Diagnostic methods for neural disorders
The invention generally relates to optical methods for the diagnosis of neuronal condition by converting a cell from a patient into a neuron and optically evaluating action potentials of that cell in vitro. The cell is transformed with an optical reporter and exhibits an optical signature in response to neural stimulation. Using genome-editing, a control cell can be made that is isogenic but-for a known mutation and a control signature obtained from the control cell. Thus, methods of the invention reveal potential neurodegenerative effects of a mutation as manifested in a patient's genetic context. The optical signature of the cell, or the difference between the signature and the control signature, is correlated to a diagnosis of the neurodegenerative disease.
1. A method for determining an effect of a compound on a neurological condition, the method comprising:
presenting a compound selected for treating a neurological condition to a cell sample exhibiting a genotype associated with the neurological condition, the cell sample comprising a plurality of neurons, wherein at least one of the plurality of neurons expresses an optical reporter of membrane electrical potential;
optically stimulating an optogenetic actuator in the sample following presentation of the compound, wherein the optogenetic actuator is a light gated ion channel;
generating an optical signal in response to the optical stimulation of the light gated ion channel using the optical reporter;
receiving, via a microscopy system, the optical signal generated by the optical reporter;
analyzing the optical signal generated by the optical reporter with respect to a control signal obtained from a control cell sample not exhibiting a genotype associated with the neurological condition; and
identifying the compound as a candidate for treatment of the neurological condition based on the analysis of said optical signal with respect to the control signal.
2. The method of claim 1 , wherein a plurality of samples are exposed to a plurality of different compounds.
3. The method of claim 1 , wherein the light gated ion channel comprises an algal channelrhodopsin being expressed by a second neuron in synaptic communication with the at least one of the plurality of neurons, and the optical reporter of membrane potential comprises a microbial rhodopsin with between 1 and 10 amino acid substitutions relative to a wild type form of the microbial rhodopsin.
4. The method of claim 3 , wherein the at least one of the plurality of neurons also expresses a genetically-encoded indicator of intracellular calcium level, and
wherein the receive optical signal includes a signal from the genetically-encoded indicator of intracellular calcium level, and further wherein the neurological condition is any one of autism, epilepsy, Alzheimer's, amyotrophic lateral sclerosis, or tuberous sclerosis.
5. The method of claim 1 , wherein each of the plurality of neurons is caused to express both the optical reporter of membrane electrical potential and the light gated ion channel.
6. The method of claim 5 , further comprising transforming the neurons with a vector that includes a nucleic acid encoding the optical reporter of membrane electrical potential and the light gated ion channel.
7. The method of claim 1 , wherein the neurological condition is selected from the group consisting of Cockayne syndrome, Down Syndrome, Dravet syndrome, familial dysautonomia, Fragile X Syndrome, Friedreich's ataxia, Gaucher disease, hereditary spastic paraplegias, Machado-Joseph disease, Phelan-McDermid syndrome (PMDS), polyglutamine (polyQ)-encoding CAG repeats, spinal muscular atrophy, Timothy syndrome, Alzheimer's disease, frontotemporal lobar degeneration, Huntington's disease, multiple sclerosis, Parkinson's disease, spinal and bulbar muscular atrophy, and amyotrophic lateral sclerosis.
8. The method of claim 1 , further comprising performing the presenting, optically stimulating, generating, and receiving steps on a control sample, wherein the neurons in the cell sample are isogenic with cells in the control sample but for a mutation.
9. The method of claim 1 , wherein receiving the optical signal comprises observing a cluster of cells with a microscope and using a computer to isolate a signal generated by the optical reporter from among a plurality of signals from the cluster of cells.
10. The method of claim 9 , wherein the computer isolates the signal by performing an independent component analysis and identifying a spike train produced by the neuron.
11. The method of claim 10 , further comprising using the microscopy system to obtain an image of a plurality of clusters of cells.