Raphaelle Luisier - Academia.edu (original) (raw)

Papers by Raphaelle Luisier

Nature Communications

Amyotrophic Lateral Sclerosis (ALS) causes motor neuron degeneration, with 97% of cases exhibitin... more Amyotrophic Lateral Sclerosis (ALS) causes motor neuron degeneration, with 97% of cases exhibiting TDP-43 proteinopathy. Elucidating pathomechanisms has been hampered by disease heterogeneity and difficulties accessing motor neurons. Human induced pluripotent stem cell-derived motor neurons (iPSMNs) offer a solution; however, studies have typically been limited to underpowered cohorts. Here, we present a comprehensive compendium of 429 iPSMNs from 15 datasets, and 271 post-mortem spinal cord samples. Using reproducible bioinformatic workflows, we identify robust upregulation of p53 signalling in ALS in both iPSMNs and post-mortem spinal cord. p53 activation is greatest with C9orf72 repeat expansions but is weakest with SOD1 and FUS mutations. TDP-43 depletion potentiates p53 activation in both post-mortem neuronal nuclei and cell culture, thereby functionally linking p53 activation with TDP-43 depletion. ALS iPSMNs and post-mortem tissue display enrichment of splicing alterations, s...

Amyotrophic Lateral Sclerosis (ALS) is characterised by progressive motor neuron degeneration, bu... more Amyotrophic Lateral Sclerosis (ALS) is characterised by progressive motor neuron degeneration, but there is marked genetic and clinical heterogeneity1. It has been challenging to identify common ALS mechanisms among this diversity; however, a systematic framework examining motor neurons across the ALS spectrum may reveal unifying insights. Here, we present the most comprehensive compendium of ALS human induced pluripotent stem cell-derived motor neurons (iPSNs) from 429 donors, spanning 10 ALS mutations and sporadic ALS, from 15 datasets, including Answer ALS and NeuroLINCS. Using gold-standard reproducible bioinformatic workflows, we identified that ALS iPSNs show a common increase in the DNA damage response, which is characterised by activation of p53 signalling. The strongest p53 activation was observed in C9orf72 repeat expansions but was also found in TARDBP, FUS and sporadic subgroups. p53 activation was replicated in an ALS postmortem spinal cord cohort of 203 samples, indica...

Amyotrophic Lateral Sclerosis (ALS) is characterised by progressive motor neuron degeneration but... more Amyotrophic Lateral Sclerosis (ALS) is characterised by progressive motor neuron degeneration but there is marked genetic and clinical heterogeneity1. Identifying common mechanisms of ALS amongst this diversity has been challenging, however, a systematic framework examining motor neurons across the ALS spectrum may reveal unifying insights. Here, we present the most comprehensive compendium of ALS human-induced pluripotent stem cell-derived motor neurons (iPSNs) from 429 donors across 15 datasets including Answer ALS and NeuroLINCS, spanning 10 ALS mutations and sporadic ALS. Using gold-standard reproducible bioinformatic workflows, we identify that ALS iPSNs show common activation of the DNA damage response and p53 signalling, which was replicated in the NYGC ALS postmortem cohort of 203 spinal cord samples. The strongest p53 activation was observed in C9orf72 repeat expansions but was also independently increased in TARDBP, FUS and sporadic subgroups. ALS iPSNs showed extensive sp...

Multichannel microscopy data from histopathological sections of 1/ Tissue sections from spinal co... more Multichannel microscopy data from histopathological sections of 1/ Tissue sections from spinal cord of SOD1- and VCP-mutant ALS mouse models together with control immunolabeled for FUS, SFPQ, ChAT and counterstained with DAPI. 2/ Tissue sections from spinal cord of Healthy and sporadic ALS donors immunolabeled for FUS or SFPQ, ChAT and counterstained with DAPI. 3/ Scripts to preprocess these images. These two series of multichannel fluorescent microscopy data have been used in several manuscripts including: Luisier R, Tyzack GE, Hall CE, Mitchell JS, Devine H, Taha DM, et al. Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS. Nat Commun 2018; 9: 2010. Tyzack GE, Luisier R, Taha DM, Neeves J, Modic M, Mitchell JS, et al. Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis. Brain 2019; 142: 2572–80.

Multichannel microscopy data from histopathological sections of 1/ SOD1- and VCP-mutant mouse mod... more Multichannel microscopy data from histopathological sections of 1/ SOD1- and VCP-mutant mouse models together with control co-stained for SFPQ, FUS, CHAT and DAPI The moues folder contains 2/ Healthy and sporadic ALS patients either co-stained for SFPQ or FUS together with CHAT and DAPI These two series of multichannel fluorescent microscopy data have been used in several manuscripts including: Luisier R, Tyzack GE, Hall CE, Mitchell JS, Devine H, Taha DM, et al. Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS. Nat Commun 2018; 9: 2010. Tyzack GE, Luisier R, Taha DM, Neeves J, Modic M, Mitchell JS, et al. Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis. Brain 2019; 142: 2572–80.

