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WO2025096017A2 - Dosage de fuite vésiculaire de neurotransmetteur - Google Patents

Dosage de fuite vésiculaire de neurotransmetteur Download PDF

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WO2025096017A2
WO2025096017A2 PCT/US2024/030991 US2024030991W WO2025096017A2 WO 2025096017 A2 WO2025096017 A2 WO 2025096017A2 US 2024030991 W US2024030991 W US 2024030991W WO 2025096017 A2 WO2025096017 A2 WO 2025096017A2
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cells
sv2c
vmat2
cell culture
fluorescent
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WO2025096017A3 (fr
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Gary W. Miller
Meghan L. BUCHER
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Columbia University in the City of New York
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Columbia University in the City of New York
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease

Definitions

  • 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.
  • 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.
  • a method of measuring vesicular leakage 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.
  • a method of identifying a compound that modulates vesicular leakage 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.
  • a method of identifying a compound that modulates vesicular leakage 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.
  • a method of treating a neurological disorder in a subject in need thereof comprising: (a) contacting a cell culture with a fluorescence 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, 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.
  • the cells of the cell culture are genetically modified cells.
  • the cells of the cell culture have reduced synaptic vesicle glycoprotein 2C (SV2C) expression compared to a cell without genetic modification.
  • the cells of the cell culture have reduced expression of synaptic vesicle localized proteins compared to a cell without genetic modification.
  • the cells of the cell culture have reduced expression of plasma membrane localized proteins compared to a cell without genetic modification.
  • the cells of the cell culture have reduced expression of organelle localized proteins compared to a cell without genetic modification.
  • 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.
  • 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.
  • 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.
  • a cell culture under a control condition comprises a cell culture cultured under standard cell culturing conditions for such a cell culture.
  • a cell culture under a control condition comprises a cell culture contacted with a control compound.
  • the control compound is a solvent.
  • the control compound is sterile ultrapure water.
  • the control compound is dimethyl sulfoxide (DMSO).
  • the test compound is a pharmacological regulator.
  • the test compound comprises tetrabenazine (TBZ).
  • the test compound is an environmental or toxicological regulator.
  • the test compound comprises l-methyl-4- phenyl- 1 ,2,3 ,6-tetrahydropyri dine (MPTP).
  • Figure 1 shows a schematic showing that synaptic vesicle glycoprotein 2C (SV2C) promotes retention of dopamine and dopamine analogues (FFN206, MPP + ) within synaptic vesicles.
  • SV2C synaptic vesicle glycoprotein 2C
  • 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.
  • 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 FFN206 uptake 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.
  • DMSO control
  • IpM tetrabenazine
  • Figure 3C shows IC50 calculation for tetrabenazine in HEK-VMAT2 vs HEK-VMAT2-SV2C cells.
  • 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).
  • Figures 5A-5B show that SV2C increases vesicular [ 3 H]-dopamine uptake and retention in isolated vesicles.
  • SV2C slows vesicular leakage of [ 3 H]-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.
  • Comparison of non-linear regressions determined the curves of best fit were significantly different for each cell line ****p ⁇ 0.0001.
  • Figures 7A-7B show that SV2C increases vesicular uptake and retention of [ 3 H]- MPP + .
  • Figure 7A shows vesicles isolated from HEK-VMAT2 and HEK-VMAT2-SV2C cells before undergoing uptake assay. Vesicles isolated from HEK-VMAT2 cells averaged
  • 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).
  • Figure 9 shows that genetic ablation of SV2C results in increased loss of nigral TH+ neurons following MPTP as compared to wild-type animals.
  • Figure 12 shows 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.
  • Figure 13 shows that fluorescence decays linearly over time in HEK-DAT- VMAT2 cells in the retention assay.
  • Figure 15 shows that SV2C enhances vesicular uptake of the fluorescent dopamine analogue FFN206.
  • Figure 16 shows that SV2C increases vesicular [ 3 H]-dopamine uptake and retention in isolated vesicles.
  • Figure 17 shows that SV2C increases vesicular uptake and retention of [ 3 H]- MPP .
  • 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.
  • Figure 19 shows that genetic ablation of SV2C does not affect vesicular capacity of [ 3 H]-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.
  • 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.
  • n 4-10 animals per genotype:treatment group.
  • 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.
