Human iPSC-derived Neurons Research Articles

Patient-derived iPSC-cerebral organoid modeling of the 17q11.2 microdeletion syndrome establishes CRLF3 as a critical regulator of neurogenesis.

SUMMARYNeurodevelopmental disorders are often caused by chromosomal microdeletions comprising numerous contiguous genes. A subset of neurofibromatosis type 1 (NF1) patients with severe developmental delays and intellectual disability harbors such a microdeletion event on chromosome 17q11.2, involving the NF1 gene and flanking regions (NF1 total gene deletion [NF1-TGD]). Using patient-derived human induced pluripotent stem cell (hiPSC)-forebrain cerebral organoids (hCOs), we identify both neural stem cell (NSC) proliferation and neuronal maturation abnormalities in NF1-TGD hCOs. While increased NSC proliferation results from decreased NF1/RAS regulation, the neuronal differentiation, survival, and maturation defects are caused by reduced cytokine receptor-like factor 3 (CRLF3) expression and impaired RhoA signaling. Furthermore, we demonstrate a higher autistic trait burden in NF1 patients harboring a deleterious germline mutation in the CRLF3 gene (c.1166T>C, p.Leu389Pro). Collectively, these findings identify a causative gene within the NF1-TGD locus responsible for hCO neuronal abnormalities and autism in children with NF1.

Quantifying scaling exponents for neurite morphology of in vitro-cultured human iPSC-derived neurons using discrete Loewner evolution: A statistical-physical approach to the neuropathology in Alzheimer's disease.

Defining the morphological disorders causing neurodegenerative diseases is an unresolved problem. In this study, we propose a statistical-physical approach to quantify neurite morphology and evaluate the pathological states induced by Alzheimer's disease (AD). We analyzed the two-dimensional morphologies of neurites of in vitro-cultured human induced-pluripotent stem cell-derived neurons, reprogrammed from both a healthy person and a patient with AD, using discrete chordal Loewner evolution. For the numerically calculated Loewner driving forces, detrended fluctuation analysis was performed, and the morphological characteristics of the neurites were quantified using short-range and long-range scaling exponents. The day in vitro (DIV)-dependent behaviors of the scaling exponents and the associated neurite-type categorizations suggested that differences between healthy and AD neurites can be observed from the early stage (DIV3) of their development. Notably, AD neurites have less long-range autocorrelations than healthy neurites, particularly in the earlier stages (DIV3-10). Immunofluorescence-staining results suggested that these differences precede significant expressions of β-amyloid and phosphorylated tau, which are known as biological factors causing AD. We expect that these results will lead to a theoretical interpretation of the neurogenerative disease, providing the physical properties of individual neurites with different morphologies.

Phenotypic manifestation of \u03b1-synuclein strains derived from Parkinson\u2019s disease and multiple system atrophy in human dopaminergic neurons

α-Synuclein is critical in the pathogenesis of Parkinson’s disease and related disorders, yet it remains unclear how its aggregation causes degeneration of human dopaminergic neurons. In this study, we induced α-synuclein aggregation in human iPSC-derived dopaminergic neurons using fibrils generated de novo or amplified in the presence of brain homogenates from Parkinson’s disease or multiple system atrophy. Increased α-synuclein monomer levels promote seeded aggregation in a dose and time-dependent manner, which is associated with a further increase in α-synuclein gene expression. Progressive neuronal death is observed with brain-amplified fibrils and reversed by reduction of intraneuronal α-synuclein abundance. We identified 56 proteins differentially interacting with aggregates triggered by brain-amplified fibrils, including evasion of Parkinson’s disease-associated deglycase DJ-1. Knockout of DJ-1 in iPSC-derived dopaminergic neurons enhance fibril-induced aggregation and neuronal death. Taken together, our results show that the toxicity of α-synuclein strains depends on aggregate burden, which is determined by monomer levels and conformation which dictates differential interactomes. Our study demonstrates how Parkinson’s disease-associated genes influence the phenotypic manifestation of strains in human neurons.

