iPSC-derived Motor Neurons Research Articles

Reduced C9orf72 expression exacerbates polyGR toxicity in patient iPSC-derived motor neurons and a Type I protein arginine methyltransferase inhibitor reduces that toxicity.

Intronic repeat expansions in the C9orf72 gene are the most frequent known single genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These repeat expansions are believed to result in both loss-of-function and toxic gain-of-function. Gain-of-function results in the production of toxic arginine-rich dipeptide repeat proteins (DPRs), namely polyGR and polyPR. Small-molecule inhibition of Type I protein arginine methyltransferases (PRMTs) has been shown to protect against toxicity resulting from polyGR and polyPR challenge in NSC-34 cells and primary mouse-derived spinal neurons, but the effect in human motor neurons (MNs) has not yet been explored. To study this, we generated a panel of C9orf72 homozygous and hemizygous knockout iPSCs to examine the contribution of C9orf72 loss-of-function toward disease pathogenesis. We differentiated these iPSCs into spinal motor neurons (sMNs). We found that reduced levels of C9orf72 exacerbate polyGR15 toxicity in a dose-dependent manner. Type I PRMT inhibition was able to partially rescue polyGR15 toxicity in both wild-type and C9orf72-expanded sMNs. This study explores the interplay of loss-of-function and gain-of-function toxicity in C9orf72 ALS. It also implicates type I PRMT inhibitors as a possible modulator of polyGR toxicity.

Downregulation of Hsp90 and the antimicrobial peptide Mtk suppresses poly(GR)-induced neurotoxicity in C9ORF72-ALS/FTD

GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat RNAs can be translated into dipeptide repeat proteins, including poly(GR), whose mechanisms of action remain largely unknown. In an RNA-seq analysis of poly(GR) toxicity in Drosophila, we found that several antimicrobial peptide genes, such as metchnikowin (Mtk), and heat shock protein (Hsp) genes are activated. Mtk knockdown in the fly eye or in all neurons suppresses poly(GR) neurotoxicity. These findings suggest a cell-autonomous role of Mtk in neurodegeneration. Hsp90 knockdown partially rescues both poly(GR) toxicity in flies and neurodegeneration in C9ORF72 motor neurons derived from induced pluripotent stem cells (iPSCs). Topoisomerase II (TopoII) regulates poly(GR)-induced upregulation of Hsp90 and Mtk. TopoII knockdown also suppresses poly(GR) toxicity in Drosophila and improves survival of C9ORF72 iPSC-derived motor neurons. These results suggest potential novel therapeutic targets for C9ORF72-ALS/FTD.

Differentiation of Human Induced Pluripotent Stem Cells into Mature and Myelinating Schwann Cells.

In the peripheral nervous system, Schwann cells (SCs) play a crucial role in axonal growth, metabolic support of neurons, and the production of myelin sheaths. Expansion of SCs after extraction from human or animal nerves is a long and often low-yielding process. We established a rapid cell culture method using a defined serum-free medium to differentiate human induced pluripotent stem cells (iPSCs) into SCs in only 21 days. The SC identity was characterized by expression of SRY-Box Transcription factor 10 (SOX10), S100b, glial fibrillary acidic protein (GFAP), P75, growth-associated protein 43 (GAP43), and early growth response 2 (EGR2) markers. The SC purity reached 87% as assessed by flow cytometry using the specific SOX10 marker, and 69% based on S100b expression. When SCs were cocultured with iPSC-derived motor neurons two-dimensionally or three-dimensionally (3D), they also expressed the markers of myelin MBP, MPZ, and gliomedin. Likewise, when they were seeded on the opposite side of a porous collagen sponge from motor neurons in the 3D model, they were able to migrate through it and colocalize with motor axons after 8 weeks of maturation. Moreover, they were shown by transmission electron microscopy to form myelin sheaths around motor axons. These results suggest that the use of autologous iPSC-derived SCs for clinical applications such as the repair of peripheral nerve damage, the treatment of spinal cord injuries, or for demyelinating diseases could be a valuable option. Impact Statement Peripheral nerve injuries can cause the complete paralysis of the upper or lower limbs, which considerably reduces the quality of life of patients. To repair this injury, many approaches have been developed by tissue engineering. Combining biomaterials with Schwann cells (SCs) has been shown to be an effective solution for stimulating nerve regeneration. However, the challenge faced concerns the strategy for obtaining autologous SCs to treat patients. A promising approach is to differentiate them from the patient's own cells, previously induced into pluripotent stem cells. We propose a fast culture method to generate functional SCs differentiated from induced pluripotent stem cells.

