Dysregulation of miRNA expression and excitation in MEF2C autism patient hiPSC-neurons and cerebral organoids.

MEF2C is a critical transcription factor in neurodevelopment, whose loss-of-function mutation in humans results in MEF2C haploinsufficiency syndrome (MHS), a severe form of autism spectrum disorder (ASD)/intellectual disability (ID). Despite prior animal studies of MEF2C heterozygosity to mimic MHS, MHS-specific mutations have not been investigated previously, particularly in a human context as hiPSCs afford. Here, for the first time, we use patient hiPSC-derived cerebrocortical neurons and cerebral organoids to characterize MHS deficits.

Single-Cell Patch-Clamp/Proteomics of Human Alzheimer's Disease iPSC-Derived Excitatory Neurons Versus Isogenic Wild-Type Controls Suggests Novel Causation and Therapeutic Targets.

Standard single-cell (sc) proteomics of disease states inferred from multicellular organs or organoids cannot currently be related to single-cell physiology. Here, a scPatch-Clamp/Proteomics platform is developed on single neurons generated from hiPSCs bearing an Alzheimer's disease (AD) genetic mutation and compares them to isogenic wild-type controls. This approach provides both current and voltage electrophysiological data plus detailed proteomics information on single-cells.

Targeted protein S-nitrosylation of ACE2 inhibits SARS-CoV-2 infection.

Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity.

Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection.

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity.

α-Synuclein Oligomers Induce Glutamate Release from Astrocytes and Excessive Extrasynaptic NMDAR Activity in Neurons, Thus Contributing to Synapse Loss.

Synaptic and neuronal loss are major neuropathological characteristics of Parkinson's disease. Misfolded protein aggregates in the form of Lewy bodies, comprised mainly of α-synuclein (αSyn), are associated with disease progression, and have also been linked to other neurodegenerative diseases, including Lewy body dementia, Alzheimer's disease, and frontotemporal dementia. However, the effects of αSyn and its mechanism of synaptic damage remain incompletely understood.

Novel Therapeutic Approach for Excitatory/Inhibitory Imbalance in Neurodevelopmental and Neurodegenerative Diseases.

Excitatory/inhibitory (E/I) balance, defined as the balance between excitation and inhibition of synaptic activity in a neuronal network, accounts in part for the normal functioning of the brain, controlling, for example, normal spike rate. In many pathological conditions, this fine balance is perturbed, leading to excessive or diminished excitation relative to inhibition, termed E/I imbalance, reflected in network dysfunction. E/I imbalance has emerged as a contributor to neurological disorders that occur particularly at the extremes of life, including autism spectrum disorder and Alzheimer's disease, pointing to the vulnerability of neuronal networks at these critical life stages.

NitroSynapsin ameliorates hypersynchronous neural network activity in Alzheimer hiPSC models.

Beginning at early stages, human Alzheimer's disease (AD) brains manifest hyperexcitability, contributing to subsequent extensive synapse loss, which has been linked to cognitive dysfunction. No current therapy for AD is disease-modifying. Part of the problem with AD drug discovery is that transgenic mouse models have been poor predictors of potential human treatment.

Mechanisms of hyperexcitability in Alzheimer's disease hiPSC-derived neurons and cerebral organoids vs isogenic controls.

Human Alzheimer's disease (AD) brains and transgenic AD mouse models manifest hyperexcitability. This aberrant electrical activity is caused by synaptic dysfunction that represents the major pathophysiological correlate of cognitive decline. However, the underlying mechanism for this excessive excitability remains incompletely understood.

Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling.

Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry.

Pharmacologically targeted NMDA receptor antagonism by NitroMemantine for cerebrovascular disease.

Stroke and vascular dementia are leading causes of morbidity and mortality. Neuroprotective therapies have been proposed but none have proven clinically tolerated and effective. While overstimulation of N-methyl-d-aspartate-type glutamate receptors (NMDARs) is thought to contribute to cerebrovascular insults, the importance of NMDARs in physiological function has made this target, at least in the view of many in 'Big Pharma,' 'undruggable' for this indication.

Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription.

