Human iPSC-derived Neurons Research Articles

Cell body shape and directional movement stability in human-induced pluripotent stem cell-derived dopaminergic neurons

Neuronal migration is necessary in the process of the formation of brain architecture. Recently, we demonstrated that human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons exhibit directional migration in vitro. However, it remains unclear how the cell shape is involved in their migration. In this study, we performed live imaging analyses using human iPSC-derived dopaminergic neurons. Our automated method, which can automatically identify the cell body shape and the cell position at specific time points, revealed that healthy iPSC-derived dopaminergic neurons migrate according to their shape. This migration behavior was out of accord in neurons derived from iPSCs carrying an RELN deletion. Our findings provide a novel theory that cell body orientation is related to the stability of movement direction for human dopaminergic neurons, under the regulation of RELN.

Human iPSC-Derived Neurons and Cerebral Organoids Establish Differential Effects of Germline NF1 Gene Mutations.

SummaryNeurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by a spectrum of distinct germline NF1 gene mutations, traditionally viewed as equivalent loss-of-function alleles. To specifically address the issue of mutational equivalency in a disease with considerable clinical heterogeneity, we engineered seven isogenic human induced pluripotent stem cell lines, each with a different NF1 patient NF1 mutation, to identify potential differential effects of NF1 mutations on human central nervous system cells and tissues. Although all mutations increased proliferation and RAS activity in 2D neural progenitor cells (NPCs) and astrocytes, we observed striking differences between NF1 mutations on 2D NPC dopamine levels, and 3D NPC proliferation, apoptosis, and neuronal differentiation in developing cerebral organoids. Together, these findings demonstrate differential effects of NF1 gene mutations at the cellular and tissue levels, suggesting that the germline NF1 gene mutation is one factor that underlies clinical variability.

Three-dimensional brain-on-chip model using human iPSC-derived GABAergic neurons and astrocytes: Butyrylcholinesterase post-treatment for acute malathion exposure.

Organophosphates (OPs) induce acute and chronic neurotoxicity, primarily by inhibiting acetylcholinesterase (AChE) activity as well as by necrosis, and apoptosis. Butyrylcholinesterase (BuChE), an exogenous bioscavenger of OPs, can be used as a treatment for OP exposure. It is prerequisite to develop in vitro brain models that can study BuChE post-treatment for acute OP exposure. In this study, we developed a three-dimensional (3D) brain-on-chip platform with human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes to simulate human brain behavior. The platform consists of two compartments: 1) a hydrogel embedded with human iPSC-derived GABAergic neurons and astrocytes and 2) a perfusion channel with dynamic medium flow. The brain tissue constructs were exposed to Malathion (MT) at various concentrations and then treated with BuChE after 20 minutes of MT exposure. Results show that the iPSC-derived neurons and astrocytes directly interacted and formed synapses in the 3D matrix, and that treatment with BuChE improved viability after MT exposure up to a concentration of 10−3 M. We conclude that the 3D brain-on-chip platform with human iPSC-derived brain cells is a suitable model to study the neurotoxicity of OP exposure and evaluate therapeutic compounds for treatment.

Distinct responses of neurons and astrocytes to TDP-43 proteinopathy in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by motor neuron loss, resulting in muscle wasting, paralysis and eventual death. A key pathological feature of ALS is cytoplasmically mislocalized and aggregated TDP-43 protein in >95% of cases, which is considered to have prion-like properties. Historical studies have predominantly focused on genetic forms of ALS, which represent ∼10% of cases, leaving the remaining 90% of sporadic ALS relatively understudied. Additionally, the role of astrocytes in ALS and their relationship with TDP-43 pathology is also not currently well understood. We have therefore used highly enriched human induced pluripotent stem cell (iPSC)-derived motor neurons and astrocytes to model early cell type-specific features of sporadic ALS. We first demonstrate seeded aggregation of TDP-43 by exposing human iPSC-derived motor neurons to serially passaged sporadic ALS post-mortem tissue (spALS) extracts. Next, we show that human iPSC-derived motor neurons are more vulnerable to TDP-43 aggregation and toxicity compared with their astrocyte counterparts. We demonstrate that these TDP-43 aggregates can more readily propagate from motor neurons into astrocytes in co-culture paradigms. We next found that astrocytes are neuroprotective to seeded aggregation within motor neurons by reducing (mislocalized) cytoplasmic TDP-43, TDP-43 aggregation and cell toxicity. Furthermore, we detected TDP-43 oligomers in these spALS spinal cord extracts, and as such demonstrated that highly purified recombinant TDP-43 oligomers can reproduce this observed cell-type specific toxicity, providing further support to a protein oligomer-mediated toxicity hypothesis in ALS. In summary, we have developed a human, clinically relevant, and cell-type specific modelling platform that recapitulates key aspects of sporadic ALS and uncovers both an initial neuroprotective role for astrocytes and the cell type-specific toxic effect of TDP-43 oligomers.

Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex

One of the main obstacles for studying the molecular and cellular mechanisms underlying human neurodevelopment in vivo is the scarcity of experimental models. The discovery that neurons can be generated from human induced pluripotent stem cells (hiPSCs) paves the way for novel approaches that are stem cell-based. Here, we developed a technique to follow the development of transplanted hiPSC-derived neuronal precursors in the cortex of mice over time. Using post-mortem immunohistochemistry we quantified the differentiation and maturation of dendritic patterns of the human neurons over a total of six months. In addition, entirely hiPSC-derived neuronal parenchyma was followed over eight months using two-photon in vivo imaging through a cranial window. We found that transplanted hiPSC-derived neuronal precursors exhibit a “protracted” human developmental programme in different cortical areas. This offers novel possibilities for the sequential in vivo study of human cortical development and its alteration, followed in “real time”.

Establishing Myelinating Cocultures Using Human iPSC-Derived Sensory Neurons to Investigate Axonal Degeneration and Demyelination.

Complex signaling between Schwann cells and axons are vital for peripheral neuron development, myelination, and repair. The interaction between these two cell types can be modeled in vitro by coculturing rodent Schwann cells and neurons together. These have in the past been used with great success to help unravel the bidirectional signaling mechanisms that lead to Schwann cell proliferation and myelination. To provide more translatable potential, we have developed myelinating cocultures using human, induced pluripotent stem cell (iPSC)-derived neurons. Under the right conditions, the human neurons are efficiently myelinated by rat Schwann cells, demonstrating successful cross-species signaling. This chapter describes all the necessary steps to generate these myelinating cocultures and methods to investigate and quantify various aspects of myelination. The myelinating cocultures can be maintained in excellent health for over 1year, facilitating their use to study developmental or chronic disease processes. With this in mind, we have used the cocultures to model a sensory neuropathy which displays clinically with both axonal and demyelinating features. In the cocultures, we found evidence of extensive axonal degeneration and demyelination demonstrated by axonal swelling and fragmentation, and myelin disintegration. The myelinating cocultures can therefore be used to study complex, human disease processes that result in both axonal and myelin-associated degenerative processes.

Carbonization of polymer precursors substrates to direct human iPSC-derived neurons differentiation and maturation

Abstract In this work, precursor polymers were processed by electrospinning and photolithography. Polyacrilonitrile (PAN) fibers were obtained by electrospinning, while SU-8 high-resolution micro-patterns on silicon wafers were produced by photolithography. The substrates were subjected to thermal stabilization and pyrolysis to obtain carbon structures. iPSCs-derived Neural Stem Cells (NSCs) were cultured on the carbonized materials to analyze their adhesion and differentiation into neurons on the designed scaffolds. Results show that NSCs recognized the substrates and differentiated into neurons on both scaffold typologies. In order to evaluate differences among substrates used, neuronal marker expression analysis was performed.

