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N-methyl-D-aspartate receptor (NMDAR) encephalitis is the most common subtype of autoimmune encephalitis characterized by a complex neuropsychiatric syndrome usually including memory impairment. Patients develop an intrathecal immune response against NMDARs with antibodies that presumably bind to the amino-terminal domain of the GluN1 subunit. The therapeutic response to immunotherapy is often delayed. Therefore, new therapeutic approaches for fast neutralization of NMDAR antibodies are needed. Here, we developed fusion constructs consisting of the Fc part of immunoglobulin G and the amino-terminal domains of either GluN1 or combinations of GluN1 with GluN2A or GluN2B. Surprisingly, both GluN1 and GluN2 subunits were required to generate high-affinity epitopes. The construct with both subunits efficiently prevented NMDAR binding of patient-derived monoclonal antibodies and of patient CSF containing high-titre NMDAR antibodies. Furthermore, it inhibited the internalization of NMDARs in rodent dissociated neurons and human induced pluripotent stem cell-derived neurons. Finally, the construct stabilized NMDAR currents recorded in rodent neurons and rescued memory defects in passive-transfer mouse models using intrahippocampal injections. Our results demonstrate that both GluN1 and GluN2B subunits contribute to the main immunogenic region of the NMDAR and provide a promising strategy for fast and specific treatment of NMDAR encephalitis, which could complement immunotherapy.

Bi-allelic mutations in GBA1, the gene that encodes β-glucocerebrosidase (GCase), cause Gaucher disease (GD), whereas mono-allelic mutations do not cause overt pathology. Yet mono- or bi-allelic GBA1 mutations are the highest known risk factor for Parkinson’s disease (PD). GCase deficiency results in the accumulation of glucosylceramide (GluCer) and its deacylated metabolite glucosylsphingosine (GluSph). Brains from patients with neuronopathic GD have high levels of GluSph, and elevation of this lipid in GBA1-associated PD has been reported. To uncover the mechanisms involved in GBA1-associated PD, we used human induced pluripotent stem cell-derived dopaminergic (DA) neurons from patients harboring heterozygote mutations in GBA1 (GBA1/PD–DA neurons). We found that compared with gene-edited isogenic controls, GBA1/PD–DA neurons exhibit mammalian target of rapamycin complex 1 (mTORC1) hyperactivity, a block in autophagy, an increase in the levels of phosphorylated α-synuclein (129) and α-synuclein aggregation. These alterations were prevented by incubation with mTOR inhibitors. Inhibition of acid ceramidase, the lysosomal enzyme that deacylates GluCer to GluSph, prevented mTOR hyperactivity, restored autophagic flux and lowered α-synuclein levels, suggesting that GluSph was responsible for these alterations. Incubation of gene-edited wild type (WT) controls with exogenous GluSph recapitulated the mTOR/α-synuclein abnormalities of GBA1/PD neurons, and these phenotypic alterations were prevented when GluSph treatment was in the presence of mTOR inhibitors. We conclude that GluSph causes an aberrant activation of mTORC1, suppressing normal lysosomal functions, including the clearance of pathogenic α-synuclein species. Our results implicate acid ceramidase in the pathogenesis of GBA1-associated PD, suggesting that this enzyme is a potential therapeutic target for treating synucleinopathies caused by GCase deficiency.

Epitranscriptomic regulation adds a layer of post-transcriptional control to brain function during development and adulthood. The identification of RNA-modifying enzymes has opened the possibility of investigating the role epitranscriptomic changes play in the disease process. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to methylate mammalian non-coding RNAs. NSun2 loss of function due to autosomal-recessive mutations has been associated with neurological abnormalities in humans. Here, we show NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex. Strikingly, we unravel decreased NSun2 protein expression and an increased ratio of pTau/NSun2 in the brains of patients with Alzheimer’s disease (AD) as demonstrated by Western blotting and immunostaining, respectively. In a well-established Drosophila melanogaster model of tau-induced toxicity, reduction of NSun2 exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. Conditional ablation of NSun2 in the mouse brain promoted a decrease in the miR-125b m6A levels and tau hyperphosphorylation. Utilizing human induced pluripotent stem cell (iPSC)-derived neuronal cultures, we confirmed NSun2 deficiency results in tau hyperphosphorylation. We also found that neuronal NSun2 levels decrease in response to amyloid-beta oligomers (AβO). Notably, AβO-induced tau phosphorylation and cell toxicity in human neurons could be rescued by overexpression of NSun2. Altogether, these results indicate that neuronal NSun2 deficiency promotes dysregulation of miR-125b and tau phosphorylation in AD and highlights a novel avenue for therapeutic targeting.

SHANK3 is a member of the SHANK family of scaffolding proteins that localize to the postsynaptic density of excitatory synapses. Mutations within the SHANK3 gene or SHANK3 haploinsufficiency is thought to be one of the major causes for Phelan-McDermid Syndrome (PMDS) that is characterized by a broad spectrum of autism-related behavioral alterations. Several approaches have already been proposed to elevate SHANK3 protein levels in PMDS patients like transcriptional activation or inhibition of SHANK3 degradation. We undertook a systematic screening approach and tested whether defined antisense oligonucleotides (ASOs) directed against the 3' untranslated region (3'-UTR) of the human SHANK3 mRNA are suitable to elevate SHANK3 protein levels. Using human induced pluripotent stem cells (hiPSCs) and hiPSCs-derived motoneurons from controls and PMDS patients we eventually identified two 18 nucleotide ASOs (ASO 4-5.2-4 and 4-5.2-6) that were able to increase SHANK3 protein levels in vitro by about 1.3- to 1.6-fold. These findings were confirmed by co-transfection of the identified ASOs with a GFP-SHANK3-3'-UTR construct in HEK293T cells using GFP protein expression as read-out. Based on these results we propose a novel approach to elevate SHANK3 protein concentrations by 3'-UTR specific ASOs. Further research is needed to test the suitability of SHANK3-specific ASOs as pharmacological compounds also in vivo.

During cellular specification, transcription factors orchestrate cellular decisions through gene regulation. By hijacking these transcriptional networks, human pluripotent stem cells (hPSCs) can be specialized into neurons with different molecular identities for the purposes of regenerative medicine and disease modeling. However, molecular fine tuning cell types to match their in vivo counterparts remains a challenge. Directing cell fates often result in blended or incomplete neuron identities. A better understanding of hPSC to neuron gene regulation is needed. Here, we used single cell RNA sequencing to resolve some of these graded molecular identities during human neurogenesis from hPSCs. Differentiation platforms were established to model neural induction from stem cells, and we characterized these differentiated cell types by 10x single cell RNA sequencing. Using single cell trajectory and co-expression analyses, we identified a co-regulated transcription factor module expressing achaete-scute family basic helix-loop-helix transcription factor 1 (ASCL1) and neuronal differentiation 1 (NEUROD1). We then tested the function of these transcription factors in neuron subtype differentiation by gene knockout in a novel human system that reports the expression of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis. ASCL1 was identified as a necessary transcription factor for regulating dopaminergic neurotransmitter selection.

Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disease marked by death of motor neurons (MNs) present in the spinal cord, brain stem and motor cortex. Despite extensive research, the reason for neurodegeneration is still not understood. To generate novel hypotheses of putative underlying molecular mechanisms, we used human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs) from SOD1- and TARDBP (TDP-43 protein)-mutant-ALS patients and healthy controls to perform high-throughput RNA-sequencing (RNA-Seq). An integrated bioinformatics approach was employed to identify differentially expressed genes (DEGs) and key pathways underlying these familial forms of the disease (fALS). In TDP43-ALS, we found dysregulation of transcripts encoding components of the transcriptional machinery and transcripts involved in splicing regulation were particularly affected. In contrast, less is known about the role of SOD1 in RNA metabolism in motor neurons. Here, we found that many transcripts relevant for mitochondrial function were specifically altered in SOD1-ALS, indicating that transcriptional signatures and expression patterns can vary significantly depending on the causal gene that is mutated. Surprisingly, however, we identified a clear downregulation of genes involved in protein translation in SOD1-ALS suggesting that ALS-causing SOD1 mutations shift cellular RNA abundance profiles to cause neural dysfunction. Altogether, we provided here an extensive profiling of mRNA expression in two ALS models at the cellular level, corroborating the major role of RNA metabolism and gene expression as a common pathomechanism in ALS.

The recent CRISPR revolution has provided researchers with powerful tools to perform genome editing in a variety of organisms. However, recent reports indicate widespread occurrence of unintended CRISPR-induced on-target effects (OnTEs) at the edited site in mice and human induced pluripotent stem cells (iPSCs) that escape standard quality controls. By altering gene expression of targeted or neighbouring genes, OnTEs can severely affect phenotypes of CRISPR-edited cells and organisms and thus lead to data misinterpretation, which can undermine the reliability of CRISPR-based studies. Here we describe a broadly applicable framework for detecting OnTEs in genome-edited cells and organisms after non-homologous end joining-mediated and homology-directed repair-mediated editing. Our protocol enables identification of OnTEs such as large deletions, large insertions, rearrangements or loss of heterozygosity (LOH). This is achieved by subjecting genomic DNA first to quantitative genotyping PCR (qgPCR), which determines the number of intact alleles at the target site using the same PCR amplicon that has been optimized for genotyping. This combination of genotyping and quantitation makes it possible to exclude clones with monoallelic OnTEs and hemizygous editing, which are often mischaracterized as correctly edited in standard Sanger sequencing. Second, occurrence of LOH around the edited locus is detected by genotyping neighbouring single-nucleotide polymorphisms (SNPs), using either a Sanger sequencing-based method or SNP microarrays. All steps are optimized to maximize simplicity and minimize cost to promote wide dissemination and applicability across the field. The entire protocol from genomic DNA extraction to OnTE exclusion can be performed in 6–9 d. CRISPR-induced on-target effects (large deletions, large insertions, rearrangements or loss of heterozygosity) occur frequently at the edited site. This protocol describes how to identify these effects using quantitative genotyping PCR and SNP genotyping.

Iron accumulation in microglia has been observed in Alzheimer's disease and other neurodegenerative disorders and is thought to contribute to disease progression through various mechanisms, including neuroinflammation. To study this interaction, we treated human induced pluripotent stem cell-derived microglia (iPSC-MG) with iron, in combination with inflammatory stimuli such as interferon gamma (IFN-gamma) and amyloid beta. Both IFN-gamma and iron treatment increased labile iron levels, but only iron treatment led to a consistent increase of ferritin levels, reflecting long-term iron storage. Therefore, in iPSC-MG, ferritin appeared to be regulated by iron revels rather than inflammation. Further investigation showed that while IFN-gamma induced pro-inflammatory activation, iron treatment dampened both classic pro- and anti-inflammatory activation on a transcriptomic level. Notably, iron-loaded microglia showed strong upregulation of cellular stress response pathways, the NRF2 pathway, and other oxidative stress pathways. Functionally, iPSC-MG exhibited altered phagocytosis and impaired mitochondrial metabolism following iron treatment. Collectively, these data suggest that in MG, in contrast to current hypotheses, iron treatment does not result in pro-inflammatory activation, but rather dampens it and induces oxidative stress.

Little is known about the early pathogenic events by which mutant superoxide dismutase 1 (SOD1) causes amyotrophic lateral sclerosis (ALS). This lack of mechanistic understanding is a major barrier to the development and evaluation of efficient therapies. Although protein aggregation is known to be involved, it is not understood how mutant SOD1 causes degeneration of motoneurons (MNs). Previous research has relied heavily on the overexpression of mutant SOD1, but the clinical relevance of SOD1 overexpression models remains questionable. We used a human induced pluripotent stem cell (iPSC) model of spinal MNs and three different endogenous ALS-associated SOD1 mutations (D90Ahom, R115Ghet or A4Vhet) to investigate early cellular disturbances in MNs. Although enhanced misfolding and aggregation of SOD1 was induced by proteasome inhibition, Cells 2022, it was not affected by activation of the stress granule pathway. Interestingly, we identified loss of mitochondrial, but not lysosomal, integrity as the earliest common pathological phenotype, which preceded elevated levels of insoluble, aggregated SOD1. A super-elongated mitochondrial morphology with impaired inner mitochondrial membrane potential was a unifying feature in mutant SOD1 iPSC-derived MNs. Impaired mitochondrial integrity was most prominent in mutant D90Ahom MNs, whereas both soluble disordered and detergent-resistant misfolded SOD1 was more prominent in R115Ghet and A4Vhet mutant lines. Taking advantage of patient-specific models of SOD1-ALS in vitro, our data suggest that mitochondrial dysfunction is one of the first crucial steps in the pathogenic cascade that leads to SOD1-ALS and also highlights the need for individualized medical approaches for SOD1-ALS.

Summary In amyotrophic lateral sclerosis (ALS) motor neurons (MNs) undergo dying-back, where the distal axon degenerates before the soma. The hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of ALS, but the mechanism of pathogenesis is largely unknown with both gain- and loss-of-function mechanisms being proposed. To better understand C9ORF72-ALS pathogenesis, we generated isogenic induced pluripotent stem cells. MNs with HRE in C9ORF72 showed decreased axonal trafficking compared with gene corrected MNs. However, knocking out C9ORF72 did not recapitulate these changes in MNs from healthy controls, suggesting a gain-of-function mechanism. In contrast, knocking out C9ORF72 in MNs with HRE exacerbated axonal trafficking defects and increased apoptosis as well as decreased levels of HSP70 and HSP40, and inhibition of HSPs exacerbated ALS phenotypes in MNs with HRE. Therefore, we propose that the HRE in C9ORF72 induces ALS pathogenesis via a combination of gain- and loss-of-function mechanisms., Highlights • Axonal trafficking is disrupted in MNs with hexanucleotide repeat expansion (HRE) • C9ORF72 knockout (KO) exacerbated phenotypes in MNs with HRE • C9ORF72 KO reduced heat shock proteins in MNs with HRE • Inhibition of heat shock proteins exacerbated ALS phenotypes in MNs with HRE, Sterneckert and colleagues generated isogenic induced pluripotent stem cell lines and demonstrated that MNs with hexanucleotide repeat expansion (HRE) in C9ORF72 show reduced axonal trafficking, which is not recapitulated by knocking out C9ORF72 in MNs from healthy individuals. In contrast, knocking out C9ORF72 exacerbated phenotypes in MNs with HRE, suggesting that both gain- and loss-of-function mechanisms contribute to C9ORF72-ALS.