  • n 2-4 animals per genotype:treatment group.
  • 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.
  • a method of measuring vesicular leakage 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.
  • a method of measuring vesicular retention or leakage 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.
  • a fluorescence signal of the fluorescent false neurotransmitter is measured in real-time.
  • real-time measurement comprises continuous or near-continuous data collection that can be used in any of the methods described herein.
  • 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).
  • the real-time measurements are collected over at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes.
  • the real-time measurements are collected over 45 minutes.
  • the cell culture comprises cells cultured in a tissue culture plate.
  • 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.
  • the multiwell plate is a black walled plate.
  • the multiwell plate is a black walled with clear flat bottom.
  • the multiwell plate is a black walled with clear flat bottom half-area 96-well plate.
  • 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.
  • the cells of the cell culture are an immortalized mammalian cell line.
  • immortalized cells lines include but not limited to PC 12, RCSN-3, BE2, SH-SY5Y, and HEK-293 cells.
  • the cells of the cell culture are induced pluripotent stem cells.
  • the cells of the cell culture are neuronal cultures differentiated from induced pluripotent stem cells.
  • the cells of the cell culture are primary neuronal cultures.
  • the cells of the cell culture are HEK-293 cells.
  • 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.
  • the cells of the cell culture express vesicular monoamine transporter 2 (VMAT2).
  • the cells of the cell culture are HEK-293 cells that express VMAT2.
  • the cells of the cell culture express synaptic vesicle glycoprotein 2C (SV2C).
  • the cells of the cell culture are HEK- 293 cells that express SV2C.
  • the cells of the cell culture express VMAT2 and SV2C.
  • the cells of the cell culture are HEK-293 cells that express VMAT2 and SV2C.
  • the cells of the cell culture express at least one of VMAT2, dopamine transporter (DAT), or synaptic vesicle glycoprotein 2C (SV2C).
  • the cells of the cell culture are HEK-293 cells that express at least one of the following proteins: VMAT2, DAT, or SV2C.
  • the cells of the cell culture are genetically modified cells.
  • the cells of the cell culture have reduced synaptic vesicle glycoprotein 2C (SV2C) expression level.
  • the level of SV2C is reduced as compared to normal, wild-type cells of the same cell type.
  • the cells of the cell culture are genetically modified to knock out SV2C.
  • 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.
  • 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.
  • CRISPR/Cas9 systems comprise a guide RNA (gRNA) or a single-molecule guide RNA (sgRNA).
  • gRNA or sgRNA comprises a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding CASP8.
  • Antisense oligonucleotides include antisense DNA, RNA, and DNA/RNA molecules, act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
  • 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.
  • 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.
  • the false neurotransmitter is a dopamine analogue.
  • 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.
  • FFN200 comprises
  • FFN270 comprises [0059] In some embodiments, dansyl-dopamine comprises
  • 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.
  • the slope is calculated by performing linear or non-linear regression.
  • 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.
  • the slope is calculated by performing linear or non-linear regression.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a method of identifying compound that modulates vesicular leakage 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.
  • the cells of the cell culture are an immortalized mammalian cell line.
  • immortalized cells lines include but not limited to PC 12, RCSN-3, BE2, SH-SY5Y, and HEK-293 cells.
  • the cells of the cell culture are induced pluripotent stem cells.
  • the cells of the cell culture are neuronal cultures differentiated from induced pluripotent stem cells.
  • the cells of the cell culture are primary neuronal cultures.
  • the cells of the cell culture are HEK-293 cells.
  • 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.
  • the cells of the cell culture express vesicular monoamine transporter 2 (VMAT2).
  • the cells of the cell culture are HEK-293 cells that express VMAT2.
  • the cells of the cell culture express synaptic vesicle glycoprotein 2C (SV2C).
  • the cells of the cell culture are HEK- 293 cells that express SV2C.
  • the cells of the cell culture express VMAT2 and SV2C.
  • the cells of the cell culture are HEK-293 cells that express VMAT2 and SV2C.
  • the cells of the cell culture express at least one of VMAT2, dopamine transporter (DAT), or synaptic vesicle glycoprotein 2C (SV2C).
  • the cells of the cell culture are HEK-293 cells that express at least one of the following proteins: VMAT2, DAT, or SV2C.
  • the cells of the cell culture are genetically modified cells.
  • the cells of the cell culture have reduced synaptic vesicle glycoprotein 2C (SV2C) expression level.