Gasdermin-E mediates mitochondrial damage in axons and neurodegeneration

Mitochondrial dysfunction and axon loss are hallmarks of neurologic diseases. Gasdermin (GSDM) proteins are executioner pore-forming molecules that mediate cell death, yet their roles in the central nervous system (CNS) are not well understood. Here, we find that one GSDM family member, GSDME, is expressed by both mouse and human neurons. GSDME plays a role in mitochondrial damage and axon loss. Mitochondrial neurotoxins induced caspase-dependent GSDME cleavage and rapid localization to mitochondria in axons, where GSDME promoted mitochondrial depolarization, trafficking defects, and neurite retraction. Frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS)-associated proteins TDP-43 and PR-50 induced GSDME-mediated damage to mitochondria and neurite loss. GSDME knockdown protected against neurite loss in ALS patient iPSC-derived motor neurons. Knockout of GSDME in SOD1G93A ALS mice prolonged survival, ameliorated motor dysfunction, rescued motor neuron loss, and reduced neuroinflammation. Weidentify GSDME as an executioner of neuronal mitochondrial dysfunction that may contribute to neurodegeneration.

Large-scale analyses of CAV1 and CAV2 suggest their expression is higher in post-mortem ALS brain tissue and affects survival.

Introduction: Caveolin-1 and Caveolin-2 (CAV1 and CAV2) are proteins associated with intercellular neurotrophic signalling. There is converging evidence that CAV1 and CAV2 (CAV1/2) genes have a role in amyotrophic lateral sclerosis (ALS). Disease-associated variants have been identified within CAV1/2 enhancers, which reduce gene expression and lead to disruption of membrane lipid rafts. Methods: Using large ALS whole-genome sequencing and post-mortem RNA sequencing datasets (5,987 and 365 tissue samples, respectively), and iPSC-derived motor neurons from 55 individuals, we investigated the role of CAV1/2 expression and enhancer variants in the ALS phenotype. Results: We report a differential expression analysis between ALS cases and controls for CAV1 and CAV2 genes across various post-mortem brain tissues and three independent datasets. CAV1 and CAV2 expression was consistently higher in ALS patients compared to controls, with significant results across the primary motor cortex, lateral motor cortex, and cerebellum. We also identify increased survival among carriers of CAV1/2 enhancer mutations compared to non-carriers within Project MinE and slower progression as measured by the ALSFRS. Carriers showed a median increase in survival of 345 days. Discussion: These results add to an increasing body of evidence linking CAV1 and CAV2 genes to ALS. We propose that carriers of CAV1/2 enhancer mutations may be conceptualised as an ALS subtype who present a less severe ALS phenotype with a longer survival duration and slower progression. Upregulation of CAV1/2 genes in ALS cases may indicate a causal pathway or a compensatory mechanism. Given prior research supporting the beneficial role of CAV1/2 expression in ALS patients, we consider a compensatory mechanism to better fit the available evidence, although further investigation into the biological pathways associated with CAV1/2 is needed to support this conclusion.

Epigenetic regulation of plastin 3 expression by the macrosatellite DXZ4 and the transcriptional regulator CHD4

