miller (2021). "Assessing Vesicular Monoamine Transport and Toxicity Using Fluorescent False Neurotransmitters." Chem Res Toxicol 34(5): 1256-1264.

[0159] Brighina, L., C. Riva, F. Bertoia, E. Saracchi, S. Fermi, S. Goldwurm and C. Ferrarese (2013). "Analysis of vesicular monoamine transporter 2 polymorphisms in Parkinson's disease." Neurobiol Aging 34(6): 1712 el 719- 1713.

[0160] Bucher, M. L., C. W. Barrett, C. J. Moon, A. D. Mortimer, E. A. Burton, J. T. Greenamyre and T. G. Hastings (2020). "Acquired dysregulation of dopamine homeostasis reproduces features of Parkinson's disease." NPJ Parkinsons Dis 6(1): 34.

[0161] Caudle, W. M., J. R. Richardson, M. Z. Wang, T. N. Taylor, T. S. Guillot, A. L. McCormack, R. E. Colebrooke, D. A. Di Monte, P. C. Emson and G. W. Miller (2007)."Reduced vesicular storage of dopamine causes progressive nigrostriatal neurodegeneration." J Neurosci 27(30): 8138-8148.

[0162] Chang, W. P. and T. C. Sudhof (2009). "SV2 renders primed synaptic vesicles competent for Ca2+ -induced exocytosis." J Neurosci 29(4): 883-897.

[0163] Ciruelas, K., D. Marcotulli and S. M. Bajjalieh (2019). "Synaptic vesicle protein 2: A multi-faceted regulator of secretion." Semin Cell Dev Biol 95: 130-141.

[0164] Dardou, D., S. Monlezun, P. Foerch, J. P. Courade, L. Cuvelier, M. De Ryck and S. N. Schiffmann (2013). "A role for Sv2c in basal ganglia functions." Brain Res 1507: 61- 73.

[0165] Dunn, A. R., C. A. Hoffman, K. A. Stout, M. Ozawa, R. K. Dhamsania and G. W. Miller (2019). "Immunochemical analysis of the expression of SV2C in mouse, macaque and human brain." Brain Res 1702: 85-95.

[0166] Dunn, A. R., K. A. Stout, M. Ozawa, K. M. Lohr, C. A. Hoffman, A. I. Bernstein, Y. Li, M. Wang, C. Sgobio, N. Sastry, H. Cai, W. M. Caudle and G. W. Miller (2017)."Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson’s disease." PNAS: E2253-2262.

[0167] Edwards, R. H. (1993). "Neural degeneration and the transport of neurotransmitters." Ann Neurol 34(5): 638-645.

[0168] Edwards, R. H. (2007). "The neurotransmitter cycle and quantal size." Neuron 55(6): 835-858.

[0169] Ehringer, H. and O. Homykiewicz (1960). "[Distribution of noradrenaline and dopamine (3-hydroxytyramine) in the human brain and their behavior in diseases of the extrapy rami dal system]." Klin Wochenschr 38: 1236-1239.

[0170] Eiden, L. E. and E. Weihe (2011). "VMAT2: a dynamic regulator of brain monoaminergic neuronal function interacting with drugs of abuse." Ann N Y Acad Sci 1216: 86-98.

[0171] Eisenhofer, G., I. J. Kopin and D. S. Goldstein (2004). "Leaky catecholamine stores: undue waste or a stress response coping mechanism?" Ann N Y Acad Sci 1018: 224- 230.

[0172] Feany, M. B., S. Lee, R. H. Edwards and K. M. Buckley (1992). "The synaptic vesicle protein SV2 is a novel type of transmembrane transporter." Cell 70(5): 861-867.

[0173] Floor, E., P. S. Leventhal, Y. Wang, L. Meng and W. Chen (1995). "Dynamic storage of dopamine in rat brain synaptic vesicles in vitro." J Neurochem 64(2): 689-699.