Identification of bioactive metabolites in human iPSC-derived dopaminergic neurons with PARK2 mutation: Altered mitochondrial and energy metabolism.

SummaryPARK2 (parkin) mutations cause early-onset Parkinson's disease (PD). Parkin is an ubiquitin E3 ligase that participates in several cellular functions, including mitochondrial homeostasis. However, the specific metabolomic changes caused by parkin depletion remain unknown. Here, we used isogenic human induced pluripotent stem cells (iPSCs) with and without PARK2 knockout (KO) to investigate the effect of parkin loss of function by comparative metabolomics supplemented with ultrastructural and functional analyses. PARK2 KO neurons displayed increased tricarboxylic acid (TCA) cycle activity, perturbed mitochondrial ultrastructure, ATP depletion, and dysregulation of glycolysis and carnitine metabolism. These perturbations were combined with increased oxidative stress and a decreased anti-oxidative response. Key findings for PARK2 KO cells were confirmed using patient-specific iPSC-derived neurons. Overall, our data describe a unique metabolomic profile associated with parkin dysfunction and show that combining metabolomics with an iPSC-derived dopaminergic neuronal model of PD is a valuable approach to obtain novel insight into the disease pathogenesis.

Genoppi is an open-source software for robust and standardized integration of proteomic and genetic data

Combining genetic and cell-type-specific proteomic datasets can generate biological insights and therapeutic hypotheses, but a technical and statistical framework for such analyses is lacking. Here, we present an open-source computational tool called Genoppi (lagelab.org/genoppi) that enables robust, standardized, and intuitive integration of quantitative proteomic results with genetic data. We use Genoppi to analyze 16 cell-type-specific protein interaction datasets of four proteins (BCL2, TDP-43, MDM2, PTEN) involved in cancer and neurological disease. Through systematic quality control of the data and integration with published protein interactions, we show a general pattern of both cell-type-independent and cell-type-specific interactions across three cancer cell types and one human iPSC-derived neuronal cell type. Furthermore, through the integration of proteomic and genetic datasets in Genoppi, our results suggest that the neuron-specific interactions of these proteins are mediating their genetic involvement in neurodegenerative diseases. Importantly, our analyses suggest that human iPSC-derived neurons are a relevant model system for studying the involvement of BCL2 and TDP-43 in amyotrophic lateral sclerosis.

Bardet-Biedl syndrome proteins regulate intracellular signaling and neuronal function in patient-specific iPSC-derived neurons

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell-derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations BBS1M390R and BBS10C91fsX95 did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of BBS1M390R hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that BBS1M390R and BBS10C91fsX95 mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS10C91fsX95 neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in BBS1M390R and BBS10C91fsX95 iPSC-derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis.

Overlapping roles of JIP3 and JIP4 in promoting axonal transport of lysosomes in human iPSC-derived neurons.

The dependence of neurons on microtubule-based motors for the movement of lysosomes over long distances raises questions about adaptations that allow neurons to meet these demands. Recently, JIP3/MAPK8IP3, a neuronally enriched putative adaptor between lysosomes and motors, was identified as a critical regulator of axonal lysosome abundance. In this study, we establish a human induced pluripotent stem cell (iPSC)-derived neuron model for the investigation of axonal lysosome transport and maturation and show that loss of JIP3 results in the accumulation of axonal lysosomes and the Alzheimer’s disease–related amyloid precursor protein (APP)-derived Aβ42 peptide. We furthermore reveal an overlapping role of the homologous JIP4 gene in lysosome axonal transport. These results establish a cellular model for investigating the relationship between lysosome axonal transport and amyloidogenic APP processing and more broadly demonstrate the utility of human iPSC–derived neurons for the investigation of neuronal cell biology and pathology.

Sensing serotonin secreted from human serotonergic neurons using aptamer-modified nanopipettes.