Dysregulated Plastin 3 (PLS3) levels associate with a wide range of skeletal and neuromuscular disorders and the most common types of solid and hematopoietic cancer. Most importantly, PLS3 overexpression protects against spinal muscular atrophy. Despite its crucial role in F-actin dynamics in healthy cells and its involvement in many diseases, the mechanisms that regulate PLS3 expression are unknown. Interestingly, PLS3 is an X-linked gene and all asymptomatic SMN1-deleted individuals in SMA-discordant families who exhibit PLS3 upregulation are female, suggesting that PLS3 may escape X chromosome inactivation. To elucidate mechanisms contributing to PLS3 regulation, we performed a multi-omics analysis in two SMA-discordant families using lymphoblastoid cell lines and iPSC-derived spinal motor neurons originated from fibroblasts. We show that PLS3 tissue-specifically escapes X-inactivation. PLS3 is located ∼500 kb proximal to the DXZ4 macrosatellite, which is essential for X chromosome inactivation. By applying molecular combing in a total of 25 lymphoblastoid cell lines (asymptomatic individuals, individuals with SMA, control subjects) with variable PLS3 expression, we found a significant correlation between the copy number of DXZ4 monomers and PLS3 levels. Additionally, we identified chromodomain helicase DNA binding protein 4 (CHD4) as an epigenetic transcriptional regulator of PLS3 and validated co-regulation of the two genes by siRNA-mediated knock-down and overexpression of CHD4. We show that CHD4 binds the PLS3 promoter by performing chromatin immunoprecipitation and that CHD4/NuRD activates the transcription of PLS3 by dual-luciferase promoter assays. Thus, we provide evidence for a multilevel epigenetic regulation of PLS3 that may help to understand the protective or disease-associated PLS3 dysregulation.

Large-scale differentiation of iPSC-derived motor neurons from ALS and control subjects

Using induced pluripotent stem cells (iPSCs) to understand the mechanisms of neurological disease holds great promise; however, there is a lack of well-curated lines from a large array of participants. Answer ALS has generated over 1,000 iPSC lines from control and amyotrophic lateral sclerosis (ALS) patients along with clinical and whole-genome sequencing data. The current report summarizes cell marker and gene expression in motor neuron cultures derived from 92 healthy control and 341 ALS participants using a 32-day differentiation protocol. This is the largest set of iPSCs to be differentiated into motor neurons, and characterization suggests that cell composition and sex are significant sources of variability that need to be carefully controlled for in future studies. These data are reported as a resource for the scientific community that will utilize Answer ALS data for disease modeling using a wider array of omics being made available for these samples.

An Optimized Workflow to Generate and Characterize iPSC-Derived Motor Neuron (MN) Spheroids.

A multitude of in vitro models based on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) have been developed to investigate the underlying causes of selective MN degeneration in motor neuron diseases (MNDs). For instance, spheroids are simple 3D models that have the potential to be generated in large numbers that can be used across different assays. In this study, we generated MN spheroids and developed a workflow to analyze them. To start, the morphological profiling of the spheroids was achieved by developing a pipeline to obtain measurements of their size and shape. Next, we confirmed the expression of different MN markers at the transcript and protein levels by qPCR and immunocytochemistry of tissue-cleared samples, respectively. Finally, we assessed the capacity of the MN spheroids to display functional activity in the form of action potentials and bursts using a microelectrode array approach. Although most of the cells displayed an MN identity, we also characterized the presence of other cell types, namely interneurons and oligodendrocytes, which share the same neural progenitor pool with MNs. In summary, we successfully developed an MN 3D model, and we optimized a workflow that can be applied to perform its morphological, gene expression, protein, and functional profiling over time.

Behind sporadic ALS: A biophysical characterization of motor neurons in health and disease.

Amyotrophic lateral sclerosis (ALS) is characterized by death of motor neurons due to accumulation of aggregates and perturbation of intracellular transport. The underlying causes of these changes remain yet mysterious. Although a fraction of cases are caused by inherited mutations, the majority are sporadic, and the risk increases with age. The cell interior is extremely crowded with organelles and macromolecular complexes. This intracellular molecular crowding crucially impacts molecular assembly and transport. We hypothesized that age-associated changes could perturb macromolecular crowding and participate in the pathogenesis of ALS. We generated and leveraged genetically encoded multimeric nanoparticles (GEMs) to quantify macromolecular crowding in human iPSC-derived motor neurons, for the first time. The diffusivity of tracer particles like GEMs is generally decreased by molecular crowding and increased by active cellular processes. GEMs’ diffusivity was different depending on the subcellular localization. We observed that GEMs’ diffusivity is sensitive to the compressive state of the cell overall, while is oriented and regulated by ATP-dependent processes only in the neurites (axons and dendrites). Mechanical and osmotic pressures can increase in the aged brain. Motor neurons from ALS patients did not show modified basal molecular crowding, but might be more sensitive to mechanical perturbations. We are now studying how molecular crowding perturbations impact aggregation and transport of TDP-43, a protein found in ALS aggregates, in human motor neurons. We are performing the first high-throughput characterization of the biophysical properties of motor neurons to decipher the relationship between intracellular biophysical changes and initiation of protein aggregation in ALS.