[0174] Foo, J. N., E. G. Y. Chew, S. J. Chung, R. Peng, C. Blauwendraat, M. A. Nalls, K. Y. Mok, W. Satake, T. Toda, Y. Chao, L. C. S. Tan, M. Tandiono, M. M. Lian, E. Y. Ng, K. M. Prakash, W. L. Au, W. Y. Meah, S. Q. Mok, A. A. Annuar, A. Y. Y. Chan, L. Chen, Y. Chen, B. S. Jeon, L. Jiang, J. L. Lim, J. J. Lin, C. Liu, C. Mao, V. Mok, Z. Pei, H. F. Shang,C. H. Shi, K. Song, A. H. Tan, Y. R. Wu, Y. M. Xu, R. Xu, Y. Yan, J. Yang, B. Zhang, W. P. Koh, S. Y. Lim, C. C. Khor, J. Liu and E. K. Tan (2020). "Identification of Risk Loci for Parkinson Disease in Asians and Comparison of Risk Between Asians and Europeans: A Genome-Wide Association Study." JAMA Neurol 77(6): 746-754.

[0175] Gainetdinov, R. R., F. Fumagalli, Y. M. Wang, S. R. Jones, A. I. Levey, G. W. Miller and M. G. Caron (1998). "Increased MPTP neurotoxicity in vesicular monoamine transporter 2 heterozygote knockout mice." J Neurochem 70(5): 1973-1978.

[0176] Gan-Or, Z., I. Amshalom, A. Bar-Shira, M. Gana-Weisz, A. Mirelman, K. Marder, S. Bressman, N. Giladi and A. Orr-Urtreger (2015). "The Alzheimer disease BINI locus as a modifier of GBA-associated Parkinson disease." J Neurol 262(11): 2443-2447.

[0177] Glatt, C. E., A. D. Wahner, D. J. White, A. Ruiz-Linares and B. Ritz (2006). "Gain-of-function haplotypes in the vesicular monoamine transporter promoter are protective for Parkinson disease in women." Hum Mol Genet 15(2): 299-305.

[0178] Goldstein, D. S., I. J. Kopin and Y. Sharabi (2014). "Catecholamine autotoxicity. Implications for pharmacology and therapeutics of Parkinson disease and related disorders." Pharmacol Ther 144(3): 268-282.

[0179] Graves, S. M., Z. Xie, K. A. Stout, E. Zampese, L. F. Burbulla, J. C. Shih, J. Kondapalli, T. Patriarchi, L. Tian, L. Brichta, P. Greengard, D. Krainc, P. T. Schumacker andD. J. Surmeier (2020). "Dopamine metabolism by a monoamine oxidase mitochondrial shuttle activates the electron transport chain." Nat Neurosci 23(1): 15-20.

[0180] Grover, S., A. A. Kumar-Sreelatha, D. R. Bobbili, P. May, C. Domenighetti, P. E.Sugier, C. Schulte, C.-P. Consortium, A. Elbaz, R. Kruger, T. Gasser, M. Sharma and C.-P.Consortium (2021). "Replication of a Novel Parkinson's Locus in a European Ancestry Population." Mov Disord 36(7): 1689-1695.

[0181] Guillot, T. S. and G. W. Miller (2009). "Protective actions of the vesicular monoamine transporter 2 (VMAT2) in monoaminergic neurons." Mol Neurobiol 39(2): 149- 170.

[0182] Guzman, J. N., J. Sanchez-Padilla, D. Wokosin, J. Kondapalli, E. Ilijic, P. T. Schumacker and D. J. Surmeier (2010). "Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1." Nature 468(7324): 696-700.

[0183] Haddad, D. and K. Nakamura (2015). "Understanding the susceptibility of dopamine neurons to mitochondrial stressors in Parkinson's disease." FEBS Lett 589(24 Pt A): 3702-3713.

[0184] Harlow, M. L., J. A. Szule, J. Xu, J. H. Jung, R. M. Marshall and U. J. McMahan (2013). "Alignment of synaptic vesicle macromolecules with the macromolecules in active zone material that direct vesicle docking." PLoS One 8(7): e69410.

[0185] Hastings, T. G., D. A. Lewis and M. J. Zigmond (1996). "Role of oxidation in the neurotoxic effects of intrastriatal dopamine injections." Proc Natl Acad Sci U S A 93(5): 1956-1961.

[0186] Hill-Bums, E. M., N. Singh, P. Ganguly, T. H. Hamza, J. Montimurro, D. M. Kay, D. Yearout, P. Sheehan, K. Frodey, J. A. McLear, M. B. Feany, S. D. Hanes, W. J. Wolfgang,C. P. Zabetian, S. A. Factor and H. Payami (2013). "A genetic basis for the variable effect of smoking / nicotine on Parkinson's disease." Pharmacogenomics J 13(6): 530-537.