Muscleblind acts as a modifier of FUS toxicity by modulating stress granule dynamics and SMN localization

Mutations in fused in sarcoma (FUS) lead to amyotrophic lateral sclerosis (ALS) with varying ages of onset, progression and severity. This suggests that unknown genetic factors contribute to disease pathogenesis. Here we show the identification of muscleblind as a novel modifier of FUS-mediated neurodegeneration in vivo. Muscleblind regulates cytoplasmic mislocalization of mutant FUS and subsequent accumulation in stress granules, dendritic morphology and toxicity in mammalian neuronal and human iPSC-derived neurons. Interestingly, genetic modulation of endogenous muscleblind was sufficient to restore survival motor neuron (SMN) protein localization in neurons expressing pathogenic mutations in FUS, suggesting a potential mode of suppression of FUS toxicity. Upregulation of SMN suppressed FUS toxicity in Drosophila and primary cortical neurons, indicating a link between FUS and SMN. Our data provide in vivo evidence that muscleblind is a dominant modifier of FUS-mediated neurodegeneration by regulating FUS-mediated ALS pathogenesis.

Control of Long-Term Synaptic Potentiation and Learning by Alternative Splicing of the NMDA Receptor Subunit GluN1

NMDA receptors (NMDARs) are critical for physiological synaptic plasticity, learning, and memory and for pathological plasticity and neuronal death. The GluN1 subunit is encoded by a single gene, GRIN1, with 8 splice variants, but whether the diversity generated by this splicing has physiological consequences remains enigmatic. Here, we generate mice lacking from the GluN1 exon 5-encoded N1 cassette (GluN1a mice) or compulsorily expressing this exon (GluN1b mice). Despite no differences in basal synaptic transmission, long-term potentiation in the hippocampus is significantly enhanced in GluN1a mice compared with that in GluN1b mice. Furthermore, GluN1a mice learn more quickly and have significantly better spatial memory performance than do GluN1b mice. In addition, in human iPSC-derived neurons in autism spectrum disorder NMDARs show characteristics of N1-lacking GluN1. Our findings indicate that alternative splicing of GluN1 is a mechanism for controlling physiological long-lasting synaptic potentiation, learning, and memory.

Mitochondrial deficits in human iPSC-derived neurons from patients with 22q11.2 deletion syndrome and schizophrenia

Schizophrenia (SZ) is a highly heterogeneous disorder in both its symptoms and risk factors. One of the most prevalent genetic risk factors for SZ is the hemizygous microdeletion at chromosome 22q11.2 (22q11DS) that confers a 25-fold increased risk. Six of the genes directly disrupted in 22qDS encode for mitochondrial-localizing proteins. Here, we test the hypothesis that stem cell-derived neurons from subjects with the 22q11DS and SZ have mitochondrial deficits relative to typically developing controls. Human iPSCs from four lines of affected subjects and five lines of controls were differentiated into forebrain-like excitatory neurons. In the patient group, we find significant reductions of ATP levels that appear to be secondary to reduced activity in oxidative phosphorylation complexes I and IV. Protein products of mitochondrial-encoded genes are also reduced. As one of the genes deleted in the 22q11.2 region is MRPL40, a component of the mitochondrial ribosome, we generated a heterozygous mutation of MRPL40 in a healthy control iPSC line. Relative to its isogenic control, this line shows similar deficits in mitochondrial DNA-encoded proteins, ATP level, and complex I and IV activity. These results suggest that in the 22q11DS MRPL40 heterozygosity leads to reduced mitochondria ATP production secondary to altered mitochondrial protein levels. Such defects could have profound effects on neuronal function in vivo.