The serotonergic system in the human brain modulates several physiological processes, and altered serotonergic neurotransmission has been implicated in the neuropathology of several psychiatric disorders. The study of serotonergic neurotransmission in psychiatry has long been restricted to animal models, but advances in cell reprogramming technology have enabled the generation of serotonergic neurons from patient-induced pluripotent stem cells (iPSCs). While iPSC-derived human serotonergic neurons offer the possibility to study serotonin (5-HT) release and uptake, particularly by 5-HT-modulating drugs such as selective serotonin reuptake inhibitors (SSRIs), a major limitation is the inability to reliably quantify 5-HT secreted from neurons in vitro. Herein, we address this technical gap via a novel sensing technology that couples 5-HT-specific DNA aptamers into nanopores (glass nanopipettes) with orifices of ~10 nm to detect 5-HT in complex neuronal culture medium with higher selectivity, sensitivity, and stability than existing methods. The 5-HT aptamers undergo conformational rearrangement upon target capture and serve as gatekeepers of ionic flux through the nanopipette opening. We generated human serotonergic neurons in vitro and detected secreted 5-HT using aptamer-coated nanopipettes in a low nanomolar range, with the possibility of detecting significantly lower (picomolar) concentrations. Furthermore, as a proof of concept, we treated human serotonergic neurons in vitro with the SSRI citalopram and detected a significant increase in extracellular 5-HT using the aptamer-modified nanopipettes. We demonstrate the utility of such methods for 5-HT detection, raising the possibility of fast quantification of neurotransmitters secreted from patient-derived live neuronal cells.

Identification and characterization of conserved noncoding cis-regulatory elements that impact Mecp2 expression and neurological functions.

While changes in MeCP2 dosage cause Rett syndrome (RTT) and MECP2 duplication syndrome (MDS), its transcriptional regulation is poorly understood. Here, we identified six putative noncoding regulatory elements of Mecp2, two of which are conserved in humans. Upon deletion in mice and human iPSC-derived neurons, these elements altered RNA and protein levels in opposite directions and resulted in a subset of RTT- and MDS-like behavioral deficits in mice. Our discovery provides insight into transcriptional regulation of Mecp2/MECP2 and highlights genomic sites that could serve as diagnostic and therapeutic targets in RTT or MDS.

The thrombin receptor links brain derived neurotrophic factor to neuron cholesterol production, resiliency and repair after spinal cord injury

Despite concerted efforts to identify CNS regeneration strategies, an incomplete understanding of how the needed molecular machinery is regulated limits progress. Here we use models of lateral compression and FEJOTA clip contusion-compression spinal cord injury (SCI) to identify the thrombin receptor (Protease Activated Receptor 1 (PAR1)) as an integral facet of this machine with roles in regulating neurite growth through a growth factor- and cholesterol-dependent mechanism. Functional recovery and signs of neural repair, including expression of cholesterol biosynthesis machinery and markers of axonal and synaptic integrity, were all increased after SCI in PAR1 knockout female mice, while PTEN was decreased. Notably, PAR1 differentially regulated HMGCS1, a gene encoding a rate-limiting enzyme in cholesterol production, across the neuronal and astroglial compartments of the intact versus injured spinal cord. Pharmacologic inhibition of cortical neuron PAR1 using vorapaxar in vitro also decreased PTEN and promoted neurite outgrowth in a cholesterol dependent manner, including that driven by suboptimal brain derived neurotrophic factor (BDNF). Pharmacologic inhibition of PAR1 also augmented BDNF-driven HMGCS1 and cholesterol production by murine cortical neurons and by human SH-SY5Y and iPSC-derived neurons. The link between PAR1, cholesterol and BDNF was further highlighted by demonstrating that the deleterious effects of PAR1 over-activation are overcome by supplementing cultures with BDNF, cholesterol or by blocking an inhibitor of adenylate cyclase, Gαi. These findings document PAR1-linked neurotrophic coupling mechanisms that regulate neuronal cholesterol metabolism as an important component of the machinery regulating CNS repair and point to new strategies to enhance neural resiliency after injury.