Digital color-coded molecular barcoding reveals dysregulation of common FUS and FMRP targets in soma and neurites of ALS mutant motoneurons

Mutations in RNA binding proteins (RBPs) have been linked to the motor neuron disease amyotrophic lateral sclerosis (ALS). Extensive auto-regulation, cross-regulation, cooperation and competition mechanisms among RBPs are in place to ensure proper expression levels of common targets, often including other RBPs and their own transcripts. Moreover, several RBPs play a crucial role in the nervous system by localizing target RNAs in specific neuronal compartments. These include the RBPs FUS, FMRP, and HuD. ALS mutations in a given RBP are predicted to produce a broad impact on such delicate equilibrium. Here we studied the effects of the severe FUS-P525L mutation on common FUS and FMRP targets. Expression profiling by digital color-coded molecular barcoding in cell bodies and neurites of human iPSC-derived motor neurons revealed altered levels of transcripts involved in the cytoskeleton, neural projection and synapses. One of the common targets is HuD, which is upregulated because of the loss of FMRP binding to its 3′UTR due to mutant FUS competition. Notably, many genes are commonly altered upon FUS mutation or HuD overexpression, suggesting that a substantial part of the effects of mutant FUS on the motor neuron transcriptome could be due to HuD gain-of-function. Among altered transcripts, we also identified other common FUS and FMRP targets, namely MAP1B, PTEN, and AP2B1, that are upregulated upon loss of FMRP binding on their 3’UTR in FUS-P525L motor neurons. This work demonstrates that the impairment of FMRP function by mutant FUS might alter the expression of several genes, including new possible biomarkers and therapeutic targets for ALS.

Deep Intronic FGF14 GAA Repeat Expansion in Late-Onset Cerebellar Ataxia

BackgroundThe late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis.MethodsWe sequenced the genomes of six persons with autosomal dominant LOCA who were members of three French Canadian families and identified a candidate pathogenic repeat expansion. We then tested for association between the repeat expansion and disease in two independent case–control series — one French Canadian (66 patients and 209 controls) and the other German (228 patients and 199 controls). We also genotyped the repeat in 20 Australian and 31 Indian index patients. We assayed gene and protein expression in two postmortem cerebellum specimens and two induced pluripotent stem-cell (iPSC)–derived motor-neuron cell lines.ResultsIn the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron of FGF14, which encodes fibroblast growth factor 14. Cosegregation of the repeat expansion with disease in the families supported a pathogenic threshold of at least 250 GAA repeats ([GAA]≥250). There was significant association between FGF14 (GAA)≥250 expansions and LOCA in the French Canadian series (odds ratio, 105.60; 95% confidence interval [CI], 31.09 to 334.20; P<0.001) and in the German series (odds ratio, 8.76; 95% CI, 3.45 to 20.84; P<0.001). The repeat expansion was present in 61%, 18%, 15%, and 10% of French Canadian, German, Australian, and Indian index patients, respectively. In total, we identified 128 patients with LOCA who carried an FGF14 (GAA)≥250 expansion. Postmortem cerebellum specimens and iPSC-derived motor neurons from patients showed reduced expression of FGF14 RNA and protein.ConclusionsA dominantly inherited deep intronic GAA repeat expansion in FGF14 was found to be associated with LOCA. (Funded by Fondation Groupe Monaco and others.)