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient-derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, results in 12% correction of the inversion and restores Factor VIII mRNA expression. In this work, we present designer-recombinases as an efficient and specific means towards treatment of monogenic diseases caused by large gene inversions., Correction of disease-causing large genomic inversions remains challenging. Here, the authors developed a dual designer-recombinase system (RecF8) that efficiently corrects a 140 kb inversion frequently found in patients with severe Hemophilia A.

Optimizing drug candidates for blood-brain barrier (BBB) penetration remains one of the key challenges in drug discovery to finally target brain disorders including neurodegenerative diseases which do not have adequate treatments so far. It has been difficult to establish state-of-the-art stem cell derived in vitro models that mimic physiological barrier properties including a 3D microvasculature in a format that is scalable to screen drugs for BBB penetration. To address this challenge, we established human induced pluripotent stem cell (iPSC)-derived brain endothelial microvessels in a standardized and scalable multi-well plate format. iPSC-derived brain microvascular endothelial cells (BMECs) were supplemented with primary cell conditioned media and grew to microvessels in 10 days. Produced microvessels show typical BBB endothelial protein expression, tight-junctions and polarized localization of efflux transporter. Microvessels exhibited physiological relevant trans-endothelial electrical resistance (TEER), were leak-tight for 10 kDa dextran-Alexa 647 and strongly limited the permeability of sodium fluorescein (NaF). Permeability tests with reference compounds confirmed the suitability of our model as platform to identify potential BBB penetrating anti-inflammatory drugs. The here presented platform recapitulates physiological properties and allows rapid screening of BBB permeable anti-inflammatory compounds that has been suggested as promising substances to cure so far untreatable neurodegenerative diseases.

Niemann–Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder caused by autosomal recessive mutations in the NPC1 gene. Patients display a wide spectrum on the clinical as well as on the molecular level, wherein a so-called “variant” biochemical phenotype can be observed. Here, we report an in vitro analysis of fibroblasts obtained from an NP-C1 patient carrying the undescribed compound heterozygous mutation p.V1023Sfs*15/p.G992R. Since NP-C1 is a neurovisceral disease and the patient suffers from severe neurological as well as hepatic symptoms, we extended our study to neural differentiated and hepatocyte-like cells derived from patient-specific induced pluripotent stem cells. We detected slightly increased intracellular cholesterol levels compared to the control cell line in fibroblasts, neural differentiated and hepatocyte-like cells, suggesting a “variant” biochemical phenotype. Furthermore, the total NPC1 protein, as well as post-ER glycoforms of the NPC1 protein, tended to be reduced. In addition, colocalization analysis revealed a mild reduction of the NPC1 protein in the lysosomes. The patient was diagnosed with NP-C1 at the age of 34 years, after an initial misdiagnosis of schizophrenia. After years of mild and unspecific symptoms, such as difficulties in coordination and concentration, symptoms progressed and the patient finally presented with ataxia, dysarthria, dysphagia, vertical supranuclear gaze palsy, and hepatosplenomegaly. Genetic testing finally pointed towards an NP-C1 diagnosis, revealing the so-far undescribed compound heterozygous mutation p.V1023Sfs*15/p.G992R in the NPC1 gene. In light of these findings, this case provides support for the p.G992R mutation being causative for a “variant” biochemical phenotype leading to an adult-onset type of NP-C1 disease.

CRISPR prime-editors are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering or DNA base-editors. However, sufficient prime-editor expression levels and availability of optimized transfection protocols may affect editing efficiencies, especially in hard-to-transfect cells like hiPSC. Here, we show that piggyBac prime-editing (PB-PE) allows for sustained expression of prime-editors. We demonstrate proof-of-concept for PB-PE in a newly designed lentiviral traffic light reporter, which allows for estimation of gene correction and defective editing resulting in indels, based on expression of two different fluorophores. PB-PE can prime-edit more than 50% of hiPSC cells after antibiotic selection. We also show that improper design of pegRNA cannot simply be overcome by extended expression, but PB-PE allows for estimation of effectiveness of selected pegRNAs after few days of cultivation time. Finally, we implemented PB-PE for efficient editing of an amyotrophic lateral sclerosis-associated mutation in the SOD1-gene of patient-derived hiPSC. Progress of genome editing can be monitored by Sanger-sequencing, whereas PB-PE vectors can be removed after editing and excised cells can be enriched by fialuridine selection. Together, we present an efficient prime-editing toolbox, which can be robustly used in a variety of cell lines even when non-optimized transfection-protocols are applied.

Current treatments for Parkinson's disease (PD) provide only symptomatic relief, with no disease-modifying therapies identified to date. Repurposing FDA-approved drugs to treat PD could significantly shorten the time needed for and reduce the costs of drug development compared with conventional approaches. We developed an efficient strategy to screen for modulators of β-glucocerebrosidase (GCase), a lysosomal enzyme that exhibits decreased activity in patients with PD, leading to accumulation of the substrate glucosylceramide and oxidized dopamine and α-synuclein, which contribute to PD pathogenesis. Using a GCase fluorescent probe and affinity-based fluorescence polarization assay, we screened 1280 structurally diverse, bioactive, and cell-permeable FDA-approved drugs and found that the antipsychotic quetiapine bound GCase with high affinity. Moreover, quetiapine treatment of induced pluripotent stem cell-derived (iPSC-derived) dopaminergic neurons from patients carrying mutations in GBA1 or LRRK2 led to increased wild-type GCase protein levels and activity and partially lowered accumulation of oxidized dopamine, glucosylceramide, and α-synuclein. Similarly, quetiapine led to activation of wild-type GCase and reduction of α-synuclein in a GBA mutant mouse model (Gba1D409V/+ mice). Together, these results suggest that repurposing quetiapine as a modulator of GCase may be beneficial for patients with PD exhibiting decreased GCase activity.

Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a rare childhood onset neurodegenerative disease caused by mutations in the FA2H gene. Patients display abnormal myelination, cerebellar atrophy and some have iron deposition in the central nervous system. Here we describe the generation of AKOSi010-A, a human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a female patient carrying the compound heterozygous p.Gly45Arg/p.His319Arg, using non-integrating Sendai virus. The generated iPSCs express pluripotency markers, can differentiate into cell types of the three germ layers and show a normal karyotype. This cell line displays a unique source to study the pathophysiology of FAHN.

Niemann-Pick type C2 (NP-C2) disease is a rare hereditary disease caused by mutations in the NPC2 gene. NPC2 is a small, soluble protein consisting of 151 amino acids, primarily expressed in late endosomes and lysosomes (LE/LY). Together with NPC1, a transmembrane protein found in these organelles, NPC2 accomplishes the exclusion of cholesterol; thus, both proteins are essential to maintain cellular cholesterol homeostasis. Consequently, mutations in the NPC2 or NPC1 gene result in pathophysiological accumulation of cholesterol and sphingolipids in LE/LY. The vast majority of Niemann-Pick type C disease patients, 95%, suffer from a mutation of NPC1, and only 5% display a mutation of NPC2. The biochemical phenotype of NP-C1 and NP-C2 appears to be indistinguishable, and both diseases share several commonalities in the clinical manifestation. Studies of the pathological mechanisms underlying NP-C2 are mostly based on NP-C2 animal models and NP-C2 patient-derived fibroblasts. Recently, we established induced pluripotent stem cells (iPSCs), derived from a donor carrying the NPC2 mutations c.58G>T/c.140G>T. Here, we present a profile of pathophysiological in vitro features, shared by NP-C1 and NP-C2, of neural differentiated cells obtained from the patient specific iPSCs. Profiling comprised a determination of the NPC2 protein level, detection of cholesterol accumulation by filipin staining, analysis of oxidative stress, and determination of autophagy. As expected, the NPC2-deficient cells displayed a significantly reduced amount of NPC2 protein, and, accordingly, we observed a significantly increased amount of cholesterol. Most notably, NPC2-deficient cells displayed only a slight increase of reactive oxygen species (ROS), suggesting that they do not suffer from oxidative stress and express catalase at a high level. As a site note, comparable NPC1-deficient cells suffer from a lack of catalase and display an increased level of ROS. In summary, this cell line provides a valuable tool to gain deeper understanding, not only of the pathogenic mechanism of NP-C2, but also of NP-C1.

