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Recent advances in molecular and cellular engineering, such as human cell reprogramming, genome editing, and patient-specific organoids, have provided unprecedented opportunities for investigating human disorders in both animals and human-based models at an improved pace and precision. This progress will inevitably lead to the development of innovative drug-screening platforms and new patient-specific therapeutics. In this review, we discuss recent advances that have been made using zebrafish and human-induced pluripotent stem cell (iPSC)–derived neurons and organoids for modeling genetic epilepsies. We also provide our prospective on how these models can potentially be combined to build new screening platforms for antiseizure and antiepileptogenic drug discovery that harness the robustness and tractability of zebrafish models as well as the patient-specific genetics and biology of iPSC-derived neurons and organoids. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13311-021-01115-5.

Visual Abstract, The ability to measure changes in neuronal activity in a quantifiable and precise manner is of fundamental importance to understand neuron development and function. Repeated monitoring of neuronal activity of the same population of neurons over several days is challenging and, typically, low-throughput. Here, we describe a new biochemical reporter assay that allows for repeated measurements of neuronal activity in a cell type-specific manner. We coupled activity-dependent elements from the Arc/Arg3.1 gene with a secreted reporter, Gaussia luciferase (Gluc), to quantify neuronal activity without sacrificing the neurons. The reporter predominantly senses calcium and NMDA receptor (NMDAR)-dependent activity. By repeatedly measuring the accumulation of the reporter in cell media, we can profile the developmental dynamics of neuronal activity in cultured neurons from male and female mice. The assay also allows for longitudinal analysis of pharmacological treatments, thus distinguishing acute from delayed responses. Moreover, conditional expression of the reporter allows for monitoring cell type-specific changes. This simple, quantitative, cost-effective, automatable, and cell type-specific activity reporter is a valuable tool to study the development of neuronal activity in normal and disease-model conditions, and to identify small molecules or protein factors that selectively modulate the activity of a specific population of neurons.

Genetic abnormalities in synaptic proteins are common in individuals with autism; however, our understanding of the cellular and molecular mechanisms disrupted by these abnormalities is limited. SHANK3 is a postsynaptic scaffolding protein of excitatory synapses that has been found mutated or deleted in most patients with 22q13 deletion syndrome and about 2% of individuals with idiopathic autism and intellectual disability. Here, we generated CRISPR/Cas9-engineered human pluripotent stem cells (PSCs) with complete hemizygous SHANK3 deletion (SHANK3

Despite the potential of stem cell-derived neural transplants for treating intractable neurological diseases, the global effects of a transplant’s electrical activity on host circuitry have never been measured directly, preventing the systematic optimization of such therapies. Here, we overcome this problem by combining optogenetics, stem cell biology, and neuroimaging to directly map stem cell-driven neural circuit formation in vivo. We engineered human induced pluripotent stem cells (iPSCs) to express channelrhodopsin-2 and transplanted resulting neurons to striatum of rats. To non-invasively visualize the function of newly formed circuits, we performed high-field functional magnetic resonance imaging (fMRI) during selective stimulation of transplanted cells. fMRI successfully detected local and remote neural activity, enabling the global graft-host neural circuit function to be assessed. These results demonstrate the potential of a novel neuroimaging-based platform that can be used to identify how a graft’s electrical activity influences the brain network in vivo.

L-type voltage gated calcium channels have an important role in neuronal development by promoting dendritic growth and arborization. A point mutation in the gene encoding Ca(V)1.2 causes Timothy syndrome, a neurodevelopmental disorder associated with autism spectrum disorders (ASDs). We report that channels with the Timothy syndrome alteration cause activity-dependent dendrite retraction in rat and mouse neurons and in induced pluripotent stem cell (iPSC)-derived neurons from individuals with Timothy syndrome. Dendrite retraction was independent of calcium permeation through the mutant channel, was associated with ectopic activation of RhoA and was inhibited by overexpression of the channel-associated GTPase Gem. These results suggest that Ca(V)1.2 can activate RhoA signaling independently of Ca(2+) and provide insights into the cellular basis of Timothy syndrome and other ASDs.

