技术资料

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants,including genetic and non-genetic factors. A key pathological signature of ALS is the cytoplasmic mislocalization and aggregation of TDP-43 in affected motor neurons,which is found in 97% of cases. Recent reports have shown that mitochondrial dysfunction plays a significant role in motor neuron degeneration in ALS,and TDP-43 modulates several mitochondrial transcripts. In this study,we used induced pluripotent stem cell-derived motor neurons from ALS patients with TDP-43 mutations and a transgenic TDP-43M337V mouse model to determine how TDP-43 mutations alter mitochondrial function and axonal transport. We detected significantly reduced mitochondrial respiration and ATP production in patient induced pluripotent stem cell-derived motor neurons,linked to an interaction between TDP-43M337V with ATPB and COX5A. A downstream reduction in speed of retrograde axonal transport in patient induced pluripotent stem cell-derived motor neurons was detected,which correlated with downregulation of the motor protein complex,DCTN1/dynein. Overexpression of DCTN1 in patient induced pluripotent stem cell-derived motor neurons significantly increased the percentage of retrograde travelling mitochondria and reduced the percentage of stationary mitochondria. This study shows that ALS induced pluripotent stem cell-derived motor neurons with mutations in TDP-43 have deficiencies in essential mitochondrial functions with downstream effects on retrograde axonal transport,which can be partially rescued by DCTN1 overexpression. Dafinca et al. show that mutations in TDP-43 lead to decreased mitochondrial oxidative phosphorylation,partially due to interactions with the ATP production machinery and COX5A. These have direct effects on axonal transport,which is reduced in amyotrophic lateral sclerosis motor neurons,and overexpression of dynactin-1 significantly increases retrograde mitochondrial dynamics. View Publication

High-throughput massively parallel reporter assays (MPRAs) and phenotype-rich in vivo transgenic mouse assays are two potentially complementary ways to study the impact of noncoding variants associated with psychiatric diseases. Here,we investigate the utility of combining these assays. Specifically,we carry out an MPRA in induced human neurons on over 50,000 sequences derived from fetal neuronal ATAC-seq datasets and enhancers validated in mouse assays. We also test the impact of over 20,000 variants,including synthetic mutations and 167 common variants associated with psychiatric disorders. We find a strong and specific correlation between MPRA and mouse neuronal enhancer activity. Four out of five tested variants with significant MPRA effects affected neuronal enhancer activity in mouse embryos. Mouse assays also reveal pleiotropic variant effects that could not be observed in MPRA. Our work provides a catalog of functional neuronal enhancers and variant effects and highlights the effectiveness of combining MPRAs and mouse transgenic assays. MPRAs and in vivo transgenic mouse assays are two potentially complementary ways to assay the impact of noncoding variants. Here,authors find a strong and specific correlation between the assays in neural cells. Mouse assays also reveal pleiotropic effects not observed in MPRA. View Publication

The transcription factor Growth Factor Independence 1B (GFI1B) recruits Lysine Specific Demethylase 1 A (LSD1/KDM1A) to stimulate gene programs relevant for megakaryocyte and platelet biology. Inherited pathogenic GFI1B variants result in thrombocytopenia and bleeding propensities with varying intensity. Whether these affect similar gene programs is unknow. Here we studied transcriptomic effects of four patient-derived GFI1B variants (GFI1BT174N,H181Y,R184P,Q287*) in MEG01 megakaryoblasts. Compared to normal GFI1B,each variant affected different gene programs with GFI1BQ287* uniquely failing to repress myeloid traits. In line with this,single cell RNA-sequencing of induced pluripotent stem cell (iPSC)-derived megakaryocytes revealed a 4.5-fold decrease in the megakaryocyte/myeloid cell ratio in GFI1BQ287* versus normal conditions. Inhibiting the GFI1B-LSD1 interaction with small molecule GSK-LSD1 resulted in activation of myeloid genes in normal iPSC-derived megakaryocytes similar to what was observed for GFI1BQ287* iPSC-derived megakaryocytes. Thus,GFI1B and LSD1 facilitate gene programs relevant for megakaryopoiesis while simultaneously repressing programs that induce myeloid differentiation. Using patient-derived induced pluripotent stem cells,the authors show that the transcription factor GFI1B and the lysine demethylase KDM1A/LSD1 promote gene programs while repressing those involved in myeloid differentiation. View Publication