Cell reports 36(11), 109697 (2021). doi:10.1016/j.celrep.2021.109697, Published by Elsevier, [New York, NY]

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions., Truong et al. developed a cell-based reporter system, EXSISERS, that enables non-invasive quantification of the protein expression levels of exon-specific isoforms via intein-mediated protein splicing.

Despite its decades' long therapeutic use in psychiatry, the biological mechanisms underlying lithium's mood-stabilizing effects have remained largely elusive. Here, we investigated the effect of lithium on tryptophan breakdown via the kynurenine pathway using immortalized human microglia cells, primary human microglia isolated from surgical specimens, and microglia-like cells differentiated from human induced pluripotent stem cells. Interferon (IFN)-gamma, but not lipopolysaccharide, was able to activate immortalized human microglia, inducing a robust increase in indoleamine-2,3-dioxygenase (IDO1) mRNA transcription, IDO1 protein expression, and activity. Further, chromatin immunoprecipitation verified enriched binding of both STAT1 and STAT3 to the IDO1 promoter. Lithium counteracted these effects, increasing inhibitory GSK3 beta(S9) phosphorylation and reducing STAT1(S727) and STAT3(Y705) phosphorylation levels in IFN-gamma treated cells. Studies in primary human microglia and hiPSC-derived microglia confirmed the anti-inflammatory effects of lithium, highlighting that IDO activity is reduced by GSK3 inhibitor SB-216763 and STAT inhibitor nifuroxazide via downregulation of P-STAT1(S727) and P-STAT3(Y705). Primary human microglia differed from immortalized human microglia and hiPSC derived microglia-like cells in their strong sensitivity to LPS, resulting in robust upregulation of IDO1 and anti-inflammatory cytokine IL-10. While lithium again decreased IDO1 activity in primary cells, it further increased release of IL-10 in response to LPS. Taken together, our study demonstrates that lithium inhibits the inflammatory kynurenine pathway in the microglia compartment of the human brain.

International audience; For some time, it has been accepted that the β-site APP cleaving enzyme 1 (BACE1) and the γ-secretase are two main players in the amyloidogenic processing of the β-amyloid precursor protein (APP). Recently, the membrane-type 5 matrix metalloproteinase (MT5-MMP/MMP-24), mainly expressed in the nervous system, has been highlighted as a new key player in APP-processing, able to stimulate amyloidogenesis and also to generate a neurotoxic APP derivative. In addition, the loss of MT5-MMP has been demonstrated to abrogate pathological hallmarks in a mouse model of Alzheimer’s disease (AD), thus shedding light on MT5-MMP as an attractive new therapeutic target. However, a more comprehensive analysis of the role of MT5-MMP is necessary to evaluate how its targeting affects neurons and glia in pathological and physiological situations. In this study, leveraging on CRISPR-Cas9 genome editing strategy, we established cultures of human-induced pluripotent stem cells (hiPSC)-derived neurons and astrocytes to investigate the impact of MT5-MMP deficiency on their phenotypes. We found that MT5-MMP-deficient neurons exhibited an increased number of primary and secondary neurites, as compared to isogenic hiPSC-derived neurons. Moreover, MT5-MMP-deficient astrocytes displayed higher surface area and volume compared to control astrocytes. The MT5-MMP-deficient astrocytes also exhibited decreased GLAST and S100β expression. These findings provide novel insights into the physiological role of MT5-MMP in human neurons and astrocytes, suggesting that therapeutic strategies targeting MT5-MMP should be controlled for potential side effects on astrocytic physiology and neuronal morphology

An intronic (G4C2)n expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia primarily through gain-of-function mechanisms: the accumulation of sense and antisense repeat RNA foci and dipeptide repeat (DPR) proteins (poly-GA/GP/GR/PA/PR) translated from repeat RNA. To therapeutically block this pathway, we screen a library of 1,430 approved drugs and known bioactive compounds in patient-derived induced pluripotent stem cell-derived neurons (iPSC-Neurons) for inhibitors of DPR expression. The clinically used guanosine/cytidine analogs decitabine, entecavir, and nelarabine reduce poly-GA/GP expression, with decitabine being the most potent. Hit compounds nearly abolish sense and antisense RNA foci and reduce expression of the repeat-containing nascent C9orf72 RNA transcript and its mature mRNA with minimal effects on global gene expression, suggesting that they specifically act on repeat transcription. Importantly, decitabine treatment reduces (G4C2)n foci and DPRs in C9orf72 BAC transgenic mice. Our findings suggest that nucleoside analogs are a promising compound class for therapeutic development in C9orf72 repeat-expansion-associated disorders.

Oxidative stress (OS) represents a state of an imbalanced amount of reactive oxygen species (ROS) and/or a hampered efficacy of the antioxidative defense system. Cells of the central nervous system are particularly sensitive to OS, as they have a massive need of oxygen to maintain proper function. Consequently, OS represents a common pathophysiological hallmark of neurodegenerative diseases and is discussed to contribute to the neurodegeneration observed amongst others in Alzheimer&rsquo, s disease and Parkinson&rsquo, s disease. In this context, accumulating evidence suggests that OS is involved in the pathophysiology of Niemann-Pick type C1 disease (NPC1). NPC1, a rare hereditary neurodegenerative disease, belongs to the family of lysosomal storage disorders. A major hallmark of the disease is the accumulation of cholesterol and other glycosphingolipids in lysosomes. Several studies describe OS both in murine in vivo and in vitro NPC1 models. However, studies based on human cells are limited to NPC1 patient-derived fibroblasts. Thus, we analyzed OS in a human neuronal model based on NPC1 patient-specific induced pluripotent stem cells (iPSCs). Higher ROS levels, as determined by DCF (dichlorodihydrofluorescein) fluorescence, indicated oxidative stress in all NPC1-deficient cell lines. This finding was further supported by reduced superoxide dismutase (SOD) activity. The analysis of mRNA and protein levels of SOD1 and SOD2 did not reveal any difference between control cells and NPC1-deficient cells. Interestingly, we observed a striking decrease in catalase mRNA and protein levels in all NPC1-deficient cell lines. As catalase is a key enzyme of the cellular antioxidative defense system, we concluded that the lack of catalase contributes to the elevated ROS levels observed in NPC1-deficient cells. Thus, a restitution of a physiological catalase level may pose an intervention strategy to rescue NPC1-deficient cells from the repercussions of oxidative stress contributing to the neurodegeneration observed in NPC1.