Alignment of Skeletal Muscle Cells Facilitates Acetylcholine Receptor Clustering and Neuromuscular Junction Formation with Co-Cultured Human iPSC-Derived Motor Neurons

In vitro neuromuscular junction (NMJ) models are powerful tools for studying neuromuscular disorders. Although linearly patterned culture surfaces have been reported to be useful for the formation of in vitro NMJ models using mouse motor neuron (MNs) and skeletal muscle (SkM) myotubes, it is unclear how the linearly patterned culture surface increases acetylcholine receptor (AChR) clustering, one of the steps in the process of NMJ formation, and whether this increases the in vitro NMJ formation efficiency of co-cultured human MNs and SkM myotubes. In this study, we investigated the effects of a linearly patterned culture surface on AChR clustering in myotubes and examined the possible mechanism of the increase in AChR clustering using gene expression analysis, as well as the effects of the patterned surface on the efficiency of NMJ formation between co-cultured human SkM myotubes and human iPSC-derived MNs. Our results suggest that better differentiation of myotubes on the patterned surface, compared to the flat surface, induced gene expression of integrin α7 and AChR ε-subunit, thereby increasing AChR clustering. Furthermore, we found that the number of NMJs between human SkM cells and MNs increased upon co-culture on the linearly patterned surface, suggesting the usefulness of the patterned surface for creating in vitro human NMJ models.

Novel gene-intergenic fusion involving ubiquitin E3 ligase UBE3C causes distal hereditary motor neuropathy.

Distal hereditary motor neuropathies (dHMNs) are a group of inherited diseases involving the progressive, length-dependent axonal degeneration of the lower motor neurons. There are currently 29 reported causative genes and four disease loci implicated in dHMN. Despite the high genetic heterogeneity, mutations in the known genes account for less than 20% of dHMN cases, with the mutations identified predominantly being point mutations or indels. We have expanded the spectrum of dHMN mutations with the identification of a 1.35 Mb complex structural variation (SV) causing a form of autosomal dominant dHMN (DHMN1 OMIM %182906). Given the complex nature of SV mutations and the importance of studying pathogenic mechanisms in a neuronal setting, we generated a patient-derived DHMN1 motor neuron model harbouring the 1.35 Mb complex insertion. The DHMN1 complex insertion creates a duplicated copy of the first 10 exons of the ubiquitin-protein E3 ligase gene (UBE3C) and forms a novel gene-intergenic fusion sense transcript by incorporating a terminal pseudo-exon from intergenic sequence within the DHMN1 locus. The UBE3C intergenic fusion (UBE3C-IF) transcript does not undergo nonsense-mediated decay and results in a significant reduction of wild-type full-length UBE3C (UBE3C-WT) protein levels in DHMN1 iPSC-derived motor neurons. An engineered transgenic Caenorhabditis elegans model expressing the UBE3C-IF transcript in GABA-ergic motor neurons shows neuronal synaptic transmission deficits. Furthermore, the transgenic animals are susceptible to heat stress, which may implicate defective protein homeostasis underlying DHMN1 pathogenesis. Identification of the novel UBE3C-IF gene-intergenic fusion transcript in motor neurons highlights a potential new disease mechanism underlying axonal and motor neuron degeneration. These complementary models serve as a powerful paradigm for studying the DHMN1 complex SV and an invaluable tool for defining therapeutic targets for DHMN1.

Safety and tolerability of bosutinib in patients with amyotrophic lateral sclerosis (iDReAM study): A multicentre, open-label, dose-escalation phase 1 trial.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by the loss of motor neurons, and development of effective medicines is urgently required. Induced pluripotent stem cell (iPSC)-based drug repurposing identified the Src/c-Abl inhibitor bosutinib, which is approved for the treatment of chronic myelogenous leukemia (CML), as a candidate for the molecular targeted therapy of ALS. An open-label, multicentre, dose-escalation phase 1 study using a 3+3 design was conducted in 4 hospitals in Japan to evaluate the safety and tolerability of bosutinib in patients with ALS. Furthermore, the exploratory efficacy was evaluated using Revised ALS Functional Rating Scale (ALSFRS-R), predictive biomarkers including plasma neurofilament light chain (NFL) were explored, and single-cell RNA sequencing of iPSC-derived motor neurons was conducted. Patients, whose total ALSFRS-R scores decreased by 1-3 points during the 12-week, received escalating doses starting from 100mg quaque die (QD) up to 400mg QD based on dose-limiting toxicity (DLT) occurrence, and all participants who received one dose of the study drug were included in the primary analysis. This trial is registered with ClinicalTrials.gov, NCT04744532, as Induced pluripotent stem cell-based Drug Repurposing for Amyotrophic Lateral Sclerosis Medicine (iDReAM) study. Between March 29, 2019 and May 7, 2021, 20 patients were enrolled, 13 of whom received bosutinib treatment and 12 were included in the safety and efficacy analyses. No DLTs were observed up to 300mg QD, but DLTs were observed in 3/3 patients of the 400mg QD cohort. In all patients receiving 100mg-400mg, the prevalent adverse events (AEs) were gastrointestinal AEs in 12 patients (92.3%), liver function related AEs in 7 patients (53.8%), and rash in 3 patients (23.1%). The safety profile was consistent with that known for CML treatment, and ALS-specific AEs were not observed. A subset of patients (5/9 patients) was found to respond well to bosutinib treatment over the 12-week treatment period. It was found that the treatment-responsive patients could be distinguished by their lower levels of plasma NFL. Furthermore, single-cell RNA sequencing of iPSC-derived motor neurons revealed the pathogenesis related molecular signature in patients with ALS showing responsiveness to bosutinib. This is the first trial of a Src/c-Abl inhibitor, bosutinib, for patients with ALS. The safety and tolerability of bosutinib up to 300mg, not 400mg, in ALS were described, and responsiveness of patients on motor function was observed. Since this was an open-label trial within a short period with a limited number of patients, further clinical trials will be required. AMED and iPS Cell Research Fund.

Damaged DNA Is an Early Event of Neurodegeneration in Induced Pluripotent Stem Cell-Derived Motoneurons with UBQLN2P497H Mutation.

Ubiquilin-2 (UBQLN2) mutations lead to familial amyotrophic lateral sclerosis (FALS)/and frontotemporal dementia (FTLD) through unknown mechanisms. The combination of iPSC technology and CRISPR-mediated genome editing technology can generate an iPSC-derived motor neuron (iPSC-MN) model with disease-relevant mutations, which results in increased opportunities for disease mechanism research and drug screening. In this study, we introduced a UBQLN2-P497H mutation into a healthy control iPSC line using CRISPR/Cas9, and differentiated into MNs to study the pathology of UBQLN2-related ALS. Our in vitro MN model faithfully recapitulated specific aspects of the disease, including MN apoptosis. Under sodium arsenite (SA) treatment, we found differences in the number and the size of UBQLN2+ inclusions in UBQLN2P497H MNs and wild-type (WT) MNs. We also observed cytoplasmic TAR DNA-binding protein (TARDBP, also known as TDP-43) aggregates in UBQLN2P497H MNs, but not in WT MNs, as well as the recruitment of TDP-43 into stress granules (SGs) upon SA treatment. We noted that UBQLN2-P497H mutation induced MNs DNA damage, which is an early event in UBQLN2-ALS. Additionally, DNA damage led to an increase in compensation for FUS, whereas UBQLN2-P497H mutation impaired this function. Therefore, FUS may be involved in DNA damage repair signaling.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes.

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Altered SYNJ2BP-mediated mitochondrial-ER contacts in motor neuron disease

Synaptojanin 2 binding protein (SYNJ2BP) is an outer mitochondrial membrane protein with a cytosolic PDZ domain that functions as a cellular signaling hub. Few studies have evaluated its role in disease. Here we use induced pluripotent stem cell (iPSC)-derived motor neurons and post-mortem tissue from patients with two hereditary motor neuron diseases, spinal and bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis type 4 (ALS4), and show that SYNJ2BP expression is increased in diseased motor neurons. Similarly, we show that SYNJ2BP expression increases in iPSC-derived motor neurons undergoing stress. Using proteomic analysis, we found that elevated SYNJ2BP alters the cellular distribution of mitochondria and increases mitochondrial-ER membrane contact sites. Furthermore, decreasing SYNJ2BP levels improves mitochondrial oxidative function in the diseased motor neurons. Together, our observations offer new insight into the molecular pathology of motor neuron disease and the role of SYNJ2BP in mitochondrial dysfunction.