Astrocyte-derived extracellular vesicles enhance the survival and electrophysiological function of human cortical neurons in vitro

Neurons derived from human induced pluripotent stem cells (hiPSCs) are powerful tools for modeling neural pathophysiology and preclinical efficacy/toxicity screening of novel therapeutic compounds. However, human neurons cultured in vitro typically do not fully recapitulate the physiology of the human nervous system, especially in terms of exhibiting morphological maturation, longevity, and electrochemical signaling ability comparable to that of adult human neurons. In this study, we investigated the potential for astrocyte-derived extracellular vesicles (EVs) to modulate survival and electrophysiological function of human neurons in vitro. Specifically, we demonstrate that EVs obtained from human astrocytes promote enhanced single cell electrophysiological function and anti-apoptotic behavior in a homogeneous population of human iPSC-derived cortical neurons. Furthermore, EV-proteomic analysis was performed to identify cargo proteins with the potential to promote the physiological enhancement observed. EV cargos were found to include neuroprotective proteins such as heat shock proteins, alpha-synuclein, and lipoprotein receptor-related protein 1 (LRP1), as well as apolipoprotein E (APOE), which negatively regulates neuronal apoptosis, and a peroxidasin homolog that supports neuronal oxidative stress management. Proteins that positively regulate neuronal excitability and synaptic development were also detected, such as potassium channel tetramerization domain containing 12 (KCTD12), glucose-6- phosphate dehydrogenase (G6PD), kinesin family member 5B (KIF5B), spectrin-alpha non-erythrocytic1 (SPTAN1). The remarkable improvements in electrophysiological function and evident inhibition of apoptotic signaling in cultured neurons exposed to these cargos may hold significance for improving preclinical in vitro screening modalities. In addition, our collected data highlight the potential for EV-based therapeutics as a potential class of future clinical treatment for tackling inveterate central and peripheral neuropathies.

Evaluation of chemical compounds that inhibit neurite outgrowth using GFP-labeled iPSC-derived human neurons

Due to the increasing number of drugs and untested environmental compounds introduced into commercial use, there is recognition for a need to develop reliable and efficient screening methods to identify compounds that may adversely impact the nervous system. One process that has been implicated in neurodevelopment is neurite outgrowth; the disruption of which can result in adverse outcomes that persist later in life. Here, we developed a green fluorescent protein (GFP) labeled neurite outgrowth assay in a high-content, high-throughput format using induced pluripotent stem cell (iPSC) derived human spinal motor neurons and cortical glutamatergic neurons. The assay was optimized for use in a 1536-well plate format. Then, we used this assay to screen a set of 84 unique compounds that have previously been screened in other neurite outgrowth assays. This library consists of known developmental neurotoxicants, environmental compounds with unknown toxicity, and negative controls. Neurons were cultured for 40 h and then treated with compounds at 11 concentrations ranging from 1.56 nM to 92 μM for 24 and 48 h. Effects of compounds on neurite outgrowth were evaluated by quantifying total neurite length, number of segments, and maximum neurite length per cell. Among the 84 tested compounds, neurite outgrowth in cortical neurons and motor neurons were selectively inhibited by 36 and 31 compounds, respectively. Colchicine, rotenone, and methyl mercuric (II) chloride inhibited neurite outgrowth in both cortical and motor neurons. It is interesting to note that some compounds like parathion and bisphenol AF had inhibitory effects on neurite outgrowth specifically in the cortical neurons, while other compounds, such as 2,2′,4,4′-tetrabromodiphenyl ether and caffeine, inhibited neurite outgrowth in motor neurons. The data gathered from these studies show that GFP-labeled iPSC-derived human neurons are a promising tool for identifying and prioritizing compounds with developmental neurotoxicity potential for further hazard characterization.

Image-Based Quantitation of Kainic Acid-Induced Excitotoxicity as a Model of Neurodegeneration in Human iPSC-Derived Neurons.