Wilson disease (WD) is a rare, monogenic disorder caused by mutations in the gene ATP7B. A loss of function of the expressed protein leads to excessive hepatic and cerebral copper storage. In this study, we present the generation of two induced pluripotent stem cell (iPSC) lines derived from fibroblasts of a clinically asymptomatic, chelator treated female WD patient carrying the common missense mutation p.H1069Q and an age-matched female healthy control subject. The generated iPSC lines expressed pluripotency markers, showed differentiation potential and retained their parental genotype. Therefore, these cells provide a valuable resource to understand the pathophysiology of WD and can be used as model systems for drug testing.

Wilson disease (WD) is an inherited, autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene. Pathogenic single nucleotide variants (SNVs) lead to functional impairment of the copper transporting ATPase ATP7B, resulting in copper accumulation and toxicity in the liver and brain. We describe the generation of two induced pluripotent stem cell (iPSC) lines derived from fibroblasts of two female WD patients. Patient 1 is compound heterozygous for p.E1064A and p.H1069Q. Patient 2 is homozygous for p.M769V. These iPSCs represent a WD model for pathophysiological studies and pharmacological screening.

Chorea acanthocytosis (ChAc), an ultra-rare devastating neurodegenerative disease, is caused by mutations in the VPS13A gene, which encodes for the protein chorein. Affected patients suffer from chorea, orofacial dyskinesia, epilepsy, parkinsonism as well as peripheral neuropathy. Although medium spinal neurons of the striatum are mainly affected, other regions are impaired as well over the course of the disease. Animal studies as well as studies on human erythrocytes suggest Lyn-kinase inhibition as valuable novel opportunity to treat ChAc. In order to investigate the peripheral neuropathy aspect, we analyzed induced pluripotent stem cell derived midbrain/hindbrain cell cultures from ChAc patients in vitro. We observed dendritic microtubule fragmentation. Furthermore, by using in vitro live cell imaging, we found a reduction in the number of lysosomes and mitochondria, shortened mitochondria, an increase in retrograde transport and hyperpolarization as measured with the fluorescent probe JC-1. Deep phenotyping pointed towards a proximal axonal deterioration as the primary axonal disease phenotype. Interestingly, pharmacological interventions, which proved to be successful in different models of ChAc, were ineffective in treating the observed axonal phenotypes. Our data suggests that treatment of this multifaceted disease might be cell type and/or neuronal subtype specific, and thus necessitates precision medicine in this ultra-rare disease.

Mutations in the VPS13A gene leading to depletion of chorein protein are causative for Chorea Acanthocytosis (ChAc), a rare devastating disease, which is characterized by neurodegeneration mainly affecting the basal ganglia as well as deformation of erythrocytes. Studies on patient blood samples highlighted a dysregulation of Actin cytoskeleton caused by downregulation of the PI3K pathway and hyper-activation of Lyn-kinase, but to what extent these mechanisms are present and relevant in the affected neurons remains elusive. We studied the effects of the absence of chorein protein on the morphology and trafficking of lysosomal and mitochondrial compartments in ChAc patient-specific induced pluripotent stem cell-derived medium spiny neurons (MSNs). Numbers of both organelle types were reduced in ChAc MSNs. Mitochondrial length was shortened and their membrane potential showed significant hyperpolarization. In contrast to previous studies, showing Lyn kinase dependency of ChAc-associated pathological events in erythrocytes, pharmacological studies demonstrate that the impairment of mitochondria and lysosomes are independent of Lyn kinase activity. These data suggest that impairment in mitochondrial and lysosomal morphologies in MSNs is not mediated by a dysregulation of Lyn kinase and thus the pathological pathways in ChAc might be – at least in part – cell-type specific.

An induced pluripotent stem cell line, HIHCNi003-A (iPSC-ALSP), was created from a skin biopsy of a patient with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) caused by a heterozygous c.2512G>C, p.Val838Leu mutation in the CSF1R gene. Skin fibroblasts were reprogrammed using episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC, and hLIN28. The iPSC-ALSP line exhibits chromosomal stability with conservation of the CSF1R mutation, expresses pluripotency markers and differentiates into endo-, meso-, and ectodermal cells in vitro.

Summary Perturbations in stress granule (SG) dynamics may be at the core of amyotrophic lateral sclerosis (ALS). Since SGs are membraneless compartments, modeling their dynamics in human motor neurons has been challenging, thus hindering the identification of effective therapeutics. Here, we report the generation of isogenic induced pluripotent stem cells carrying wild-type and P525L FUS-eGFP. We demonstrate that FUS-eGFP is recruited into SGs and that P525L profoundly alters their dynamics. With a screening campaign, we demonstrate that PI3K/AKT/mTOR pathway inhibition increases autophagy and ameliorates SG phenotypes linked to P525L FUS by reducing FUS-eGFP recruitment into SGs. Using a Drosophila model of FUS-ALS, we corroborate that induction of autophagy significantly increases survival. Finally, by screening clinically approved drugs for their ability to ameliorate FUS SG phenotypes, we identify a number of brain-penetrant anti-depressants and anti-psychotics that also induce autophagy. These drugs could be repurposed as potential ALS treatments., Highlights • Generation of isogenic WT and P525L FUS-eGFP reporter iPSCs • P525L FUS-eGFP SGs are more numerous, more intense, and larger than WT • Increasing PI3K/AKT/mTOR-regulated autophagy reduces FUS-eGFP recruitment to SGs • Brain-penetrant drugs that induce autophagy ameliorate the FUS SG phenotype, Sterneckert and colleagues generate isogenic FUS-eGFP reporter iPSCs that enable the identification of stress granule (SG) phenotypes specifically induced by the ALS mutation FUS P525L. Compound screening shows that modulation of the PI3K/AKT/mTOR pathway regulating autophagy ameliorates SG phenotypes. A second screen identifies similarly acting brain-penetrant US FDA-approved drugs that could be repurposed to treat ALS.

Established disease models have helped unravel the mechanistic underpinnings of pathological phenotypes in Parkinson’s disease (PD), the second most common neurodegenerative disorder. However, these discoveries have been limited to relatively simple cellular systems and animal models, which typically manifest with incomplete or imperfect recapitulation of disease phenotypes. The advent of induced pluripotent stem cells (iPSCs) has provided a powerful scientific tool for investigating the underlying molecular mechanisms of both familial and sporadic PD within disease-relevant cell types and patient-specific genetic backgrounds. Overwhelming evidence supports mitochondrial dysfunction as a central feature in PD pathophysiology, and iPSC-based neuronal models have expanded our understanding of mitochondrial dynamics in the development and progression of this devastating disorder. The present review provides a comprehensive assessment of mitochondrial phenotypes reported in iPSC-derived neurons generated from PD patients’ somatic cells, with an emphasis on the role of mitochondrial respiration, morphology, and trafficking, as well as mitophagy and calcium handling in health and disease. Furthermore, we summarize the distinguishing characteristics of vulnerable midbrain dopaminergic neurons in PD and report the unique advantages and challenges of iPSC disease modeling at present, and for future mechanistic and therapeutic applications.