The MAPT gene encodes Tau protein,a member of the large family of microtubule-associated proteins. Tau forms large insoluble aggregates that are toxic to neurons in several neurological disorders,and neurofibrillary Tau tangles represent a key pathological hallmark of Alzheimer’s disease (AD) and other tauopathies. Lowering Tau expression levels constitutes a potential treatment for AD but the mechanisms that regulate Tau expression at the transcriptional or translational level are not well understood. Natural antisense transcripts (NATs) are a particular class of long non-coding RNAs (lncRNAs) that can regulate expression of their overlapping protein-coding genes at the epigenetic,transcriptional,or translational level. We and others identified a long non-coding RNA associated with the MAPT gene,named MAPT antisense 1 (MAPT-AS1). We confirmed that MAPT-AS1 is expressed in neurons in human post mortem brain tissue. To study the role of MAPT-AS1 on MAPT expression regulation,we modulated the expression of this lncRNA in human neuroblastoma cell lines and in human induced pluripotent stem cell (iPSC) derived neurons. In contrast to previous reports,we observed no changes on MAPT mRNA or Tau protein levels upon modulation of MAPT-AS1 levels in these cellular models. Our data suggest that MAPT-AS1 does not regulate Tau expression levels in human neurons in vitro. Thus,MAPT-AS1 does not represent a valuable therapeutic target to lower Tau expression in patients affected by tauopathies including AD. View Publication

Cardiac ventricular pressure overload affects patients with congenital heart defects and can cause cardiac insufficiency. Grafts of stem cell–derived cardiomyocytes are proposed as a complementary treatment to surgical repair of the cardiac defect,aiming to support ventricular function. Here,we report successful engraftment of human induced pluripotent stem cell–derived cardiac lineage cells into the heart of immunosuppressed rhesus macaques with a novel surgical model of right ventricular pressure overload. The human troponin+ grafts were detected in low-dose (2 × 106 cells/kg) and high-dose (10 × 106 cells/kg) treatment groups up to 12 weeks post-injection. Transplanted cells integrated and progressively matched the organization of the surrounding host myocardium. Ventricular tachycardia occurred in five out of 16 animals receiving cells,with episodes of incessant tachycardia observed in two animals; ventricular tachycardia events resolved within 19 days. Our results demonstrate that grafted cardiomyocytes mature and integrate into the myocardium of nonhuman primates modeling right ventricular pressure overload. View Publication

SummaryMutations in the DMD gene lead to Duchenne muscular dystrophy (DMD),a severe neuromuscular disorder affecting young boys as they acquire motor functions. DMD is typically diagnosed at 2–4 years of age,but the absence of dystrophin has negative impacts on skeletal muscles before overt symptoms appear in patients,which poses a serious challenge in current standards of care. Here,we investigated the consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Dystrophin deficiency was linked to marked dysregulations of cell junction proteins involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether,this work demonstrates that in vitro,dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development,which should be considered in future therapeutic strategies for DMD. Graphical abstract Highlights•Myogenic differentiation of DMD hiPSCs diverges at the somite stage•Cell junction formation is dysregulated in DMD somite cells•Somite cells from DMD hiPSCs have impaired epithelialization properties•Migration velocity of DMD-mutant somite progenitors is upregulated Natural sciences; Biological sciences; Biochemistry; Cell biology; Stem cells research; Developmental biology. View Publication