[0187] Hu, G., A. Henke, R. J. Karpowicz, Jr., M. S. Senders, F. Farrimond, R. Edwards,D. Sulzer and D. Sarnes (2013). "New fluorescent substrate enables quantitative and high- throughput examination of vesicular monoamine transporter 2 (VMAT2)." ACS Chem Biol 8(9): 1947-1954.

[0188] Isingrini, E., C. Guinaudie, L. Perret, E. Guma, V. Gorgievski, I. D. Blum, J. Colby-Milley, M. Bairachnaya, S. Melia, A. Adamantidis, K. F. Storch and B. Giros (2023). "Behavioral and Transcriptomic Changes Following Brain-Specific Loss of Noradrenergic Transmission." Biomolecules 13(3).

[0189] Jacobsen, J. C., C. Wilson, V. Cunningham, E. Glamuzina, D. O. Prosser, D. R. Love, T. Burgess, J. Taylor, B. Swan, R. Hill, S. P. Robertson, R. G. Snell and K. Lehnert (2016). "Brain dopamine-serotonin vesicular transport disease presenting as a severe infantile hypotonic parkinsonian disorder." J Inherit Metab Dis 39(2): 305-308.

[0190] Lohr, K. M., A. I. Bernstein, K. A. Stout, A. R. Dunn, C. R. Lazo, S. P. Alter, M. Wang, Y. Li, X. Fan, E. J. Hess, H. Yi, L. M. Vecchio, D. S. Goldstein, T. S. Guillot, A. Salahpour and G. W. Miller (2014). "Increased vesicular monoamine transporter enhances dopamine release and opposes Parkinson disease-related neurodegeneration in vivo." Proc Natl Acad Sci U S A 111(27): 9977-9982.

[0191] Lohr, K. M., M. Chen, C. A. Hoffman, M. J. McDaniel, K. A. Stout, A. R. Dunn, M. Wang, A. I. Bernstein and G. W. Miller (2016). "Vesicular Monoamine Transporter 2 (VMAT2) Level Regulates MPTP Vulnerability and Clearance of Excess Dopamine in Mouse Striatal Terminals." Toxicol Sci 153(1): 79-88.

[0192] Lohr, K. M., K. A. Stout, A. R. Dunn, M. Wang, A. Salahpour, T. S. Guillot and G. W. Miller (2015). "Increased Vesicular Monoamine Transporter 2 (VMAT2; Slcl8a2) Protects against Methamphetamine Toxicity." ACS Chem Neurosci 6(5): 790-799.

[0193] Madeo, M., A. D. Kovacs and D. A. Pearce (2014). "The human synaptic vesicle protein, SV2A, functions as a galactose transporter in Saccharomyces cerevisiae." J Biol Chem 289(48): 33066-33071.

[0194] Matsuda, W., T. Furuta, K. C. Nakamura, H. Hioki, F. Fujiyama, R. Arai and T. Kaneko (2009). "Single nigrostriatal dopaminergic neurons form widely spread and highly dense axonal arborizations in the neostriatum." J Neurosci 29(2): 444-453.

[0195] Meiser, J., D. Weindl and K. Hiller (2013). "Complexity of dopamine metabolism." Cell Commun Signal 11(1): 34.

[0196] Miller, G. W., J. D. Erickson, J. T. Perez, S. N. Penland, D. C. Mash, D. B. Rye and A. I. Levey (1999). "Immunochemical analysis of vesicular monoamine transporter (VMAT2) protein in Parkinson's disease." Exp Neurol 156(1): 138-148.

[0197] Olsen, A. L., S. G. Clemens and M. B. Feany (2023). "Nicotine-Mediated Rescue of alpha-Synuclein Toxicity Requires Synaptic Vesicle Glycoprotein 2 in Drosophila." Mov Disord 38(2): 244-255.

[0198] Pifl, C., A. Rajput, H. Reither, J. Blesa, C. Cavada, J. A. Obeso, A. H. Rajput and O. Hornykiewicz (2014). "Is Parkinson's disease a vesicular dopamine storage disorder? Evidence from a study in isolated synaptic vesicles of human and nonhuman primate striatum." J Neurosci 34(24): 8210-8218.

[0199] Pissadaki, E. K. and J. P. Bolam (2013). "The energy cost of action potential propagation in dopamine neurons: clues to susceptibility in Parkinson's disease." Front Comput Neurosci 7: 13.

[0200] Przedborski, S., V. Jackson-Lewis, R. Djaldetti, G. Liberatore, M. Vila, S. Vukosavic and G. Aimer (2000). "The parkinsonian toxin MPTP: action and mechanism." Restor Neurol Neurosci 16(2): 135-142.

[0201] Reigada, D., I. Diez-Perez, P. Gorostiza, A. Verdaguer, I. Gomez de Aranda, O. Pineda, J. Vilarrasa, J. Marsal, J. Blasi, J. Aleu and C. Solsona (2003). "Control of neurotransmitter release by an internal gel matrix in synaptic vesicles." Proc Natl Acad Sci U S A 100(6): 3485-3490.

[0202] Saida, K., R. Maroofian, T. Sengoku, T. Mitani, A. T. Pagnamenta, D. Marafi, M. S. Zaki, T. J. O'Brien, E. G. Karimiani, R. Kaiyrzhanov, M. Takizawa, S. Ohori, H. Y. Leong, G. Akay, H. Galehdari, M. Zamani, R. Romy, C. J. Carroll, M. B. Toosi, F. Ashrafzadeh, S. Imannezhad, H. Malek, N. Ahangari, H. Tomoum, V. K. Gowda, V. M. Srinivasan, D. Murphy, N. Dominik, H. M. Elbendary, K. Rafat, S. Yilmaz, S. Kanmaz, M. Serin, D. Krishnakumar, A. Gardham, A. Maw, T. S. Rao, S. Alsubhi, M. Srour, D. Buhas, T. Jewett, R. E. Goldberg, H. Shamseldin, E. Frengen, D. Misceo, P. Stromme, J. R. Magliocco Ceroni, C. A. Kim, G. Yesil, E. Sengenc, S. Guler, M. Hull, M. Parnes, D. Aktas, B. Anlar, Y. Bayram, D. Pehlivan, J. E. Posey, S. Alavi, S. A. Madani Manshadi, H. Alzaidan, M. Al- Owain, L. Alabdi, F. Abdulwahab, F. Sekiguchi, K. Hamanaka, A. Fujita, Y. Uchiyama, T. Mizuguchi, S. Miyatake, N. Miyake, R. M. Elshafie, K. Salayev, U. Guliyeva, F. S.Alkuraya, J. G. Gleeson, K. G. Monaghan, K. G. Langley, H. Yang, M. Motavaf, S. Safari, M. Alipour, K. Ogata, A. E. X. Brown, J. R. Lupski, H. Houlden and N. Matsumoto (2023)."Brain monoamine vesicular transport disease caused by homozygous SLC18A2 variants: A study in 42 affected individuals." Genet Med 25(1): 90-102.

[0203] Sala, G., L. Brighina, E. Saracchi, S. Fermi, C. Riva, V. Carrozza, M. Pirovano and C. Ferrarese (2010). "Vesicular monoamine transporter 2 mRNA levels are reduced in platelets from patients with Parkinson's disease." J Neural Transm (Vienna) 117(9): 1093- 1098.

[0204] Stove, S. I., A. A. Skjevik, K. Teigen and A. Martinez (2022). "Inhibition of VMAT2 by beta2-adrenergic agonists, antagonists, and the atypical antipsychotic ziprasidone." Commun Biol 5(1): 1283.

[0205] Sulzer, D. and L. Zecca (2000). "Intraneuronal dopamine-quinone synthesis: a review." Neurotox Res 1(3): 181-195.

[0206] Taylor, T. N., S. P. Alter, M. Wang, D. S. Goldstein and G. W. Miller (2014)."Reduced vesicular storage of catecholamines causes progressive degeneration in the locus ceruleus." Neuropharmacology 76 Pt A(0 0): 97-105.

[0207] Taylor, T. N., W. M. Caudle and G. W. Miller (2011). "VMAT2-Deficient Mice Display Nigral and Extranigral Pathology and Motor and Nonmotor Symptoms of Parkinson's Di sease . " Parkinsons Dis 2011: 124165.