Leucine-rich repeat kinase 2 (LRRK2), a multi-domain protein, is a key causative factor in Parkinson's disease (PD). Identification of novel substrates and the molecular mechanisms underlying the effects of LRRK2 are essential for understanding the pathogenesis of PD. In this study, we showed that LRRK2 played an important role in neuronal cell death by directly phosphorylating and activating apoptosis signal-regulating kinase 1 (ASK1). LRRK2 phosphorylated ASK1 at Thr832 that is adjacent to Thr845, which serves as an autophosphorylation site. Moreover, results of binding and kinase assays showed that LRRK2 acted as a scaffolding protein by interacting with each components of the ASK1-MKK3/6-p38 MAPK pathway through its specific domains and increasing the proximity to downstream targets. Furthermore, LRRK2-induced apoptosis was suppressed by ASK1 inhibition in neuronal stem cells derived from patients with PD. These results clearly indicate that LRRK2 acts as an upstream kinase in the ASK1 pathway and plays an important role in the pathogenesis of PD.

Mutations in the GBA gene, encoding lysosomal glucocerebrosidase, represent the major predisposing factor for Parkinson’s disease (PD), and modulation of the glucocerebrosidase activity is an emerging PD therapy. However, little is known about mechanisms regulating GBA expression. We explored the existence of a regulatory network involving GBA, its expressed pseudogene GBAP1, and microRNAs. The high level of sequence identity between GBA and GBAP1 makes the pseudogene a promising competing-endogenous RNA (ceRNA), functioning as a microRNA sponge. After selecting microRNAs potentially targeting both transcripts, we demonstrated that miR-22-3p binds to and down-regulates GBA and GBAP1, and decreases their endogenous mRNA levels up to 70%. Moreover, over-expression of GBAP1 3′-untranslated region was able to sequester miR-22-3p, thus increasing GBA mRNA and glucocerebrosidase levels. The characterization of GBAP1 splicing identified multiple out-of-frame isoforms down-regulated by the nonsense-mediated mRNA decay, suggesting that GBAP1 levels and, accordingly, its ceRNA effect, are significantly modulated by this degradation process. Using skin-derived induced pluripotent stem cells of PD patients with GBA mutations and controls, we observed a significant GBA up-regulation during dopaminergic differentiation, paralleled by down-regulation of miR-22-3p. Our results describe the first microRNA controlling GBA and suggest that the GBAP1 non-coding RNA functions as a GBA ceRNA.

Ataxin-3, the disease protein in Machado-Joseph disease, is known to be proteolytically modified by various enzymes including two major families of proteases, caspases and calpains. This processing results in the generation of toxic fragments of the polyglutamine-expanded protein. Although various approaches were undertaken to identify cleavage sites within ataxin-3 and to evaluate the impact of fragments on the molecular pathogenesis of Machado-Joseph disease, calpain-mediated cleavage of the disease protein and the localization of cleavage sites remained unclear. Here, we report on the first precise localization of calpain cleavage sites in ataxin-3 and on the characterization of the resulting breakdown products. After confirming the occurrence of calpain-derived fragmentation of ataxin-3 in patient-derived cell lines and post-mortem brain tissue, we combined in silico prediction tools, western blot analysis, mass spectrometry, and peptide overlay assays to identify calpain cleavage sites. We found that ataxin-3 is primarily cleaved at two sites, namely at amino acid positions D208 and S256 and mutating amino acids at both cleavage sites to tryptophan nearly abolished ataxin-3 fragmentation. Furthermore, analysis of calpain cleavage-derived fragments showed distinct aggregation propensities and toxicities of C-terminal polyglutamine-containing breakdown products. Our data elucidate the important role of ataxin-3 proteolysis in the pathogenesis of Machado-Joseph disease and further emphasize the relevance of targeting this disease pathway as a treatment strategy in neurodegenerative disorders.

Axonal degeneration is a key pathology of neurodegenerative diseases, including hereditary spastic paraplegia (HSP), a disorder characterized by spasticity in the lower limbs. Treatments for HSP and other neurodegenerative diseases are mainly symptomatic. While iPSC-derived neurons are valuable for drug discovery and target identification, these applications require robust differentiation paradigms and rapid phenotypic read-outs ranging between hours and a few days. Using spastic paraplegia type 4 (SPG4, the most frequent HSP subtype) as an exemplar, we here present three rapid phenotypic assays for uncovering neuronal process pathologies in iPSC-derived glutamatergic cortical neurons. Specifically, these assays detected a 51% reduction in neurite outgrowth and a 60% increase in growth cone area already 24 hours after plating; axonal swellings, a hallmark of HSP pathology, was discernible after only 5 days. Remarkably, the identified phenotypes were neuron subtype-specific and not detectable in SPG4-derived GABAergic forebrain neurons. We transferred all three phenotypic assays to a 96-well setup, applied small molecules and found that a liver X receptor (LXR) agonist rescued all three phenotypes in HSP neurons, providing a potential drug target for HSP treatment. We expect this multiparametric and rapid phenotyping approach to accelerate development of therapeutic compounds for HSP and other neurodegenerative diseases.

Niemann-Pick disease Type C (NPC) is a rare progressive neurodegenerative disorder with an incidence of 1:120,000 caused by mutations in the NPC1 or NPC2 gene. Only 5% of NPC patients suffer from mutations of the NPC2 gene. Here we demonstrate the generation of a Niemann-Pick disease Type C2 (NPC2) patient-derived induced pluripotent stem cell line. This cell line is capable to differentiate into derivatives of the neuronal lineage, providing a valuable tool to study pathogenic mechanisms of NPC2.

Summary How mutations in the microtubule-associated protein tau (MAPT) gene cause frontotemporal dementia (FTD) remains poorly understood. We generated and characterized multiple induced pluripotent stem cell (iPSC) lines from patients with MAPT IVS10+16 and tau-A152T mutations and a control subject. In cortical neurons differentiated from these and other published iPSC lines, we found that MAPT mutations do not affect neuronal differentiation but increase the 4R/3R tau ratio. Patient neurons had significantly higher levels of MMP-9 and MMP-2 and were more sensitive to stress-induced cell death. Inhibitors of MMP-9/MMP-2 protected patient neurons from stress-induced cell death and recombinant MMP-9/MMP-2 were sufficient to decrease neuronal survival. In tau-A152T neurons, inhibition of the ERK pathway decreased MMP-9 expression. Moreover, ectopic expression of 4R but not 3R tau-A152T in HEK293 cells increased MMP-9 expression and ERK phosphorylation. These findings provide insights into the molecular pathogenesis of FTD and suggest a potential therapeutic target for FTD with MAPT mutations., Highlights • New iPSC lines with MAPT p.A152T and IVS10+16 mutations are generated • MMP-2 and MMP-9 production is elevated in human neurons with MAPT mutations • Elevated MMP-2 and MMP-9 contribute to stress-induced neuronal cell death • Mutant tau increases ERK phosphorylation that activates MMP-9 expression, In this article, Gao and colleagues generated multiple iPSC lines from patients with MAPT IVS10+16 and tau-A152T mutations and found that significantly higher levels of MMP-9/MMP-2 in patient neurons contribute to stress-induced neuronal cell death. 4R but not 3R tau-A152T activates ERK, which in turn increases MMP-9 expression. These results provide insights into the molecular pathogenesis of FTD.