Here, we describe a new mechanism by which glutamate (Glu) and trace metals reciprocally modulate activity of the Cav2.3 channel by profoundly shifting its voltage-dependent gating. We show that zinc and copper, at physiologically relevant concentrations, occupy an extracellular binding site on the surface of Cav2.3 and hold the threshold for activation of these channels in a depolarized voltage range. Abolishing this binding by chelation or the substitution of key amino acid residues in IS1–IS2 (H111) and IS2–IS3 (H179 and H183) loops potentiates Cav2.3 by shifting the voltage dependence of activation toward more negative membrane potentials. We demonstrate that copper regulates the voltage dependence of Cav2.3 by affecting gating charge movements. Thus, in the presence of copper, gating charges transition into the “ON” position slower, delaying activation and reducing the voltage sensitivity of the channel. Overall, our results suggest a new mechanism by which Glu and trace metals transiently modulate voltage-dependent gating of Cav2.3, potentially affecting synaptic transmission and plasticity in the brain.

Channel STIMulation The STIM1 protein functions as a calcium sensor and regulates entry of calcium into cells across the plasma membrane. When cell surface receptors are stimulated and cause release of calcium from internal stores in the endoplasmic reticulum (ER), STIM proteins in the ER membrane interact with the Orai channel pore protein in the plasma membrane to allow calcium entry from the outside of the cell (see the Perspective by Cahalan ). Park et al. (p. 101 ) and Wang et al. (p. 105 ) now show that STIM also acts to suppress conductance by another calcium channel—the voltage-operated Ca V 1.2 channel. STIM1 appeared to interact directly with Ca V 1.2 channels in multiple cell types, including vascular smooth muscle cells, neurons, and cultured cells derived from T lymphocytes. The interaction inhibited opening of the Ca V 1.2 channels and caused depletion of the channel from the cell surface.

Molecular diversity of T-type/Ca(v)3 Ca2+ channels is created by expression of three genes and alternative splicing of those genes. Prompted by the important role of the I-II linker in gating and surface expression of Ca(v)3 channels, we describe here the properties of a novel variant that partially deletes this loop. The variant is abundantly expressed in rat brain, even exceeding transcripts with the complete exon 8. Electrophysiological analysis of the Delta8b variant revealed enhanced current density compared to Ca(v)3.1a, but similar gating. Luminometry experiments revealed an increase in the expression of Delta8b channels at the plasma membrane. We conclude that alternative splicing of Ca(v)3 channels regulates surface expression and may underlie disease states in which T-channel current density is increased.

Voltage-gated Ca(2+) channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca(2+) over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca(2+) permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca(2+)-channel alpha1 subunits, Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3 functionally expressed in Xenopus oocytes. Our data show that all Ca(v)3 channels select Ca(2+) over Na(+) by affinity. Ca(v)3.1 and Ca(v)3.2 discriminate Ca(2+), Sr(2+) and Ba(2+) based on the ion's effects on the open channel probability, whilst Ca(v)3.3 discriminates based on the ion's intrapore binding affinity. All Ca(v)3s were characterized by much smaller difference in the K(D) values for Na(+) current blockade by Ca(2+) (K(D1) approximately 6 microM) and for Ca(2+) current saturation (K(D2) approximately 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na(+)/Ca(2+) current at close to physiological Ca(2+) concentrations, which was the strongest for Ca(v)3.3, smaller for Ca(v)3.2 and the smallest for Ca(v)3.1. In addition to intrapore Ca(2+) binding site(s) Ca(v)3.2, but not Ca(v)3.1 and Ca(v)3.3, is likely to possess an extracellular Ca(2+) binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca(2+) current in native cells.

Studies of Parkinson’s disease (PD) have been greatly hindered by lack of access to affected human dopaminergic (DA) neurons. Here, we report generation of induced pluripotent stem cells that carry the p.G2019S mutation (G2019S-iPSCs) in the Leucine-Rich Repeat Kinase-2 (LRRK2) gene, the most common PD-related mutation. We demonstrate that these G2019S-iPSCs were able to differentiate into DA neurons and showed increased expression of key oxidative stress response genes and α-synuclein protein. Moreover, G2019S-mutant DA neurons were more sensitive to caspase-3 activation, caused by exposure to hydrogen peroxide, MG-132, and 6-hydroxydopamine, compared to unaffected DA neurons. These findings suggest that G2019S-iPSC-derived DA neurons exhibit early phenotypes linked to PD. Due to high penetrance of the LRRK2 mutation and its clinical resemblance to sporadic PD, these neurons may provide a valuable platform for identification of novel pharmacological agents and diagnostics for modeling and alleviation of a subset of disease phenotypes.