Parkinson's disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs).

Aβ induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and synaptic loss.

Synaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer's disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here, using FRET-based glutamate sensor imaging, we show that amyloid-β peptide (Aβ) engages α7 nicotinic acetylcholine receptors to induce release of astrocytic glutamate, which in turn activates extrasynaptic NMDA receptors (eNMDARs) on neurons.

High-frequency hippocampal oscillations activated by optogenetic stimulation of transplanted human ESC-derived neurons.

After transplantation, individual stem cell-derived neurons can functionally integrate into the host CNS; however, evidence that neurons derived from transplanted human embryonic stem cells (hESCs) can drive endogenous neuronal network activity in CNS tissue is still lacking. Here, using multielectrode array recordings, we report activation of high-frequency oscillations in the β and γ ranges (10-100 Hz) in the host hippocampal network via targeted optogenetic stimulation of transplanted hESC-derived neurons.

MEF2C enhances dopaminergic neuron differentiation of human embryonic stem cells in a parkinsonian rat model.

Human embryonic stem cells (hESCs) can potentially differentiate into any cell type, including dopaminergic neurons to treat Parkinson's disease (PD), but hyperproliferation and tumor formation must be avoided. Accordingly, we use myocyte enhancer factor 2C (MEF2C) as a neurogenic and anti-apoptotic transcription factor to generate neurons from hESC-derived neural stem/progenitor cells (NPCs), thus avoiding hyperproliferation. Here, we report that forced expression of constitutively active MEF2C (MEF2CA) generates significantly greater numbers of neurons with dopaminergic properties in vitro.

Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions.

Human induced pluripotent stem cells (hiPSCs) have been generated by reprogramming a number of different somatic cell types using a variety of approaches. In addition, direct reprogramming of mature cells from one lineage to another has emerged recently as an alternative strategy for generating cell types of interest. Here we show that a combination of a microRNA (miR-124) and two transcription factors (MYT1L and BRN2) is sufficient to directly reprogram postnatal and adult human primary dermal fibroblasts (mesoderm) to functional neurons (ectoderm) under precisely defined conditions.

Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors.

Human embryonic stem cells (hESCs) hold enormous promise for regenerative medicine. Typically, hESC-based applications would require their in vitro differentiation into a desirable homogenous cell population. A major challenge of the current hESC differentiation paradigm is the inability to effectively capture and, in the long-term, stably expand primitive lineage-specific stem/precursor cells that retain broad differentiation potential and, more importantly, developmental stage-specific differentiation propensity.

Direct reprogramming of mouse fibroblasts to neural progenitors.

The simple yet powerful technique of induced pluripotency may eventually supply a wide range of differentiated cells for cell therapy and drug development. However, making the appropriate cells via induced pluripotent stem cells (iPSCs) requires reprogramming of somatic cells and subsequent redifferentiation. Given how arduous and lengthy this process can be, we sought to determine whether it might be possible to convert somatic cells into lineage-specific stem/progenitor cells of another germ layer in one step, bypassing the intermediate pluripotent stage.

Excitatory glycine responses of CNS myelin mediated by NR1/NR3 "NMDA" receptor subunits.

NMDA receptors are typically excited by a combination of glutamate and glycine. Here we describe excitatory responses in CNS myelin that are gated by a glycine agonist alone and mediated by NR1/NR3 "NMDA" receptor subunits. Response properties include activation by d-serine, inhibition by the glycine-site antagonist CNQX, and insensitivity to the glutamate-site antagonist d-APV.

Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation.

Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs).

Balance between synaptic versus extrasynaptic NMDA receptor activity influences inclusions and neurotoxicity of mutant huntingtin.

Huntington's disease is caused by an expanded CAG repeat in the gene encoding huntingtin (HTT), resulting in loss of striatal and cortical neurons. Given that the gene product is widely expressed, it remains unclear why neurons are selectively targeted. Here we show the relationship between synaptic and extrasynaptic activity, inclusion formation of mutant huntingtin protein (mtHtt) and neuronal survival.

Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes.

Background: Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.

Methodology/principal Findings: Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage.