Video-based pooled screening yields improved far-red genetically encoded voltage indicators.

Video-based screening of pooled libraries is a powerful approach for directed evolution of biosensors because it enables selection along multiple dimensions simultaneously from large libraries. Here we develop a screening platform, Photopick, which achieves precise phenotype-activated photoselection over a large field of view (2.3 × 2.3 mm, containing >103 cells, per shot). We used the Photopick platform to evolve archaerhodopsin-derived genetically encoded voltage indicators (GEVIs) with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). These GEVIs gave improved signals in cultured neurons and in live mouse brains. By combining targeted in vivo optogenetic stimulation with high-precision voltage imaging, we characterized inhibitory synaptic coupling between individual cortical NDNF (neuron-derived neurotrophic factor) interneurons, and excitatory electrical synapses between individual hippocampal parvalbumin neurons. The QuasAr6 GEVIs are powerful tools for all-optical electrophysiology and the Photopick approach could be adapted to evolve a broad range of biosensors.

Developing antisense oligonucleotides for a TECPR2 mutation-induced, ultra-rare neurological disorder using patient-derived cellular models.

Mutations in the TECPR2 gene are the cause of an ultra-rare neurological disorder characterized by intellectual disability, impaired speech, motor delay, and hypotonia evolving to spasticity, central sleep apnea, and premature death (SPG49 or HSAN9; OMIM: 615031). Little is known about the biological function of TECPR2, and there are currently no available disease-modifying therapies for this disease. Here we describe implementation of an antisense oligonucleotide (ASO) exon-skipping strategy targeting TECPR2 c.1319delT (p.Leu440Argfs∗19), a pathogenic variant that results in a premature stop codon within TECPR2 exon 8. We used patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived neurons homozygous for the p.Leu440Argfs∗19 mutation to model the disease in vitro. Both patient-derived fibroblasts and neurons showed lack of TECPR2 protein expression. We designed and screened ASOs targeting sequences across the TECPR2 exon 8 region to identify molecules that induce exon 8 skipping and thereby remove the premature stop signal. TECPR2 exon 8 skipping restored in-frame expression of a TECPR2 protein variant (TECPR2ΔEx8) containing 1,300 of 1,411 amino acids. Optimization of ASO sequences generated a lead candidate (ASO-005-02) with ∼27 nM potency in patient-derived fibroblasts. To examine potential functional rescue induced by ASO-005-02, we used iPSC-derived neurons to analyze the neuronal localization of TECPR2ΔEx8 and showed that this form of TECPR2 retains the distinct, punctate neuronal expression pattern of full-length TECPR2. Finally, ASO-005-02 had an acceptable tolerability profile in vivo following a single 20-mg intrathecal dose in cynomolgus monkeys, showing some transient non-adverse behavioral effects with no correlating histopathology. Broad distribution of ASO-005-02 and induction of TECPR2 exon 8 skipping was detected in multiple central nervous system (CNS) tissues, supporting the potential utility of this therapeutic strategy for a subset of patients suffering from this rare disease.

Probing Synaptic Signaling with Optogenetic Stimulation and Genetically Encoded Calcium Reporters.

Optogenetics provides a powerful approach for investigating neuronal electrophysiology at the scale required for drug discovery applications. Probing synaptic function with high throughput using optogenetics requires robust tools that enable both precise stimulation of and facile readout of synaptic activity. Here we describe two functional assays to achieve this end: (1) a pre-synaptic calcium assay that utilizes the channelrhodopsin, CheRiff, patterned optogenetic stimulus, and the pre-synaptically targeted calcium reporter jRGECO1a to monitor pre-synaptic changes in calcium influx and (2) a synaptic transmission assay in which CheRiff and cytosolic jRGECO1a are expressed in non-overlapping sets of neurons, enabling pre-synaptic stimulation and post-synaptic readout of activity. This chapter describes the methodology and practical considerations for implementation of these two assays.

Simultaneous voltage and calcium imaging and optogenetic stimulation with high sensitivity and a wide field of view.

Transmembrane voltage and intracellular calcium concentration are coupled parameters essential to the function of neurons, cardiomyocytes, and other excitable cells. Here we introduce the Firefly-HR microscope for simultaneous optogenetic stimulation and voltage and calcium imaging with fluorescent proteins using three spectrally distinct visible color bands. Firefly-HR combines patterned stimulation, near-total internal reflection laser excitation through a prism located between the sample and a water-immersion objective, and concurrent imaging of three color channels. The microscope has efficient light collection, low fluorescent background, and a large field of view (0.24 x 1.2 mm @ 1000 frames/sec). We characterize optical crosstalk and demonstrate capabilities with three applications: (1) probing synaptically connected neuronal microcircuits, (2) examining the coupling between neuronal action potentials and calcium influx, and (3) studying the pharmacology of paced human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) via simultaneous recordings of voltage, calcium, and contraction.

All-Optical Electrophysiology for Disease Modeling and Pharmacological Characterization of Neurons.

A key challenge for establishing a phenotypic screen for neuronal excitability is measurement of membrane potential changes with high throughput and accuracy. Most approaches for probing excitability rely on low-throughput, invasive methods or lack cell-specific information. These limitations stimulated the development of novel strategies for characterizing the electrical properties of cultured neurons. Among these was the development of optogenetic technologies (Optopatch) that allow for stimulation and recording of membrane voltage signals from cultured neurons with single-cell sensitivity and millisecond temporal resolution. Neuronal activity is elicited using blue light activation of the channelrhodopsin variant 'CheRiff'. Action potentials and synaptic signals are measured with 'QuasAr', a rapid and sensitive voltage-indicating protein with near-infrared fluorescence that scales proportionately with transmembrane potential. This integrated technology of optical stimulation and recording of electrical signals enables investigation of neuronal electrical function with unprecedented scale and precision. © 2017 by John Wiley & Sons, Inc.