  • the level of SV2C is reduced as compared to normal, wild-type cells of the same cell type.
  • the cells of the cell culture are genetically modified to knock out SV2C.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 comprises l-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).
  • tetrabenazine comprises
  • tetrabenazine comprises
  • MPTP comprises
  • a cell culture under a control condition comprises a cell culture cultured under standard cell culturing conditions for such a cell culture.
  • a cell culture under a control condition comprises a cell culture contacted with a control compound.
  • the control compound is a solvent.
  • the control compound is sterile ultrapure water.
  • 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.
  • 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.
  • 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.
  • the control cell is cultured in a well of the same multi-well plate as the cells contacted with a test compound.
  • the multi -well plate is a 384 well plate.
  • 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.
  • 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.
  • the false neurotransmitter targets dopamine.
  • the false neurotransmitter is a fluorescent false neurotransmitter.
  • the false neurotransmitter could be any fluorescent compound that mimics a neurotransmitter.
  • 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).
  • 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).
  • the fluorescent false neurotransmitter comprises false fluorescent neurotransmitter 206 (FFN206).
  • the fluorescent false neurotransmitter consists essentially of false fluorescent neurotransmitter 206 (FFN206).
  • the fluorescent false neurotransmitter consists of false fluorescent neurotransmitter 206 (FFN206).
  • the false neurotransmitter is a fluorescent neurotransmitter ligand.
  • 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.
  • 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.
  • 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.
  • 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 false neurotransmitter 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.
  • 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.
  • 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.
  • 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 libraries can 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the cells of the cell culture are an immortalized mammalian cell line.
  • the cells of the cell culture are induced pluripotent stem cells.
  • the cells of the cell culture are neuronal cultures differentiated from induced pluripotent stem cells.
  • the cells of the cell culture are primary neuronal cultures.
  • 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.
  • the cell culture is derived from a sample from the subject.
  • 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.
  • the cells of the cell culture express vesicular monoamine transporter 2 (VMAT2).
  • the cells of the cell culture are HEK-293 cells that express VMAT2.
  • the cells of the cell culture express synaptic vesicle glycoprotein 2C (SV2C).
  • the cells of the cell culture are HEK- 293 cells that express SV2C.
  • the cells of the cell culture express VMAT2 and SV2C.
  • the cells of the cell culture are HEK-293 cells that express VMAT2 and SV2C.
  • the cells of the cell culture express at least one of VMAT2, dopamine transporter (DAT), or synaptic vesicle glycoprotein 2C (SV2C).
  • the cells of the cell culture are HEK-293 cells that express at least one of the following proteins: VMAT2, DAT, or SV2C.
  • 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.
  • 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.
  • 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).
  • TTZ tetrabenazine
  • MPTP l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine
  • a cell culture under a control condition comprises a cell culture cultured under standard cell culturing conditions for such a cell culture.
  • a cell culture under a control condition comprises a cell culture contacted with a control compound.
  • the control compound is a solvent.
  • the control compound is sterile ultrapure water.
  • 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.
  • 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.
  • 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.
  • the method is to treat Parkinson’s disease.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • immortalized cells lines include but not limited to PC12, RCSN-3, BE2, SH-SY5Y, and HEK-293 cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the therapeutic applications described herein can be applied to a human.
  • 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.
  • EXAMPLE 1 Synaptic vesicle glycoprotein 2C enhances vesicular storage of dopamine and counters dopaminergic toxicity
  • 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.
  • VMAT2 vesicular sequestration via vesicular monoamine transporter 2
  • VMAT2 vesicular monoamine transporter 2
  • SV2C 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.
  • SV2C has been implicated as a modifier of Parkinson’s disease risk.
  • SV2C mediates the retention of dopamine and dopamine analogues within vesicles.
  • FFN206 false fluorescent neurotransmitter 206
  • 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.