THE ROLE OF CAPRIN-1 PROTEIN DYSREGULATION IN SYNAPSE DECLINE LEADING TO PROGRESSION OF TAUOPATHIES

Many neurodegenerative diseases are characterized by accumulation of proteins such as tau, a microtubule stabilization protein. Toxic tau forms tangles and affects neuronal synapse function, an early step of neurodegeneration. To focus on synapse function, we highlighted Caprin-1 protein. Through gene ontology, Caprin-1 is related to RNA granule proteins, important for transport and local translation in dendrites. Caprin-1 is of interest for neurodegeneration because it is a memory related protein, transports mRNA in RNA granules, and knock out mice demonstrate memory deficits. As we age, the performance of local translation in the dendrites is compromised and leads to synapse dysfunction. We hypothesize Caprin-1 binds to Tau and becomes disrupted. This leads to the dissolution of RNA granules, inhibition of mRNA transport in dendrites, suppression of translation, and failure of synapse. Early western blot data showed reduced Caprin-1 in PS19 Tau+, supporting our model that Caprin-1 is disrupted in disease models. Through immunohistochemistry, we investigated the localization of Caprin-1 in the mouse hippocampus. We observed Caprin-1 localization to dendrites of CA1 neurons in the hippocampus. Furthermore, Caprin-1 exhibited colocalization with Rps6, an RNA granule marker. This suggests Caprin-1 associates with RNA granules in mouse hippocampus. Finally, we investigated the localization of Caprin-1 in human iPSC-derived neurons. Similar to the mouse hippocampus, we observed localization of Caprin-1 to dendrites of human neurons. In future directions, we will examine whether pathogenic tau alters the association of Caprin-1 with RNA granules and the mechanisms by which pathogenic tau negatively effects synapse function.

A combinational treatment of carotenoids decreases Aβ secretion in human neurons via β-secretase inhibition

Alzheimer’s disease (AD) is the most common cause of dementia and is characterized neuropathologically by the presence of amyloid plaques and neurofibrillary tangles. Amyloid-β (Aβ) peptides, major components of amyloid plaques and crucial pathogenic molecules in terms of the amyloid hypothesis, are derived from successive proteolytic processing of amyloid-β precursor protein (APP). In this study, we established a human neuronal culture system using induced pluripotent stem cells (iPSCs) to evaluate the possible effects of natural compounds on the amyloid phenotype. Unexpectedly, we found that combinational treatment of carotenoids, but not docosahexaenoic acid, significantly decreased Aβ secretion from iPSC-derived human cortical neurons. Importantly, the effects of the carotenoids resulted from specific inhibition of BACE1 activity and not from expression changes in APP or BACE1. Therefore, these results indicate a novel beneficial function of carotenoids in the anti-amyloidogenic processing of APP. Collectively, this study will shed light on neuronal protection by a novel mechanism during the pathogenesis of AD.

Biallelic Mutations in TSC2 Lead to Abnormalities Associated with Cortical Tubers in Human iPSC-Derived Neurons.

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in TSC1 or TSC2 Patients frequently have epilepsy, autism spectrum disorder, and/or intellectual disability, as well as other systemic manifestations. In this study, we differentiated human induced pluripotent stem cells (iPSCs) from a female patient with TSC with one or two mutations in TSC2 into neurons using induced expression of NGN2 to examine neuronal dysregulation associated with the neurological symptoms in TSC. Using this method, neuronal differentiation was comparable between the three genotypes of iPSCs. We observed that TSC2+/- neurons show mTOR complex 1 (mTORC1) hyperactivation and associated increased cell body size and process outgrowth, as well as exacerbation of the abnormalities by loss of the second allele of TSC2 in TSC2-/- neurons. Interestingly, iPSC-derived neurons with either a single or biallelic mutation in TSC2 demonstrated hypersynchrony and downregulation of FMRP targets. However, only neurons with biallelic mutations of TSC2 demonstrated hyperactivity and transcriptional dysregulation observed in cortical tubers. These data demonstrate that loss of one allele of TSC2 is sufficient to cause some morphological and physiological changes in human neurons but that biallelic mutations in TSC2 are necessary to induce gene expression dysregulation present in cortical tubers. Finally, we found that treatment of iPSC-derived neurons with rapamycin reduced neuronal activity and partially reversed gene expression abnormalities, demonstrating that mTOR dysregulation contributes to both phenotypes. Therefore, biallelic mutations in TSC2 and associated molecular dysfunction, including mTOR hyperactivation, may play a role in the development of cortical tubers.SIGNIFICANCE STATEMENT In this study, we examined neurons derived from induced pluripotent stem cells with two, one, or no functional TSC2 (tuberous sclerosis complex 2) alleles and found that loss of one or both alleles of TSC2 results in mTORC1 hyperactivation and specific neuronal abnormalities. However, only biallelic mutations in TSC2 resulted in elevated neuronal activity and upregulation of cell adhesion genes that is also observed in cortical tubers. These data suggest that loss of heterozygosity of TSC1 or TSC2 may play an important role in the development of cortical tubers, and potentially epilepsy, in patients with TSC.