Gaucher disease is the most common autosomal recessive lysosomal storage disorder, caused by mutations in the β-glucocerebrosidase gene GBA. Here we describe generation of iPSC from skin-derived fibroblasts from two unrelated individuals with neuronopathic forms of Gaucher disease. The donor for line iPSC-GBA-1, a 21 month old girl, carried the recurring GBA mutation c.1448 T > C, p.Leu483Pro at homozygous state; fibroblasts for line iPS-GBA-2 were obtained from a 4 year old girl compound heterozygous for the GBA mutations c.667 T > C, p.Trp223Arg and c.1226A > G, p.Asn409Ser. iPSCs were developed using integration free episomal vectors (OCT4, KLF4; L-MYC, SOX2 (OSKM) and LIN28).Resource tableUnlabelled TableUnique stem cell lines identifierHIHRSi001-AHIHRSi002-AAlternative names of stem cell linesiPSC-GBA-1 (HIHRSi001-A)iPSC-GBA-2 (HIHRSi002-A)InstitutionHertie Institute for Clinical Brain Research and German Center for Neurodegenerative Diseases (DZNE), Tübingen, GermanyContact information of distributorRebecca Schülerebecca.schuele-freyer@uni-tuebingen.deType of cell linesInduced pluripotent stem cell (iPSC)OriginHumanCell sourceFibroblastsClonalityClonalMethod of reprogrammingNon-integrating episomal plasmidsMultiline rationaleTwo cell lines carrying individual GBA mutationsGene modificationYesType of modificationinherited mutationAssociated diseaseGaucher Disease, neuronopathic (OMIM # 230900, 23100)Gene/locusGBAiPSC-GBA-1: NM_000157.3(GBA): c.[1448 T > C]; [1448 T > C] | p.[Leu483Pro]; [Leu483Pro] (originally published as Leu444Pro (Tsuji et al., 1987))iPSC-GBA-2: NM_000157.3(GBA): c.[667 T > C];[1226A > G] | p.[Trp223Arg];[Asn409Ser] (Trp223Arg originally published as Trp184Arg (Choy et al., 2000))Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateJune 2017Cell line repository/bankN/AEthical approvalInstitutional Review Board (“Ethikkommission”) University of Tübingen Medical School, Germany, approval number 819/2016A (2016/12/21)

Alzheimer's disease (AD) is most the frequent neurodegenerative disease, and the APOE ε4 allele is the most prominent risk factor for late-onset AD. Here, we present an iPSC line generated from peripheral blood cells of a male AD patient employing Sendai virus vectors encoding the transcription factors OCT4, SOX2, KLF4 and c-MYC. The characterized iPSC line expresses typical human pluripotency markers and shows differentiation into all three germ layers, complete reprogramming vector clearance, a normal SNP genotype and maintenance of the APOE ε4/ε4 allele.

STUB1/CHIP is a central component of cellular protein homeostasis and interacts with key proteins involved in the pathogenesis of many neurodegenerative diseases. Here, we reprogrammed human skin fibroblasts from a 12-year-old male patient with recessive spinocerebellar ataxia type 16 (OMIM #615768), carrying compound heterozygous mutations (c.355C>T, c.880A>T) in STUB1. Genomic integrity of the iPSC line HIHCNi001-A without transgene integration and genomic aberration but with maintained disease-relevant mutations was proven by SNP array analysis and Sanger sequencing while pluripotency was verified by the expression of important pluripotency markers and the capacity to differentiate into cells of all three germ layers.

The pathological cascade leading from primary storage to neural cell dysfunction and death in metachromatic leukodystrophy (MLD) has been poorly elucidated in human-derived neural cell systems. In the present study, we have modeled the progression of pathological events during the differentiation of patient-specific iPSCs to neuroepithelial progenitor cells (iPSC-NPCs) and mature neurons, astrocytes, and oligodendrocytes at the morphological, molecular, and biochemical level. We showed significant sulfatide accumulation and altered sulfatide composition during the differentiation of MLD iPSC-NPCs into neuronal and glial cells. Changes in sulfatide levels and composition were accompanied by the expansion of the lysosomal compartment, oxidative stress, and apoptosis. The neuronal and glial differentiation capacity of MLD iPSC-NPCs was significantly impaired. We showed delayed appearance and/or reduced levels of oligodendroglial and astroglial markers as well as reduced number of neurons and disorganized neuronal network. Restoration of a functional Arylsulfatase A (ARSA) enzyme in MLD cells using lentiviral-mediated gene transfer normalized sulfatide levels and composition, globally rescuing the pathological phenotype. Our study points to MLD iPSC-derived neural progeny as a useful in vitro model to assess the impact of ARSA deficiency along NPC differentiation into neurons and glial cells. In addition, iPSC-derived neural cultures allowed testing the impact of ARSA reconstitution/overexpression on disease correction and, importantly, on the biology and functional features of human NPCs, with important therapeutic implications.

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS., Abnormal cytoplasmic aggregates of FUS are a hallmark of some forms of amyotrophic lateral sclerosis (ALS). Here, using neurons derived from patients with FUS-ALS, the authors demonstrate that impairment of PARP-dependent DNA damage signaling is an event that occurs upstream of neurodegeneration and cytoplasmic aggregate formation in FUS-ALS.

A skin biopsy of a patient with spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease (MJD)) caused by a CAG trinucleotide repeat expansion in the ATXN3 gene, was used to generate an induced pluripotent stem cell line, HIHCNi002-A (iPSC-SCA3). Skin fibroblasts were reprogrammed using episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC, and hLIN28. The iPSC-SCA3 line exhibits chromosomal stability with conservation of the ATXN3 repeat expansion, expresses pluripotency markers and differentiates into endo-, meso-, and ectodermal cells in vitro.

TAR DNA-binding protein 43 (TDP43) is a cause of familiar and sporadic amyotrophic lateral sclerosis (ALS). The diverse postulated mechanisms by which TDP43 mutations cause the disease are not fully understood. Human wildtype and TDP43 S393L and G294V mutant spinal motor neuron cultures were differentiated from patient-derived iPSCs. Mutant hTDP43 and wildtype motor neuron cultures did not differ in neuron differentiation capacity during early maturation stage. During aging we detected a dramatic neurodegeneration including neuron loss and pathological neurofilament abnormalities in TDP43 mutant cultures only. Additionally mitochondria and lysosomes of aging spinal motor neurons revealed robust TDP43 mutation dependent abnormal phenotypes in size, shape, speed and motility which all appeared without TDP43 mislocalization or aggregation formation. Furthermore, D-sorbitol – known to induce stress granules and cytoplasmic mislocalization of TDP43 – rescued axonal trafficking phenotypes without any signs of TDP43 mislocalization or aggregation formation. Our data indicate TDP43 mutation-dependent but cytosolic aggregation-independent mechanisms of motor neuron degeneration in TDP43 ALS.