[0208] Taylor, T. N., W. M. Caudle, K. R. Shepherd, A. Noorian, C. R. Jackson, P. M. luvone, D. Weinshenker, J. G. Greene and G. W. Miller (2009). "Nonmotor symptoms of Parkinson's disease revealed in an animal model with reduced monoamine storage capacity." J Neurosci 29(25): 8103-8113.

[0209] Vautrin, J. (2009). "SV2 frustrating exocytosis at the semi-diffusor synapse." Synapse 63(4): 319-338.

[0210] Xiong, N., N. Li, E. Martin, J. Yu, J. Li, J. Liu, D. Y. Lee, O. Isacson, J. Vance, H. Qing, T. Wang and Z. Lin (2016). "hVMAT2: A Target of Individualized Medication for Parkinson's Disease." Neurotherapeutics 13(3): 623-634.

[0211] Yang, X., P. Xu, Q. Zhao, R. An, H. Jia, Z. Liu and Y. Xu (2015). "Polymorphism in the Vesicular Monoamine Transporter 2 Gene Decreases the Risk of Parkinson's Disease in Han Chinese Men." Parkinsons Dis 2015: 903164.

[0212] Yee, S. W. and K. M. Giacomini (2021). "Emerging Roles of the Human Solute Carrier 22 Family." Drug Metab Dispos 50(9): 1193-1210.

Claims

What is claimed:

1. A method of measuring vesicular leakage, the method comprising: a) contacting a cell culture with a fluorescent false neurotransmitter, b) measuring a fluorescence signal of the fluorescent false neurotransmitter in realtime, and c) determining a rate of fluorescence decay of the fluorescence signal.

2. A method of identifying a compound that modulates vesicular leakage, the method comprising: a) contacting a cell culture with a fluorescent false neurotransmitter, b) measuring a fluorescence signal of the fluorescent false neurotransmitter, c) contacting the cell culture with a test compound, d) measuring a fluorescence signal of the fluorescent false neurotransmitter in realtime, e) determining a rate of fluorescence decay of the fluorescence signal, and f) identifying the test compound as a compound that modulates vesicular leakage if the rate of fluorescence decay determined in step e) is higher or lower than a rate of fluorescence decay of a cell culture under a control condition.

3. A method of identifying a compound that modulates vesicular leakage, the method comprising: a) contacting a cell culture with a test compound, b) contacting the cell with a fluorescent false neurotransmitter, c) measuring a fluorescent signal of the fluorescent false neurotransmitter in real-time, d) determining a rate of fluorescence decay of the fluorescence signal, and e) identifying the test compound as a compound that modulates vascular leakage if the rate of fluorescence decay determined in step c) is higher or lower than a rate of fluorescence decay of a cell culture under a control condition.

4. The method of claims 1-3, wherein cells of the cell culture are genetically modified cells.

5. The method of claims 1-3, wherein the cells of the cell culture have reduced synaptic vesicle glycoprotein 2C (SV2C) expression level.

6. The method of claims 1-3, wherein the fluorescent false neurotransmitter comprises a dopamine analogue.

7. The method of claims 1-3, wherein the fluorescent false neurotransmitter comprises false fluorescent neurotransmitter 206 (FFN206).

8. The method of claims 1-3, wherein the rate of fluorescence decay of the fluorescence signal is determined by: i. for each fluorescence signal value of the real-time measurement, subtracting a background fluorescence signal, and calculating a percent fluorescence signal change relative to the fluorescence signal measured in step b), and ii. plotting the percentage of fluorescence signal change versus time; and iii. calculating the slope of the percentage of fluorescence signal change versus time.

9. The method of claims 1-3, wherein the rate of fluorescence decay of the fluorescence signal is determined by: i. for each fluorescence signal value of the real-time measurement, subtracting a background fluorescence signal, ii. plotting the fluorescent signal value versus time; and iii. calculating the slope of the fluorescence signal value.

10. The method of claims 2-3, wherein a cell culture under a control condition comprises a cell culture contacted with dimethyl sulfoxide (DMSO).

11. The method of claims 2-3, wherein the test compound comprises tetrabenazine (TBZ).

12. The method of claims 2-3, wherein the test compound comprises l-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP).

13. The method of claim 3, wherein the neurological disorder is Parkinson’s disease.

Images