Drug repurposing for Alzheimer\u2019s disease based on transcriptional profiling of human iPSC-derived cortical neurons

Alzheimer’s disease is a complex disorder encompassing multiple pathological features with associated genetic and molecular culprits. However, target-based therapeutic strategies have so far proved ineffective. The aim of this study is to develop a methodology harnessing the transcriptional changes associated with Alzheimer’s disease to develop a high content quantitative disease phenotype that can be used to repurpose existing drugs. Firstly, the Alzheimer’s disease gene expression landscape covering severe disease stage, early pathology progression, cognitive decline and animal models of the disease has been defined and used to select a set of 153 drugs tending to oppose disease-associated changes in the context of immortalised human cancer cell lines. The selected compounds have then been assayed in the more biologically relevant setting of iPSC-derived cortical neuron cultures. It is shown that 51 of the drugs drive expression changes consistently opposite to those seen in Alzheimer’s disease. It is hoped that the iPSC profiles will serve as a useful resource for drug repositioning within the context of neurodegenerative disease and potentially aid in generating novel multi-targeted therapeutic strategies.

Are human iPSC-derived neurons a good tool to detect seizurogenic drugs?

Are human iPSC-derived neurons a good tool to detect seizurogenic drugs?

Detection of drug-induced seizure-like activities using MEA system in cultured human iPSC-derived neurons: Report from multi-site pilot study of the HESI NeuTox committee in collaboration with CSAHi and iNCENS

Detection of drug-induced seizure-like activities using MEA system in cultured human iPSC-derived neurons: Report from multi-site pilot study of the HESI NeuTox committee in collaboration with CSAHi and iNCENS

Multiple BACE1 inhibitors abnormally increase the BACE1 protein level in neurons by prolonging its half-life

IntroductionThere is keen interest in elucidating the biological mechanisms underlying recent failures of β-site amyloid precursor protein–cleaving enzyme-1 (BACE1) inhibitors in Alzheimer's disease trials. MethodsWe developed a highly sensitive and specific immunoassay for BACE1 in cell lines and iPSC-derived human neurons to systematically analyze the effects of eight clinically relevant BACE1 inhibitors. ResultsSeven of 8 inhibitors elevated BACE1 protein levels. Among protease inhibitors tested, the elevation was specific to BACE1 inhibitors. The inhibitors did not increase BACE1 transcription but extended the protein's half-life. BACE1 became elevated at concentrations below the IC50 for amyloid β (Aβ). DiscussionElevation of BACE1 by 7 of 8 BACE1 inhibitors raises new concerns about advancing such β-secretase inhibitors for AD. Chronic elevation could lead to intermittently uninhibited BACE1 when orally dosed inhibitors reach trough levels, abnormally increasing substrate processing. Compounds such as roburic acid that lower Aβ by dissociating β/γ secretase complexes are better candidates because they neither inhibit β- and γ-secretase nor increase BACE1 levels.

Synaptic Dysfunction in Human Neurons With Autism-Associated Deletions in PTCHD1-AS