Excitotoxicity is a feature of many neurodegenerative diseases and acquired forms of neural injury that is characterized by disruption of neuronal morphology. This is typically seen as beading and fragmentation of neurites when exposed to excitotoxins such as the AMPA receptor agonist kainic acid, with the extent to which these occur used to quantitate neurodegeneration. Induced pluripotent stem cells (iPSCs) provide a means to generate human neurons in vitro for mechanistic studies and can thereby be used to investigate how cells respond to excitotoxicity and to identify or test potential neuroprotective agents. To facilitate such studies, we have optimized a protocol for human iPSC differentiation to mature neurons in a 96-well plate format that enables image-based quantitation of changes to neuron morphology when exposed to kainic acid. Our protocol assays neuron morphology across seven excitotoxin concentrations with multiple control conditions and is ideally suited to comparison of neurons generated through differentiation of two isogenic iPSC lines in a single plate. We have included detailed step-by-step protocols for neural stem cell differentiation, neuronal maturation and exposure to kainic acid treatment, as well as different approaches to image-based quantitation that involve immunofluorescence or phase-contrast microscopy.

ERICH3: vesicular association and antidepressant treatment response

Selective serotonin reuptake inhibitors (SSRIs) are standard of care for major depressive disorder (MDD) pharmacotherapy, but only approximately half of these patients remit on SSRI therapy. Our previous genome-wide association study identified a single-nucleotide polymorphism (SNP) signal across the glutamate-rich 3 (ERICH3) gene that was nearly genome-wide significantly associated with plasma serotonin (5-HT) concentrations, which were themselves associated with SSRI response for MDD patients enrolled in the Mayo Clinic PGRN-AMPS SSRI trial. In this study, we performed a meta-analysis which demonstrated that those SNPs were significantly associated with SSRI treatment outcomes in four independent MDD trials. However, the function of ERICH3 and molecular mechanism(s) by which it might be associated with plasma 5-HT concentrations and SSRI clinical response remained unclear. Therefore, we characterized the human ERICH3 gene functionally and identified ERICH3 mRNA transcripts and protein isoforms that are highly expressed in central nervous system cells. Coimmunoprecipitation identified a series of ERICH3 interacting proteins including clathrin heavy chain which are known to play a role in vesicular function. Immunofluorescence showed ERICH3 colocalization with 5-HT in vesicle-like structures, and ERICH3 knock-out dramatically decreased 5-HT staining in SK-N-SH cells as well as 5-HT concentrations in the culture media and cell lysates without changing the expression of 5-HT synthesizing or metabolizing enzymes. Finally, immunofluorescence also showed ERICH3 colocalization with dopamine in human iPSC-derived neurons. These results suggest that ERICH3 may play a significant role in vesicular function in serotonergic and other neuronal cell types, which might help explain its association with antidepressant treatment response.

De Novo VPS4A Mutations Cause Multisystem Disease with Abnormal Neurodevelopment

SummaryThe endosomal sorting complexes required for transport (ESCRTs) are essential for multiple membrane modeling and membrane-independent cellular processes. Here we describe six unrelated individuals with de novo missense variants affecting the ATPase domain of VPS4A, a critical enzyme regulating ESCRT function. Probands had structural brain abnormalities, severe neurodevelopmental delay, cataracts, growth impairment, and anemia. In cultured cells, overexpression of VPS4A mutants caused enlarged endosomal vacuoles resembling those induced by expression of known dominant-negative ATPase-defective forms of VPS4A. Proband-derived fibroblasts had enlarged endosomal structures with abnormal accumulation of the ESCRT protein IST1 on the limiting membrane. VPS4A function was also required for normal endosomal morphology and IST1 localization in iPSC-derived human neurons. Mutations affected other ESCRT-dependent cellular processes, including regulation of centrosome number, primary cilium morphology, nuclear membrane morphology, chromosome segregation, mitotic spindle formation, and cell cycle progression. We thus characterize a distinct multisystem disorder caused by mutations affecting VPS4A and demonstrate that its normal function is required for multiple human developmental and cellular processes.

