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Principal Investigator: Graham Thomas Dempsey
Organization: Q-STATE BIOSCIENCES, INC.
Fiscal Year: 2019
Award: $777,880
Funding agency: National Institute of Mental Health
Project Summary: Synaptic dysfunction has been implicated in many neurological diseases including epilepsy,
Alzheimer’s, Parkinson’s, autism spectrum disorder (ASD), schizophrenia, depression, ADHD and Huntington’s.
Despite the prevalence and severity of these disorders, the development of new therapeutics has lagged. This
is due, in part, to challenges in replicating relevant biology in robust, scalable in vitro assays. Current methods
of measuring synaptic function, which stimulate presynaptic cells and record from postsynaptic cells, lack
sufficient throughput for drug screening. The Optopatch platform recently developed at Q-State Biosciences,
comprised of engineered optogenetic proteins, custom microscopes, and software, makes it possible to
simultaneously stimulate (blue light) and record (red light) electrical activity from ~100 neurons with 1 millisecond
temporal resolution, single-cell spatial resolution and high signal-to-noise ratio. Additionally, patterned blue light
can be used to probe synaptic connections by stimulating individual neurons while recording postsynaptic
potentials (PSPs) in all remaining cells. In Phase I, we developed synaptic assays in primary rodent neurons for:
1. Presynaptic calcium – The red calcium sensing protein jRGECO1a is targeted to presynaptic boutons by
fusion with synaptophysin. Neural activity is stimulated with blue light via a channelrhodopsin, CheRiff.
2. Postsynaptic calcium – jRGECO1a is targeted to postsynaptic spines by fusion with PSD95. Distinct
subsets of neurons express either actuator or reporter. Action potentials triggered in presynaptic cells
generate calcium signals in postsynaptic cells.
3. Postsynaptic voltage – CheRiff and the red voltage sensing protein QuasAr are expressed in distinct
subsets of neurons. Presynaptic cell stimulation leads to PSPs recorded in QuasAr-expressing
postsynaptic cells. Pharmacological probes isolate excitatory signaling through either AMPA or NMDA
channels or inhibitory signaling through GABAA channels. Inhibitory neurons can be labeled with a
fluorescent tag expressed under control of the Dlx1/2 promoter, to resolve different synapse classes:
excitatory (E) → inhibitory (I), E →E, I →E, and I → I.
In the follow-on Phase II project, we propose to: (1) transition the assays to human induced pluripotent stem cell
derived neurons, testing multiple strategies to increase the synaptic maturation of the cells, (2) expand assays
in rodent cells to include plasticity, particularly long-term potentiation (LTP) and spike timing dependent plasticity,
and (3) apply these assays in disease models of ASD using knockout of three synaptic proteins, SHANK3,
SYNGAP1, and GRIN2B, whose loss causes severe ASD in all cases. The most robust phenotype will be used
to (4) screen a library of approved drugs to demonstrate assay throughput and sensitivity and identify candidates
for potential repurposing. The establishment of ASD-associated cellular phenotypes for HTS would provide a
foundation for drug discovery for these serious and poorly treated diseases.