  • 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)).
  • 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.
  • VMAT2 vesicular monoamine transporter 2
  • vesicular monoamine transporter 2 minimizes the cytosolic pool of dopamine by sequestering dopamine into synaptic vesicles.
  • VMAT2 vesicular monoamine transporter 2
  • 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.
  • VMAT2 immunoreactivity demonstrates decreased VMAT2 immunoreactivity
  • 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)).
  • SV2s synaptic vesicle glycoproteins
  • 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 SV2B having 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)).
  • 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.
  • SV2C may be a novel neurotransmitter transporter (Feany M. et. al. Cell 70(5) P861-867 (1992)).
  • 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.
  • GWAS genome-wide association study
  • 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)).
  • our lab has demonstrated aberrant SV2C staining patterns in post-mortem brain tissue from people with Parkinson’s disease (Dunn A. et. al.
  • HEK293 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.
  • VMAT2 human vesicular monoamine transporter 2
  • HEK-VMAT2C human synaptic vesicle glycoprotein 2C
  • 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).
  • 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 antibody overnight 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)).
  • 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.
  • PBS phosphate buffered saline
  • 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.
  • primary antibody solution PBS with 10% normal goat serum, 0.1% triton-x, and 10% bovine serum albumin
  • Mouse anti-SV2C (1 :500 (Sigma, MABN367)
  • Rabbit anti-VMAT2 (1 :500 (Miller lab)
  • 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.
  • 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.
  • mice 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.
  • 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.
  • 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.
  • VMAT2 human vesicular monoamine transporter 2
  • FFN206 fluorescent false neurotransmitter 206
  • FFN206 The accumulation of FFN206 can be blocked with VMAT2 inhibitors and by dissipating the proton gradient VMAT2 requires to load substrate within subcellular compartments.
  • VMAT2 inhibitors Black C. et. al. Chem. Res. Toxicol. 34, 5, 1256-1264 (2021)).
  • FFN206 accumulates within vesicular compartments, the fluorescent signal is concentrated allowing for visualization and measurement of the substrate by microscopy and plate reader analysis.
  • the uptake of FFN206 was measured in HEK-VMAT2-SV2C cells and in HEK-VMAT2 cells.
  • 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.
  • a TBZ dose-response was performed to determine whether HEK-VMAT2-SV2C cells are resistant to TBZ-induced inhibition.
  • FFN206 fluorescence fluorescence over time.
  • 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. By removing 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • SV2C does not appear to affect VMAT2 expression in HEK-VMAT2 cell lines
  • 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.
  • 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.
  • 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.
  • Retention, or leakage, assays were performed in a similar manner to FFN206 retention assays where vesicles were incubated with [ 3 H]-dopamine before sequestration was blocked with the VMAT2 inhibitors TBZ or reserpine and the amount of [ 3 H]-dopamine remaining within the vesicles was measured at multiple time-points.
  • HEK293 cells are not neurons and do not contain the same factors as neurons, these findings were true in vesicles isolated from mouse brain.
  • 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.
  • the baseline amount of [ 3 H]- dopamine uptake synaptic vesicles derived from the brain of SV2C-KO mice displayed enhanced rate of [ 3 H]-dopamine leak compared to those from wild-type animals (Figure 6.).
  • 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.
  • TBZ VMAT2 inhibitor tetrabenazine
  • 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.
  • 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.
  • 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.
  • 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 of transmitter molecules into the synaptic cleft upon endocytosis (de Toledo G. et. al. Nature 363, 554-558 (1993); Harlow M. et. al.
  • 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.
  • Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson’s disease.”
  • VMAT2 a dynamic regulator of brain monoaminergic neuronal function interacting with drugs of abuse.
  • VMAT2 vesicular monoamine transporter 2
  • VMAT2 Vulnerability and Clearance of Excess Dopamine in Mouse Striatal Terminals.

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Abstract

L'invention concerne des procédés de mesure de fuite vésiculaire en utilisant un faux neurotransmetteur fluorescent et des procédés d'identification d'un composé qui module la fuite vésiculaire en utilisant un faux neurotransmetteur fluorescent.
PCT/US2024/030991 2023-05-24 2024-05-24 Dosage de fuite vésiculaire de neurotransmetteur Pending WO2025096017A2 (fr)

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