Longitudinal Characterization of Transcriptomic, Functional, and Morphological Features in Human iPSC-Derived Neurons and Their Application to Investigate Translational Progranulin Disease Biology

The disease biology of frontotemporal lobe dementia (FTD) is complex and not fully understood, with limited translational value appreciated from animal models to date. Human cellular systems that can recapitulate phenotypic features of disease offer promise as translational tools to not only increase our understanding of disease processes but also increase the probability of success of translating novel treatment options to patients. However not all researchers may necessarily have access to well-characterized induced pluripotent stem cell (iPSC)-derived human neurons. As an example, we therefore comprehensively profiled phenotypic features over time in one commercially-available IPSC-derived human neuron cell line. This included systems-level assessments of neurite outgrowth dynamics, neuronal network function, and genome-wide gene expression. By investigating progranulin biology as an example we then demonstrated the utility of these cells as a tool to investigate human disease biology. For example, by using the siRNA-mediated knockdown of the progranulin (GRN) gene, we demonstrated the establishment of an isogenic human cellular model to facilitate translational FTD research. We reproduced findings from rodent neurons by demonstrating that recombinant progranulin (rPGRN) mediated neuroprotection. Contrary to previous rodent data, in our human cellular models, growth factor treatment showed no consistent sensitivity to modulate neurite outgrowth dynamics. Our study further provides the first evidence that rRPGRN modulated neuronal firing and synchrony in human neurons. Taken together, our datasets are a valuable systems-level resource demonstrating the utility of the tested commercially-available human iPSC neurons for investigating basic human neurobiology, translational neuroscience, and drug discovery applications in neurodegenerative and other CNS diseases.

Proteomic analysis of human iPSC-derived sensory neurons implicates cell stress and microtubule dynamics dysfunction in bortezomib-induced peripheral neurotoxicity

The neurotoxic effects of the chemotherapeutic agent bortezomib on dorsal root ganglia sensory neurons are well documented, yet the mechanistic underpinnings that govern these cellular processes remain incompletely understood. In this study, system-wide proteomic changes were identified in human induced pluripotent stem cell-derived sensory neurons (iSNs) exposed to a clinically relevant dose of bortezomib. Label-free mass spectrometry facilitated the identification of approximately 2800 iSN proteins that exhibited differential levels in the setting of bortezomib. A significant proportion of these proteins affect the cellular processes of microtubule dynamics, cytoskeletal and cytoplasmic organization, and molecular transport, and pathway analysis revealed an enrichment of proteins in signaling pathways attributable to the unfolded protein response and the integrated stress response. Alterations in microtubule-associated proteins suggest a multifaceted relationship exists between bortezomib-induced proteotoxicity and microtubule cytoskeletal architecture, and MAP2 was prioritized as a topmost influential candidate. We observed a significant reduction in the overall levels of MAP2c in somata without discernable changes in neurites. As MAP2 is known to affect cellular processes including axonogenesis, neurite extension and branching, and neurite morphology, its altered levels are suggestive of a prominent role in bortezomib-induced neurotoxicity.

Upregulation of Cellular Palmitoylation Mitigates α-Synuclein Accumulation and Neurotoxicity.

