Biology subjects

Farinelli, F.

Publications and source records attributed to Farinelli, F..

4 recordsLinked to original sources

bioRxiv2025

Automated high-throughput patch clamp electrophysiology of hiPSC-derived neuronal models

The advent of human induced pluripotent stem cells (hiPSCs) and their differentiation into neurons and brain organoids has revolutionized our ability to model brain disorders in a human context. However, current technologies to assay the electrophysiological properties of human neurons in these models remain limited by throughput, as single-cell manual patch clamp is laborious and resource intensive. Here, we provide methods to perform high-throughput automated patch-clamp (APC) on hiPSC-derived neurons. We describe how to dissociate and perform voltage-clamp recordings on human neurons from three well-established protocols - 2D directed differentiation of cortical neurons, NGN2-induced neurons, and 3D cortical organoids - using the Nanion Syncropatch 384, a commercially available high-throughput APC system. Using this approach, we investigated the biophysical properties of voltage-gated sodium channels (VGSCs) and provide direct comparisons between manual and APC recordings across all three hiPSC-derived model systems. We demonstrate the capability of this automated system for pharmacological analysis of native human VGSC isoforms, which will enable compound screening approaches. Lastly, we provide methods to sort specific cellular populations within these hiPSC models using fluorescence-activated cell sorting (FACS) followed by APC. These methods and results provide a transformative and novel high-throughput technique for quantifying passive and active membrane properties in cell-type specific and/or genetically modified hiPSC-derived neurons.

neuroscience↗

bioRxiv2023

TCF4 mutations disrupt synaptic function through dysregulation of RIMBP2 in patient-derived cortical neurons

Genetic variation in the transcription factor 4 (TCF4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of ASD called Pitt Hopkins Syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models is shown to regulate various aspects of neuronal development and function. However, our understanding of how disease-causing mutations in TCF4 confer pathophysiology in a human context is lacking. Here we show that cortical neurons derived from patients with TCF4 mutations have deficits in spontaneous synaptic transmission, network excitability and homeostatic plasticity. Transcriptomic analysis indicates these phenotypes result from altered expression of genes involved in presynaptic neurotransmission and identifies the presynaptic binding protein, RIMBP2 as the most differentially expressed gene in PTHS neurons. Remarkably, TCF4-dependent deficits in spontaneous synaptic transmission and network excitability were rescued by increasing RIMBP2 expression in presynaptic neurons. Together, these results identify TCF4 as a critical transcriptional regulator of human synaptic development and plasticity and specifically identifies dysregulation of presynaptic function as an early pathophysiology in PTHS.

neuroscience↗

bioRxiv2022

Constitutive Inhibition of Transient Receptor Potential CanonicalType 6 (TRPC6) by O-GlcNAcylation at Threonine-221

Transient receptor potential canonical type 6 (TRPC6) is a non-voltage gated cation channel that principally conducts calcium to regulate signaling in cardiac, vascular, neuronal and other cells. Abnormally increased TRPC6 expression/conductance and genetic gain of function mutations contribute to fibrosis, hypertrophy, proteinuria, and edema, notably linked to its stimulation of nuclear factor of activated T-cells (NFAT) signaling. Hyperglycemia (HG) also activates TRPC6/NFAT as a cause of diabetic renal disease. While prior work linked HG-TRPC6 activation to oxidant stress, the role of another major HG modification - O-GlcNAcylation, is unknown. Here we show TRPC6 is constitutively O-GlcNAcylated, TRPC6 and O-GlcNAc transferase proteins interact, this modification potently suppresses basal channel conductance and NFAT activity, and it is unaltered by HG. Proteomics identifies O-GlcNAcylation at Ser14, Thr70, and Thr221 in the N-terminus ankyrin-4 (AR4) and neighboring linker (LH1) domains of TRPC6. Of these, T221 is most impactful as a T221A mutation increases basal NFAT activity 11-fold, TRPC6 conductance 75-80% vs wild-type, and when expressed in cardiomyocytes amplifies NFAT-pro-hypertrophic gene expression. T221 is highly conserved and mutating homologs in TRPC3 and TRPC7 also markedly elevates basal NFAT activity. Molecular models predict electrostatic interactions between T221 O-GlcNAc and Ser199, Glu200, and Glu246, and we find similarly elevated NFAT activity from alanine substitutions at these coordinating sites as well. Thus, O-GlcNAcylation at T221 and its interaction with coordinating residues in AR4-LH1 is required for basal TRPC6 channel conductance and regulation of NFAT.

biochemistry↗

bioRxiv2021

Electrophysiological measures from human iPSC-derived neurons are associated with schizophrenia clinical status and predict individual cognitive performance

Neurons derived from human induced pluripotent stem cells (hiPSCs) have been used to model basic cellular aspects of neuropsychiatric disorders, but the relationship between the emergent phenotypes and the clinical characteristics of donor individuals has been unclear. We analyzed RNA expression and indices of cellular function in hiPSC-derived neural progenitors and cortical neurons generated from 13 individuals with high polygenic risk scores (PRS) for schizophrenia and a clinical diagnosis of schizophrenia, along with 15 neurotypical individuals with low PRS. We identified electrophysiological measures associated with diagnosis that implicated altered Na+ channel function and GABA-ergic neurotransmission. Importantly, electrophysiological measures predicted cardinal clinical and cognitive features found in these schizophrenia patients. The identification of basic neuronal physiological properties related to core clinical characteristics of illness is a potentially critical step in generating leads for novel therapeutics.

neuroscience↗