14 research outputs found
Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis
Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process
Astrocyte response to motor neuron injury promotes structural synaptic plasticity via STAT3-regulated TSP-1 expression.
The role of remote astrocyte (AC) reaction to central or peripheral axonal insult is not clearly understood. Here we use a transgenic approach to compare the direct influence of normal with diminished AC reactivity on neuronal integrity and synapse recovery following extracranial facial nerve transection in mice. Our model allows straightforward interpretations of AC-neuron signalling by reducing confounding effects imposed by inflammatory cells. We show direct evidence that perineuronal reactive ACs play a major role in maintaining neuronal circuitry following distant axotomy. We reveal a novel function of astrocytic signal transducer and activator of transcription-3 (STAT3). STAT3 regulates perineuronal astrocytic process formation and re-expression of a synaptogenic molecule, thrombospondin-1 (TSP-1), apart from supporting neuronal integrity. We demonstrate that, through this new pathway, TSP-1 is responsible for the remote AC-mediated recovery of excitatory synapses onto axotomized motor neurons in adult mice. These data provide new targets for neuroprotective therapies via optimizing AC-driven plasticity.This is the final version. It was first published in Nature Communications here: http://www.nature.com/ncomms/2014/140711/ncomms5294/abs/ncomms5294.html
Aberrant cytoplasmic intron retention is a blueprint for RNA binding protein mislocalization in VCP-related amyotrophic lateral sclerosis
We recently described aberrantly increased cytoplasmic SFPQ intron-retaining transcripts (IRTs) and concurrent SFPQ protein mislocalization as new hallmarks of amyotrophic lateral sclerosis (ALS). However, the generalizability and potential roles of cytoplasmic IRTs in health and disease remain unclear. Here, using time-resolved deep sequencing of nuclear and cytoplasmic fractions of human induced pluripotent stem cells undergoing motor neurogenesis, we reveal that ALS-causing VCP gene mutations lead to compartment-specific aberrant accumulation of IRTs. Specifically, we identify > 100 IRTs with increased cytoplasmic abundance in ALS samples. Furthermore, these aberrant cytoplasmic IRTs possess sequence-specific attributes and differential predicted binding affinity to RNA binding proteins. Remarkably, TDP-43, SFPQ and FUS-RNA binding proteins known for nuclear-to-cytoplasmic mislocalization in ALS-abundantly and specifically bind to this aberrant cytoplasmic pool of IRTs. Our data are therefore consistent with a novel role for cytoplasmic IRTs in regulating compartment-specific protein abundance. This study provides new molecular insight into potential pathomechanisms underlying ALS and highlights aberrant cytoplasmic IRTs as potential therapeutic targets
Reactive astrocytes in ALS display diminished intron retention
Reactive astrocytes are implicated in amyotrophic lateral sclerosis (ALS), although the mechanisms controlling reactive transformation are unknown. We show that decreased intron retention (IR) is common to human-induced pluripotent stem cell (hiPSC)-derived astrocytes carrying ALS-causing mutations in VCP, SOD1 and C9orf72. Notably, transcripts with decreased IR and increased expression are overrepresented in reactivity processes including cell adhesion, stress response and immune activation. This was recapitulated in public-datasets for (i) hiPSC-derived astrocytes stimulated with cytokines to undergo reactive transformation and (ii) in vivo astrocytes following selective deletion of TDP-43. We also re-examined public translatome sequencing (TRAP-seq) of astrocytes from a SOD1 mouse model, which revealed that transcripts upregulated in translation significantly overlap with transcripts exhibiting decreased IR. Using nucleocytoplasmic fractionation of VCP mutant astrocytes coupled with mRNA sequencing and proteomics, we identify that decreased IR in nuclear transcripts is associated with enhanced nonsense mediated decay and increased cytoplasmic expression of transcripts and proteins regulating reactive transformation. These findings are consistent with a molecular model for reactive transformation in astrocytes whereby poised nuclear reactivity-related IR transcripts are spliced, undergo nuclear-to-cytoplasmic translocation and translation. Our study therefore provides new insights into the molecular regulation of reactive transformation in astrocytes
Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS
Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed regulators of RNA processing. To understand the molecular impact of ALS-causing mutations on neuronal development and disease, we analysed transcriptomes during in vitro differentiation of motor neurons (MNs) from human control and patient-specific VCP mutant induced-pluripotent stem cells (iPSCs). We identify increased intron retention (IR) as a dominant feature of the splicing programme during early neural differentiation. Importantly, IR occurs prematurely in VCP mutant cultures compared with control counterparts. These aberrant IR events are also seen in independent RNAseq data sets from SOD1- and FUS-mutant MNs. The most significant IR is seen in the SFPQ transcript. The SFPQ protein binds extensively to its retained intron, exhibits lower nuclear abundance in VCP mutant cultures and is lost from nuclei of MNs in mouse models and human sporadic ALS. Collectively, we demonstrate SFPQ IR and nuclear loss as molecular hallmarks of familial and sporadic ALS
Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS.
Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateralĀ sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS
Automated and unbiased discrimination of ALS from control tissue at single cell resolution
Histopathological analysis of tissue sections is invaluable in neurodegeneration research. However, cellātoācell variation in both the presence and severity of a given phenotype is a key limitation of this approach, reducing the signal to noise ratio and leaving unresolved the potential of singleācell scoring for a given disease attribute. Here, we tested different machine learning methods to analyse highācontent microscopy measurements of hundreds of motor neurons (MNs) from amyotrophic lateral sclerosis (ALS) postāmortem tissue sections. Furthermore, we automated the identification of phenotypically distinct MN subpopulations in VCPā and SOD1āmutant transgenic mice, revealing common morphological cellular phenotypes. Additionally we established scoring metrics to rank cells and tissue samples for both disease probability and severity. By adapting this paradigm to human postāmortem tissue, we validated our core finding that morphological descriptors robustly discriminate ALS from control healthy tissue at single cell resolution. Determining disease presence, severity and unbiased phenotypes at single cell resolution might prove transformational in our understanding of ALS and neurodegeneration more broadly
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Delineating Astrocytic Cytokine Responses in a Human Stem Cell Model of Neural Trauma.
Neuroinflammation has been shown to mediate the pathophysiological response following traumatic brain injury (TBI). Accumulating evidence implicates astrocytes as key immune cells within the central nervous system (CNS), displaying both pro- and anti-inflammatory properties. The aim of this study was to investigate how in vitro human astrocyte cultures respond to cytokines across a concentration range that approximates the aftermath of human TBI. To this end, enriched cultures of human induced pluripotent stem cell (iPSC)-derived astrocytes were exposed to interleukin-1Ī² (IL-1Ī²) (1-10,000āpg/mL), IL-4 (1-10,000āpg/mL), IL-6 (100-1,000,000āpg/mL), IL-10 (1-10,000āpg/mL) and tumor necrosis factor (TNF)-Ī± (1-10,000āpg/mL). After 1, 24, 48 and 72āh, cultures were fixed and immunolabeled, and the secretome/supernatant was analyzed at 24, 48, and 72āh using a human cytokine/chemokine 39-plex Luminex assay. Data were compared to previous in vitro studies of neuronal cultures and clinical TBI studies. The secretome revealed concentration-, time- and/or both concentration- and time-dependent production of downstream cytokines (29, 21, and 17 cytokines, respectively, p<0.05). IL-1Ī² exposure generated the most profound downstream response (27 cytokines), IL-6 and TNF had intermediate responses (13 and 11 cytokines, respectively), whereas IL-4 and IL-10 only led to weak responses over time or in escalating concentration (8 and 8 cytokines, respectively). Notably, expression of IL-1Ī², IL-6, and TNF cytokine receptor mRNA was higher in astrocyte cultures than in neuronal cultures. Several secreted cytokines had temporal trajectories, which corresponded to those seen in the aftermath of human TBI. In summary, iPSC-derived astrocyte cultures exposed to cytokine concentrations reflecting those in TBI generated an increased downstream cytokine production, particularly IL-1Ī². Although more work is needed to better understand how different cells in the CNS respond to the neuroinflammatory milieu after TBI, our data shows that iPSC-derived astrocytes represent a tractable model to study cytokine stimulation in a cell type-specific manner.EPT is supported by post-doctoral scholarships from the Swedish Society for Medical Research and Swedish Society of Medicine (grant no. SLS-587221). KLHC is supported by the National Institute for Health Research Biomedical Research Centre, Cambridge (Neuroscience Theme; Brain Injury and Repair Theme). PJH is supported by a Research Professorship from the National Institute for Health Research (NIHR), the NIHR Cambridge Biomedical Research Centre, the NIHR Global Health Research Group on Neurotrauma a European Union Seventh Framework Program grant (CENTER-TBI; grant no. 602150), and the Royal College of Surgeons of England. RP is supported by an MRC / MNDA Lady Edith Wolfson Senior Clinical Fellowship (MR/S006591/1). We also acknowledge support from the
University College London Hospital Biomedical Research Centre. AH is supported by the Medical Research Council/Royal College of Surgeons of England Clinical Research Training Fellowship, Royal College of Surgeons of England Pump Priming Grant and the National Institute for Health Research Biomedical Research Centre, Cambridge. The Luminex 200 analyser was purchased with Medical Research Council (MRC) funding (G0600986 ID79068). We acknowledge the DPUK/MRC platform for provision of the Opera Phenix for high-throughput iPSC analysis