Synucleinopathies, including Parkinson's disease (PD), are characterized by α-synuclein (αS) cytoplasmic inclusions. αS-dependent vesicle-trafficking defects are important in PD pathogenesis, but their mechanisms are not well understood. Protein palmitoylation, post-translational addition of the fatty acid palmitate to cysteines, promotes trafficking by anchoring specific proteins to the vesicle membrane. αS itself cannot be palmitoylated as it lacks cysteines, but it binds to membranes, where palmitoylation occurs, via an amphipathic helix. We hypothesized that abnormal αS membrane-binding impairs trafficking by disrupting palmitoylation. Accordingly, we investigated the therapeutic potential of increasing cellular palmitoylation. We asked whether upregulating palmitoylation by inhibiting the depalmitoylase acyl-protein-thioesterase-1 (APT1) ameliorates pathologic αS-mediated cellular phenotypes and sought to identify the mechanism. Using human neuroblastoma cells, rat neurons, and iPSC-derived PD patient neurons, we examined the effects of pharmacologic and genetic downregulation of APT1 on αS-associated phenotypes. APT1 inhibition or knockdown decreased αS cytoplasmic inclusions, reduced αS serine-129 phosphorylation (a PD neuropathological marker), and protected against αS-dependent neurotoxicity. We identified the APT1 substrate microtubule-associated-protein-6 (MAP6), which binds to vesicles in a palmitoylation-dependent manner, as a key mediator of these effects. Mechanistically, we found that pathologic αS accelerated palmitate turnover on MAP6, suggesting that APT1 inhibition corrects a pathological αS-dependent palmitoylation deficit. We confirmed the disease relevance of this mechanism by demonstrating decreased MAP6 palmitoylation in neurons from αS gene triplication patients. Our findings demonstrate a novel link between the fundamental process of palmitoylation and αS pathophysiology. Upregulating palmitoylation represents an unexplored therapeutic strategy for synucleinopathies. © 2020 International Parkinson and Movement Disorder Society.

Toward Brain-on-a-Chip: Human Induced Pluripotent Stem Cell-Derived Guided Neuronal Networks in Tailor-Made 3D Nanoprinted Microscaffolds.

Brain-on-a-chip (BoC) concepts should consider three-dimensional (3D) scaffolds to mimic the 3D nature of the human brain not accessible by conventional planar cell culturing. Furthermore, the essential key to adequately address drug development for human pathophysiological diseases of the nervous system, such as Parkinson's or Alzheimer's, is to employ human induced pluripotent stem cell (iPSC)-derived neurons instead of neurons from animal models. To address both issues, we present electrophysiologically mature human iPSC-derived neurons cultured in BoC applicable microscaffolds prepared by direct laser writing. 3D nanoprinted tailor-made elevated cavities interconnected by freestanding microchannels were used to create defined neuronal networks-as a proof of concept-with two-dimensional topology. The neuronal outgrowth in these nonplanar structures was investigated, among others, in terms of neurite length, size of continuous networks, and branching behavior using z-stacks prepared by confocal microscopy and cross-sectional scanning electron microscopy images prepared by focused ion beam milling. Functionality of the human iPSC-derived neurons was demonstrated with patch clamp measurements in both current- and voltage-clamp mode. Action potentials and spontaneous excitatory postsynaptic currents-fundamental prerequisites for proper network signaling-prove full integrity of these artificial neuronal networks. Considering the network formation occurring within only a few days and the versatile nature of direct laser writing to create even more complex scaffolds for 3D network topologies, we believe that our study offers additional approaches in human disease research to mimic the complex interconnectivity of the human brain in BoC studies.

Single-Cell Transcriptomics of Parkinson’s Disease Human In Vitro Models Reveals Dopamine Neuron-Specific Stress Responses

The advent of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized Parkinson's disease (PD) research, but single-cell transcriptomic analysis suggests unresolved cellular heterogeneity within these models. Here, we perform the largest single-cell transcriptomic study of human iPSC-derived dopaminergic neurons to elucidate gene expression dynamics in response to cytotoxic and genetic stressors. We identify multiple neuronal subtypes with transcriptionally distinct profiles and differential sensitivity to stress, highlighting cellular heterogeneity in dopamine invitro models. We validate this disease model by showing robust expression of PD GWAS genes and overlap with postmortem adult substantia nigra neurons. Importantly, stress signatures are ameliorated using felodipine, an FDA-approved drug. Using isogenic SNCA-A53T mutants, we find perturbations in glycolysis, cholesterol metabolism, synaptic signaling, and ubiquitin-proteasomal degradation. Overall, our study reveals cell type-specific perturbations in human dopamine neurons, which will further our understanding of PD and have implications for cell replacement therapies.