BackgroundThe Xp22.11 locus that encompasses PTCHD1, DDX53, and the long noncoding RNA PTCHD1-AS is frequently disrupted in male subjects with autism spectrum disorder (ASD), but the functional consequences of these genetic risk factors for ASD are unknown. MethodsTo evaluate the functional consequences of PTCHD1 locus deletions, we generated induced pluripotent stem cells (iPSCs) from unaffected control subjects and 3 subjects with ASD with microdeletions affecting PTCHD1-AS/PTCHD1, PTCHD1-AS/DDX53, or PTCHD1-AS alone. Function of iPSC-derived cortical neurons was assessed using molecular approaches and electrophysiology. We also compiled novel and known genetic variants of the PTCHD1 locus to explore the roles of PTCHD1 and PTCHD1-AS in genetic risk for ASD and other neurodevelopmental disorders. Finally, genome editing was used to explore the functional consequences of deleting a single conserved exon of PTCHD1-AS. ResultsiPSC-derived neurons from subjects with ASD exhibited reduced miniature excitatory postsynaptic current frequency and N-methyl-D-aspartate receptor hypofunction. We found that 35 ASD-associated deletions mapping to the PTCHD1 locus disrupted exons of PTCHD1-AS. We also found a novel ASD-associated deletion of PTCHD1-AS exon 3 and showed that exon 3 loss altered PTCHD1-AS splicing without affecting expression of the neighboring PTCHD1 coding gene. Finally, targeted disruption of PTCHD1-AS exon 3 recapitulated diminished miniature excitatory postsynaptic current frequency, supporting a role for the long noncoding RNA in the etiology of ASD. ConclusionsOur genetic findings provide strong evidence that PTCHD1-AS deletions are risk factors for ASD, and human iPSC-derived neurons implicate these deletions in the neurophysiology of excitatory synapses and in ASD-associated synaptic impairment.

An Epilepsy-Associated KCNT1 Mutation Enhances Excitability of Human iPSC-Derived Neurons by Increasing Slack KNa Currents.

Mutations in the KCNT1 (Slack, KNa1.1) sodium-activated potassium channel produce severe epileptic encephalopathies. Expression in heterologous systems has shown that the disease-causing mutations give rise to channels that have increased current amplitude. It is not known, however, whether such gain of function occurs in human neurons, nor whether such increased KNa current is expected to suppress or increase the excitability of cortical neurons. Using genetically engineered human induced pluripotent stem cell (iPSC)-derived neurons, we have now found that sodium-dependent potassium currents are increased several-fold in neurons bearing a homozygous P924L mutation. In current-clamp recordings, the increased KNa current in neurons with the P924L mutation acts to shorten the duration of action potentials and to increase the amplitude of the afterhyperpolarization that follows each action potential. Strikingly, the number of action potentials that were evoked by depolarizing currents as well as maximal firing rates were increased in neurons expressing the mutant channel. In networks of spontaneously active neurons, the mean firing rate, the occurrence of rapid bursts of action potentials, and the intensity of firing during the burst were all increased in neurons with the P924L Slack mutation. The feasibility of an increased KNa current to increase firing rates independent of any compensatory changes was validated by numerical simulations. Our findings indicate that gain-of-function in Slack KNa channels causes hyperexcitability in both isolated neurons and in neural networks and occurs by a cell-autonomous mechanism that does not require network interactions.SIGNIFICANCE STATEMENTKCNT1 mutations lead to severe epileptic encephalopathies for which there are no effective treatments. This study is the first demonstration that a KCNT1 mutation increases the Slack current in neurons. It also provides the first explanation for how this increased potassium current induces hyperexcitability, which could be the underlining factor causing seizures.

Small-Molecule Modulation of TDP-43 Recruitment to Stress Granules Prevents Persistent TDP-43 Accumulation in ALS/FTD

Stress granules (SGs) form during cellular stress and are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). To yield insights into the role of SGs in pathophysiology, we performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and human iPSC-derived motor neurons (iPS-MNs). One major class of active molecules contained extended planar aromatic moieties, suggesting a potential to intercalate in nucleic acids. Accordingly, we show that several hit compounds can prevent the RNA-dependent recruitment of the ALS-associated RNA-binding proteins (RBPs) TDP-43, FUS, and HNRNPA2B1 into SGs. We further demonstrate that transient SG formation contributes to persistent accumulation of TDP-43 into cytoplasmic puncta and that our hit compounds can reduce this accumulation in iPS-MNs from ALS patients. We propose that compounds with planar moieties represent a promising starting point to develop small-molecule therapeutics for treating ALS/FTD.