MODELS FOR PARKINSON'S DISEASE STUDIES
Disease models for Parkinson's disease are disclosed comprising a dopaminergic neuron having one or more phenotypic or genotypic characteristics of Parkinson's disease where the neuron expresses an optical reporter of, and an optical activator of, electrical activity and that exhibits an optical signature in response to neural stimulation. Cells may optionally include an indicator of intracellular calcium levels. Transformed neurons may be optically evaluated for action potentials to track the development of disease, evaluate potential therapies, diagnosis of disease, and to identify mutations and genes associated with disease development and progression.
1 . A method for screening a compound for Parkinson's disease treatment, the method comprising:
presenting a compound to a sample comprising a dopaminergic neuron having one or more phenotypic or genotypic characteristics of Parkinson's disease, wherein the dopaminergic neuron expresses an optical reporter of membrane electrical potential and a light-gated ion channel;
receiving, via a microscopy system, an optical signal generated by the optical reporter in response to optical stimulation of the sample following presentation of said compound; and
identifying the compound as a candidate for Parkinson's disease treatment based on said optical signal.
2 . The method of claim 1 , wherein the phenotypic characteristic is selected from the group consisting of diffuse cytoplasmic accumulations of SNCA protein, fewer and simpler processes than a disease-free dopaminergic neuron, immature appearance relative to the disease-free dopaminergic neuron, signs of degeneration, short or absent neurites, vacuolated soma, a fragmented nucleus and cleaved caspase-3.
3 . The method of claim 1 , wherein the genotypic characteristic comprises a mutation in a gene selected from the group consisting of SNCA, PARK11, GIGYF2, PARK13, UCHL1, LRRK2, PARK2, PARK7, PINK1, ATP13A2, GBA, APOE, PARK 16, PARK3, PARK10, PARK12, HTRA2, PLA2G6, FBXO7, EIF4G1, and VPS35.
4 . The method of claim 1 , wherein the microscopy system comprises a digital micromirror device that provides the optical stimulation.
5 . The method of claim 1 , wherein the microscopy system further comprises a charge-coupled device camera configured to capture the optical signal from the dopaminergic neuron.
6 . The method of claim 1 , wherein the dopaminergic neuron also expresses a protein that reports a change in an intracellular calcium level.
7 . The method of claim 6 , wherein the dopaminergic neuron is stimulated by a second neuron that expresses the light-gated ion channel.
8 . The method of claim 7 , wherein the second neuron also expresses the optical reporter of change in membrane potential.
9 . The method of claim 8 , wherein:
the light-gated ion channel comprises an algal channelrhodopsin; and
the protein that reports changes in intracellular calcium levels comprises a GCaMP variant.
10 . The method of claim 6 , wherein the protein that reports a change in an intracellular calcium level is selected from the group consisting of jRCaMP1a, jRGECO1a and RCaMP2.
11 . The method of claim 1 , wherein the dopaminergic neuron is an hiPSC-derived dopaminergic neuron.
12 . The method of claim 6 , further comprising detecting a change in AP waveform and a change in the intracellular calcium level upon exposure of the dopaminergic neuron to the compound.
13 . The method of claim 1 , further comprising spatially patterning a plurality of neurons in a cell culture on a substrate.
14 . The method of claim 1 , wherein the compound comprises an A2A receptor antagonist.
15 . The method of claim 1 , wherein the identifying step comprises comparing the optical signal of the sample to an optical signal obtained from a control cell.
16 . The method of claim 1 , wherein the optical reporter of membrane electrical potential comprises a microbial rhodopsin.
17 . The method of claim 16 , wherein the microbial rhodopsin comprises QuasAr1 or QuasAr2.
18 . The method of claim 16 , wherein the microbial rhodopsin is expressed from a gene that is integrated into the dopaminergic neuron.
19 . The method of claim 1 , wherein the light-gated ion channel comprises a blue-shifted actuator.
20 . The method of claim 19 , wherein the blue-shifted actuator comprises TsChR or PsChR.
21 . The method of claim 6 , wherein the light-gated ion channel comprises a blue-shifted actuator with an excitation maximum at a wavelength <450 nm and the protein that reports the change in the intracellular calcium level comprises a red-shifted calcium indicator with an excitation maximum between 520 nm and 570 nm inclusive.
22 . A cell culture comprising:
a first dopaminergic neuron that expresses a light-gated ion channel;
a second dopaminergic neuron electrically contiguous with the first dopaminergic neuron, wherein the second dopaminergic neuron expresses a genetically-encoded optical reporter of activity; and
wherein at least one of the first dopaminergic neuron or the second dopaminergic neuron comprises one or more phenotypic or genotypic characteristics of Parkinson's disease.
23 . The cell culture of claim 22 , wherein the phenotypic characteristic is selected from the group consisting of diffuse cytoplasmic accumulations of SNCA protein, fewer and simpler processes than a disease-free dopaminergic neuron, immature appearance relative to the disease-free dopaminergic neuron, signs of degeneration, short or absent neurites, vacuolated soma, a fragmented nucleus and cleaved caspase-3.
24 . The cell culture of claim 22 , wherein the genotypic characteristic comprises a mutation in a gene selected from the group consisting of SNCA, PARK11, GIGYF2, PARK13, UCHL1, LRRK2, PARK2, PARK7, PINK1, ATP13A2, GBA, APOE, PARK 16, PARK3, PARK10, PARK12, HTRA2, PLA2G6, FBXO7, EIF4G1, and VPS35.
25 . The cell culture of claim 22 , wherein the light-gated ion channel comprises a channelrhodopsin.
26 . The cell culture of claim 22 , wherein the second dopaminergic neuron expresses a genetically encoded Ca++ indicator.
27 . The cell culture of claim 26 , wherein the genetically encoded Ca++ indicator comprises at least one selected from the list consisting of GCaMP6f, jRCaMP1a, jRGECO1a, and RCaMP2.
28 . The cell culture of claim 22 , wherein the first dopaminergic neuron is spatially segregated from and in electrical contact with the second dopaminergic neuron.