Human iPSC co-culture model to investigate the interaction between microglia and motor neurons

Motor neuron diseases such as amyotrophic lateral sclerosis are primarily characterized by motor neuron degeneration with additional involvement of non-neuronal cells, in particular, microglia. In previous work, we have established protocols for the differentiation of iPSC-derived spinal motor neurons and microglia. Here, we combine both cell lineages and establish a novel co-culture of iPSC-derived spinal motor neurons and microglia, which is compatible with motor neuron identity and function. Co-cultured microglia express key identity markers and transcriptomically resemble primary human microglia, have highly dynamic ramifications, are phagocytically competent, release relevant cytokines and respond to stimulation. Further, they express key amyotrophic lateral sclerosis-associated genes and release disease-relevant biomarkers. This novel and authentic human model system facilitates the study of physiological motor neuron-microglia crosstalk and will allow the investigation of non-cell-autonomous phenotypes in motor neuron diseases such as amyotrophic lateral sclerosis.

P97/VCP ATPase inhibitors can rescue p97 mutation-linked motor neuron degeneration.

Mutations in p97/VCP cause two motor neuron diseases: inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia and familial amyotrophic lateral sclerosis. How p97 mutations lead to motor neuron degeneration is, however, unknown. Here we used patient-derived induced pluripotent stem cells to generate p97 mutant motor neurons. We reduced the genetic background variation by comparing mutant motor neurons to its isogenic wild type lines. Proteomic analysis reveals that p97R155H/+ motor neurons upregulate several cell cycle proteins at Day 14, but this effect diminishes by Day 20. Molecular changes linked to delayed cell cycle exit are observed in p97 mutant motor neurons. We also find that two p97 inhibitors, CB-5083 and NMS-873, restore some dysregulated protein levels. In addition, two p97 inhibitors and a food and drug administration-approved cyclin-dependent kinase 4/6 inhibitor, Abemaciclib, can rescue motor neuron death. Overall, we successfully used iPSC-derived motor neurons, identified dysregulated proteome and transcriptome and showed that p97 inhibitors rescue phenotypes in this disease model.

Aryl Hydrocarbon Receptor (AhR)-Mediated Signaling in iPSC-Derived Human Motor Neurons.

Exposure to environmental pollutants and endogenous metabolites that induce aryl hydrocarbon receptor (AhR) expression has been suggested to affect cognitive development and, particularly in boys, also motor function. As current knowledge is based on epidemiological and animal studies, in vitro models are needed to better understand the effects of these compounds in the human nervous system at the molecular level. Here, we investigated expression of AhR pathway components and how they are regulated by AhR ligands in human motor neurons. Motor neurons generated from human induced pluripotent stem cells (hiPSCs) were characterized at the molecular level and by electrophysiology. mRNA levels of AhR target genes, CYP1A1 and CYP1B1 (cytochromes P450 1A1/1B1), and AhR signaling components were monitored in hiPSCs and in differentiated neurons following treatment with AhR ligands, 2,3,7,8,-tetrachlodibenzo-p-dioxin (TCDD), L-kynurenine (L-Kyn), and kynurenic acid (KA), by RT-qPCR. Changes in AhR cellular localization and CYP1A1 activity in neurons treated with AhR ligands were also assessed. The neurons we generated express motor neuron-specific markers and are functional. Transcript levels of CYP1B1, AhR nuclear translocators (ARNT1 and ARNT2) and the AhR repressor (AhRR) change with neuronal differentiation, being significantly higher in neurons than hiPSCs. In contrast, CYP1A1 and AhR transcript levels are slightly lower in neurons than in hiPSCs. The response to TCDD treatment differs in hiPSCs and neurons, with only the latter showing significant CYP1A1 up-regulation. In contrast, TCDD slightly up-regulates CYP1B1 mRNA in hiPSCs, but downregulates it in neurons. Comparison of the effects of different AhR ligands on AhR and some of its target genes in neurons shows that L-Kyn and KA, but not TCDD, regulate AhR expression and differently affect CYP1A1 and CYP1B1 expression. Finally, although TCDD does not significantly affect AhR transcript levels, it induces AhR protein translocation to the nucleus and increases CYP1A1 activity. This is in contrast to L-Kyn and KA, which either do not affect or reduce, respectively, CYP1A1 activity. Expression of components of the AhR signaling pathway are regulated with neuronal differentiation and are differently affected by TCDD, suggesting that pluripotent stem cells might be less sensitive to this toxin than neurons. Crucially, AhR signaling is affected differently by TCDD and other AhR ligands in human motor neurons, suggesting that they can provide a valuable tool for assessing the impact of environmental pollutants.