Terms: <4-Aminobutanoic Acid><4-Aminobutyric Acid><4-amino-butanoic acid><AD/HD><ADHD><Action Potentials><Address><Alzheimer><Alzheimer Type Dementia><Alzheimer disease><Alzheimer sclerosis><Alzheimer syndrome><Alzheimer's><Alzheimer's Disease><Alzheimers Dementia><Alzheimers disease><Aminalon><Aminalone><Assay><Attention deficit hyperactivity disorder><Autism><Autistic Disorder><Bioassay><Biologic Assays><Biologic Models><Biologic Sciences><Biological Assay><Biological Models><Biological Sciences><Biology><Bioscience><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Calcium><Calcium Ion Signaling><Calcium Signaling><Cas nuclease technology><Cell Body><Cell Communication and Signaling><Cell Maturation><Cell Signaling><Cells><Chemicals><Co-culture><Cocultivation><Coculture><Coculture Techniques><Collaborations><Complex><Computer software><Coupled><Custom><DLG4><DLG4 gene><Data Set><Dataset><Depressed mood><Development><Disease><Disease model><Disorder><Drug Screening><Drugs><Dysfunction><Early Infantile Autism><Electrophysiology><Electrophysiology (science)><Engineering><Environment><Epilepsy><Epileptic Seizures><Epileptics><Exhibits><Fire - disasters><Fires><Foundations><Frequencies><Functional disorder><GABA><Genes><Genetic><Grant><Growth Agents><Growth Factor><Growth Substances><Health><High Throughput Assay><Human><Huntington Chorea><Huntington Disease><Huntington's><Huntington's Disease><Huntington's Disease Pathway><Huntingtons Disease><Hyperactivity Disorder NOS><Hyperkinetic Syndrome><Image><Individual><Industrialization><Infantile Autism><Intracellular Communication and Signaling><Ions><Kanner's Syndrome><Kinetics><Knock-out><Knockout><Label><Libraries><Life Sciences><Light><Link><Long-Term Potentiation><Longterm Potentiation><Measurement><Measures><Mediating><Medication><Mental Depression><Mental disorders><Mental health disorders><Methods><Mice><Mice Mammals><Microscope><Model System><Modeling><Modern Man><Msec><Murine><Mus><N Methyl D aspartic Acid><N methyl D aspartate><N-Methyl-D-aspartate><N-Methylaspartate><NMDA><Nerve Cells><Nerve Unit><Nervous System Diseases><Neural Cell><Neural Transmission><Neurocyte><Neurodevelopmental Disorder><Neurologic><Neurologic Disorders><Neurological><Neurological Development Disorder><Neurological Disorders><Neuromodulator><Neurons><Neurophysiology / Electrophysiology><Noise><Optics><PSD95><Paralysis Agitans><Parkinson><Parkinson Disease><Parkinson's><Parkinson's disease><Parkinsons disease><Patients><Pattern><Pharmaceutic Preparations><Pharmaceutical Agent><Pharmaceutical Preparations><Pharmaceuticals><Pharmacologic Substance><Pharmacological Substance><Pharmacology><Phase><Phenotype><Photoradiation><Physiologic><Physiological><Physiopathology><Position><Positioning Attribute><Predominantly Hyperactive-Impulsive Type Attention-Deficit Disorder><Predominantly Hyperactive-Impulsive Type Hyperactivity Disorder><Prevalence><Primary Parkinsonism><Primary Senile Degenerative Dementia><Proteins><Proteins Growth Factors><Protocol><Protocols documentation><Psychiatric Disease><Psychiatric Disorder><Reporter><Resolution><Rodent><Rodentia><Rodents Mammals><SAP90><SBIR><Schizophrenia><Schizophrenic Disorders><Seizure Disorder><Severities><Signal Transduction><Signal Transduction Systems><Signaling><Small Business Innovation Research><Small Business Innovation Research Grant><Software><Spinal Column><Spine><Synapses><Synaptic><Synaptic Transmission><Synaptic Vesicle P38 Membrane Protein><Synaptic Vesicle Protein P38><Synaptic plasticity><Synaptophysin><Testing><Therapeutic><Validation><Vertebral column><Work><attention deficit hyperactive disorder><autism spectrum disorder><autistic spectrum disorder><backbone><base><biological signal transduction><commercialization><dementia of the Alzheimer type><dementia praecox><depressed><depression><developmental><disease phenotype><disease-causing mutation><disorder model><drug discovery><drug/agent><electrophysiological><epilepsia><epileptiform><epileptogenic><excitatory neuron><fire><functional status><gamma-Aminobutyric Acid><high throughput screening><iPS><iPSC><iPSCs><imaging><improved><in vitro Assay><in vitro Model><induced pluripotent stem cell><inhibitory neuron><interest><loss of function mutation><mental illness><millisecond><mouse model><murine model><nervous system disorder><neural><neurological disease><neuronal><neuropsychiatric disease><neuropsychiatric disorder><new drug class><new drug treatments><new drugs><new therapeutics><new therapy><next generation therapeutics><novel drug class><novel drug treatments><novel drugs><novel therapeutics><novel therapy><optical><optogenetics><pathophysiology><postsynaptic><presynaptic><primary degenerative dementia><programs><promoter><promotor><psychiatric illness><psychological disorder><quasar><relating to nervous system><response><sadness><schizophrenic><screening><senile dementia of the Alzheimer type><synapse><synapse function><synaptic function><temporal measurement><temporal resolution><therapeutic candidate><time measurement><voltage><γ-Aminobutyric Acid>