The ability to generate patient-specific induced pluripotent stem cells (iPSCs) provides a unique opportunity for modeling heart disease in vitro. In this study, we generated iPSCs from a patient with dilated cardiomyopathy (DCM) caused by a missense mutation S635A in RNA-binding motif protein 20 (RBM20) and investigated the functionality and cell biology of cardiomyocytes (CMs) derived from patient-specific iPSCs (RBM20-iPSCs). The RBM20-iPSC-CMs showed abnormal distribution of sarcomeric α-actinin and defective calcium handling compared to control-iPSC-CMs, suggesting disorganized myofilament structure and altered calcium machinery in CMs of the RBM20 patient. Engineered heart muscles (EHMs) from RBM20-iPSC-CMs showed that not only active force generation was impaired in RBM20-EHMs but also passive stress of the tissue was decreased, suggesting a higher visco-elasticity of RBM20-EHMs. Furthermore, we observed a reduced titin (TTN) N2B-isoform expression in RBM20-iPSC-CMs by demonstrating a reduction of exon skipping in the PEVK region of TTN and an inhibition of TTN isoform switch. In contrast, in control-iPSC-CMs both TTN isoforms N2B and N2BA were expressed, indicating that the TTN isoform switch occurs already during early cardiogenesis. Using next generation RNA sequencing, we mapped transcriptome and splicing target profiles of RBM20-iPSC-CMs and identified different cardiac gene networks in response to the analyzed RBM20 mutation in cardiac-specific processes. These findings shed the first light on molecular mechanisms of RBM20-dependent pathological cardiac remodeling leading to DCM. Our data demonstrate that iPSC-CMs coupled with EHMs provide a powerful tool for evaluating disease-relevant functional defects and for a deeper mechanistic understanding of alternative splicing-related cardiac diseases. peerReviewed

SummaryThe LRRK2 mutation G2019S is the most common genetic cause of Parkinson’s disease (PD). To better understand the link between mutant LRRK2 and PD pathology, we derived induced pluripotent stem cells from PD patients harboring LRRK2 G2019S and then specifically corrected the mutant LRRK2 allele. We demonstrate that gene correction resulted in phenotypic rescue in differentiated neurons and uncovered expression changes associated with LRRK2 G2019S. We found that LRRK2 G2019S induced dysregulation of CPNE8, MAP7, UHRF2, ANXA1, and CADPS2. Knockdown experiments demonstrated that four of these genes contribute to dopaminergic neurodegeneration. LRRK2 G2019S induced increased extracellular-signal-regulated kinase 1/2 (ERK) phosphorylation. Transcriptional dysregulation of CADPS2, CPNE8, and UHRF2 was dependent on ERK activity. We show that multiple PD-associated phenotypes were ameliorated by inhibition of ERK. Therefore, our results provide mechanistic insight into the pathogenesis induced by mutant LRRK2 and pointers for the development of potential new therapeutics.

Numerous genes and molecular pathways are implicated in neurodegenerative proteinopathies, but their inter-relationships are poorly understood. We systematically mapped molecular pathways underlying the toxicity of alpha-synuclein (α-syn), a protein central to Parkinson's disease. Genome-wide screens in yeast identified 332 genes that impact α-syn toxicity. To “humanize” this molecular network, we developed a computational method, TransposeNet. This integrates a Steiner prize-collecting approach with homology assignment through sequence, structure, and interaction topology. TransposeNet linked α-syn to multiple parkinsonism genes and druggable targets through perturbed protein trafficking and ER quality control as well as mRNA metabolism and translation. A calcium signaling hub linked these processes to perturbed mitochondrial quality control and function, metal ion transport, transcriptional regulation, and signal transduction. Parkinsonism gene interaction profiles spatially opposed in the network (ATP13A2/PARK9 and VPS35/PARK17) were highly distinct, and network relationships for specific genes (LRRK2/PARK8, ATXN2, and EIF4G1/PARK18) were confirmed in patient induced pluripotent stem cell (iPSC)-derived neurons. This cross-species platform connected diverse neurodegenerative genes to proteinopathy through specific mechanisms and may facilitate patient stratification for targeted therapy. Keywords: alpha-synuclein; iPS cell; Parkinson’s disease; stem cell; mRNA translation; RNA-binding protein; LRRK2; VPS35; vesicle trafficking; yeast

Intronic hexanucleotide expansions in C9ORF72 are common in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non-ATG-initiated translation are responsible for the pathophysiology. We determined the interactome of C9ORF72 in motor neurons and found that C9ORF72 was present in a complex with cofilin and other actin binding proteins. Phosphorylation of cofilin was enhanced in C9ORF72-depleted motor neurons, in patient-derived lymphoblastoid cells, induced pluripotent stem cell-derived motor neurons and post-mortem brain samples from ALS patients. C9ORF72 modulates the activity of the small GTPases Arf6 and Rac1, resulting in enhanced activity of LIM-kinases 1 and 2 (LIMK1/2). This results in reduced axonal actin dynamics in C9ORF72-depleted motor neurons. Dominant negative Arf6 rescues this defect, suggesting that C9ORF72 acts as a modulator of small GTPases in a pathway that regulates axonal actin dynamics.

Induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts from a 60-year-old cerebrotendinous xanthomatosis (CTX) patient, carrying a homozygous mutation c. [1183C > A]; p. R395S in CYP27A1 . Episomal plasmids encoding the pluripotency genes OCT4, SOX2, KLF4, L-MYC and LIN28 were introduced via electroporation. The generated line iPS-CTX-R395S has no sign of plasmid integration or chromosomal aberration and retained the mutation site in CYP27A1 . Furthermore, iPSCs express pluripotency markers and are able to differentiate in all germ layers in vitro . The generated line may be a useful tool for disease modelling of CTX.

Chorea-acanthocytosis (ChAc) is a fatal neurological disorder characterized by red blood cell acanthocytes and striatal neurodegeneration. Recently, severe cell membrane disturbances based on depolymerized cortical actin and an elevated Lyn kinase activity in erythrocytes from ChAc patients were identified. How this contributes to the mechanism of neurodegeneration is still unknown. To gain insight into the pathophysiology, we established a ChAc patient-derived induced pluripotent stem cell model and an efficient differentiation protocol providing a large population of human striatal medium spiny neurons (MSNs), the main target of neurodegeneration in ChAc. Patient-derived MSNs displayed enhanced neurite outgrowth and ramification, whereas synaptic density was similar to controls. Electrophysiological analysis revealed a pathologically elevated synaptic activity in ChAc MSNs. Treatment with the F-actin stabilizer phallacidin or the Src kinase inhibitor PP2 resulted in the significant reduction of disinhibited synaptic currents to healthy control levels, suggesting a Src kinase- and actin-dependent mechanism. This was underlined by increased G/F-actin ratios and elevated Lyn kinase activity in patient-derived MSNs. These data indicate that F-actin stabilization and Src kinase inhibition represent potential therapeutic targets in ChAc that may restore neuronal function.SIGNIFICANCE STATEMENTChorea-acanthocytosis (ChAc) is a fatal neurodegenerative disease without a known cure. To gain pathophysiological insight, we newly established a humanin vitromodel using skin biopsies from ChAc patients to generate disease-specific induced pluripotent stem cells (iPSCs) and developed an efficient iPSC differentiation protocol providing striatal medium spiny neurons. Using patch-clamp electrophysiology, we detected a pathologically enhanced synaptic activity in ChAc neurons. Healthy control levels of synaptic activity could be restored by treatment of ChAc neurons with the F-actin stabilizer phallacidin and the Src kinase inhibitor PP2. Because Src kinases are involved in bridging the membrane to the actin cytoskeleton by membrane protein phosphorylation, our data suggest an actin-dependent mechanism of this dysfunctional phenotype and potential treatment targets in ChAc.

Human skin fibroblasts were isolated from a 48-year-old patient carrying compound heterozygous mutations (c.610+364G>A and c.1311A>G) in OPA1, responsible for early onset optic atrophy complicated by ataxia and pyramidal signs (Behr syndrome; OMIM #210000). Fibroblasts were reprogrammed using episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated transgene-free line iPS-OPA1-BEHR showed no additional genomic aberrations, maintained the disease-relevant mutations, expressed important pluripotency markers and was capable to differentiate into cells of all three germ layers in vitro. The generated iPS-OPA1-BEHR line might be a useful platform to study the pathomechanism of early onset complicated optic atrophy syndromes.