An intronic G4C2 repeat expansion in the C9ORF72 gene is the major known cause for Amyotrophic Lateral Sclerosis (ALS),with current evidence for both,loss of function and pathological gain of function disease mechanisms. We screened 96 200 small molecules in C9ORF72 patient iPS neurons for modulation of nuclear G4C2 RNA foci and identified 82 validated hits,including the Brd4 inhibitor JQ1 as well as novel analogs of Spliceostatin-A,a known modulator of SF3B1,the branch point binding protein of the U2-snRNP. Spliceosome modulation by these SF3B1 targeted compounds recruits SRSF1 to nuclear G4C2 RNA,mobilizing it from RNA foci into nucleocytoplasmic export. This leads to increased repeat-associated non-canonical (RAN) translation and ultimately,enhanced cell toxicity. Our data (i) provide a new pharmacological entry point with novel as well as known,publicly available tool compounds for dissection of C9ORF72 pathobiology in C9ORF72 ALS models,(ii) allowing to differentially modulate RNA foci versus RAN translation,and (iii) suggest that therapeutic RNA foci elimination strategies warrant caution due to a potential storage function,counteracting translation into toxic dipeptide repeat polyproteins. Instead,our data support modulation of nuclear export via SRSF1 or SR protein kinases as possible targets for future pharmacological drug discovery. View Publication

Differentiation approaches to obtain mesenchymal stem cells (MSCs) have gradually developed over the last few decades. The problem is that different protocols give different MSC types,making further research difficult. Here,we tried three different approaches to differentiate embryonic stem cells (ESCs) from early mesoderm to MSCs using serum-containing or xeno-free differentiation medium and observed differences in the cells’ morphology,doubling rate,ability to form colonies,surface marker analysis,and multilineage differentiation potential of the obtained cell lines. We concluded that the xeno-free medium best fits the criteria of MSCs’ morphology,growth kinetics,and surface marker characterization. In contrast,the serum-containing medium gives better potential for further MSC differentiation into osteogenic,chondrogenic,and adipogenic lineages. View Publication

A large number of synaptic proteins have been recurrently associated with complex brain disorders. One of these proteins,the Traf and Nck interacting kinase (TNIK),is a postsynaptic density (PSD) signaling hub,with many variants reported in neurodevelopmental disorder (NDD) and psychiatric disease. While rodent models of TNIK dysfunction have abnormal spontaneous synaptic activity and cognitive impairment,the role of mutations found in patients with TNIK protein deficiency and TNIK protein kinase activity during early stages of neuronal and synapse development has not been characterized. Here,using hiPSC-derived excitatory neurons,we show that TNIK mutations dysregulate neuronal activity in human immature synapses. Moreover,the lack of TNIK protein kinase activity impairs MAPK signaling and protein phosphorylation in structural components of the PSD. We show that the TNIK interactome is enriched in NDD risk factors and TNIK lack of function disrupts signaling networks and protein interactors associated with NDD that only partially overlap to mature mouse synapses,suggesting a differential role of TNIK in immature synapsis in NDD. View Publication

N6-methyladenosine (m6A) is an abundant internal RNA modification that can impact gene expression at both post-transcriptional and transcriptional levels. However,the landscapes and functions of m6A in human brains and neurodegenerative diseases,including Alzheimer’s disease (AD),are under-explored. Here,we examined RNA m6A methylome using total RNA-seq and meRIP-seq in middle frontal cortex of post-mortem brains from individuals with or without AD,which revealed m6A alteration on both mRNAs and various noncoding RNAs. Notably,many promoter-antisense RNAs (paRNAs) displayed cell-type-specific expression and changes in AD,including one produced adjacent to MAPT that encodes the Tau protein. MAPT-paRNA is highly expressed in neurons,and m6A positively controls its expression. In iPSC-derived human excitatory neurons,MAPT-paRNA does not impact the nearby MAPT mRNA,but instead promotes expression of hundreds of neuronal and synaptic genes,and is protective against excitotoxicity. Analysis of single nuclei RNA-DNA interactome in human brains supports that brain paRNAs interact with both cis- and trans-chromosomal target genes to impact their transcription. These data reveal landscapes and functions of noncoding RNAs and m6A in brain gene regulation and AD pathogenesis. This study characterised the landscapes and changes of RNA m6A in brains of individuals with or without Alzheimer’s disease,and revealed roles of a promoter antisense RNA next to MAPT in neuronal gene regulation that promote neuronal survival. View Publication