Brain Pathology, 2021

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive neurodegenerative disease, whic... more Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive neurodegenerative disease, which remains incurable due to an incomplete understanding of the underlying molecular pathogenesis. Using the VCP mutant mouse transgenic model of ALS and human post-mortem tissue from sporadic ALS (sALS) patients, we previously showed that spinal motor neurons (MNs) exhibit reduced nuclear to cytoplasmic (N/C) ratios of FUS (Fused in Sarcoma) and SFPQ (Splicing factor Proline and Glutamine rich) proteins. Furthermore, we showed that SFPQ is also mislocalized in SOD1-mutant ALS models, while FUS is not (1,2). Although these studies revealed novel molecular hallmarks of ALS, they also present important limitations shared by the majority of such traditional neuropathological studies. Firstly, key observations are based on manual

Non-genotoxic carcinogens (NGC) form a group of molecules that do not directly bind DNA (1) but t... more Non-genotoxic carcinogens (NGC) form a group of molecules that do not directly bind DNA (1) but that produce perturbations in the gene expression and epigenetic state of cells which facilitate tumor formation, typically through the promotion of preexisting neoplastic cells into neoplasms. The molecular events underlying the NGC-induced transformation of normal hepatocytes to altered hepatocellular foci are still unclear and no acute early molecular markers for NGC are available for drugs under development. According to regulatory expectations, drug safety is tested in both short term in vitro and long-term in vivo studies in several experimental animals (rodent and non-rodent species) prior testing on human. As the safety assessment in experimental animals has been very successful in predicting toxicity of biologically active chemicals in humans, differences in species biochemistry or pathophysiology be- tween human and rodents have raised doubts regarding the appropriateness of ext...

bioRxiv, 2021

Intron retention (IR) is now recognized as a dominant splicing event during motor neuron (MN) dev... more Intron retention (IR) is now recognized as a dominant splicing event during motor neuron (MN) development, however the role and regulation of intron-retaining transcripts (IRTs) localized to the cytoplasm remain particularly understudied. By resolving the spatiotemporal dynamics of IR underlying distinct stages of MN lineage restriction, we identify a cytoplasmic group of IRTs that is not associated with reduced expression of their own genes but instead with an upregulation of predicted target genes of specific miRNAs, the motifs of which are enriched within the intronic sequences of this group. Next, we show that ALS-causing VCP mutations lead to a selective increase in IR of this particular class of introns. This in turn temporally coincides with an increase in the expression level of predicted target genes of these miRNAs, providing a potential mechanistic insight into ALS pathogenesis. Altogether, we propose a novel role for the cytoplasmic intronic sequences in regulating miRNA...

Scientific Reports, 2020

Human mammary epithelial cells can proliferate and reorganize into polarized multi-cellular const... more Human mammary epithelial cells can proliferate and reorganize into polarized multi-cellular constructs in-vitro, thereby functioning as an important model system in recapitulating key steps of in-vivo morphogenesis. Current approaches to constructing such three-dimensional mimics of the in-vivo microenvironment have involved the use of complex and ill-defined naturally derived matrices, whose properties are difficult to manipulate independently, and which have therefore limited our ability to understand the extrinsic regulation of morphogenesis. Here, we employ an automated, high-throughput approach to array modular building blocks of synthetic components, and develop a systematic approach to analyze colonies resulting from these varied microenvironmental combinations. This methodology allows us to systematically map the relationship between microenvironmental properties and ensuing morphogenetic phenotypes. Our analysis reveals that apico-basal polarity of mammary epithelial cells ...

Brain, 2021

We recently described aberrantly increased cytoplasmic SFPQ intron-retaining transcripts (IRTs) a... more 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 dat...

SUMMARYWe recently described aberrant cytoplasmic SFPQ intron-retaining transcripts (IRTs) and co... more SUMMARYWe recently described aberrant cytoplasmic SFPQ intron-retaining transcripts (IRTs) and concurrent SFPQ protein mislocalization as a new hallmark 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 hiPSCs 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 (but not nuclear) abundance in ALS samples. Furthermore, these aberrant cytoplasmic IRTs possess sequence-specific attributes and differential predicted binding affinity to RNA binding proteins (RBPs). Remarkably, TDP-43, SFPQ and FUS – RBPs known for nuclear-to-cytoplasmic mislocalization in ALS – avidly and specifically bind to this aberrant cytoplasmic pool of IRTs, as opposed to any individual IRT....

SUMMARYHistopathological analysis of tissue sections is an invaluable resource in neurodegenerati... more SUMMARYHistopathological analysis of tissue sections is an invaluable resource in neurodegeneration research. Importantly, cell-to-cell variation in both the presence and severity of a given phenotype is however a key limitation of this approach, reducing the signal to noise ratio and leaving unresolved the potential of single-cell scoring for a given disease attribute. Here, we developed an image processing pipeline for automated identification and profiling of motor neurons (MNs) in amyotrophic lateral sclerosis (ALS) pathological tissue sections. This approach enabled unbiased analysis of hundreds of cells, from which hundreds of features were readily extracted. Next by testing different machine learning methods, we automated the identification of phenotypically distinct MN subpopulations in VCP- and SOD1-mutant transgenic mice, revealing common aberrant phenotypes in cellular shape. Additionally we established scoring metrics to rank cells and tissue samples for both disease pro...

Amyotrophic lateral sclerosis (ALS)-causing mutations clearly implicate ubiquitously expressed an... more Amyotrophic lateral sclerosis (ALS)-causing mutations clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins (RBPs), which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RBPs mislocalize without necessarily becoming constituents of ALS cytoplasmic inclusions themselves remains unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RBP Fused in Sarcoma (FUS), in an unaggregated state, may occur more widely in ALS that previously recognized. To address this hypothesis, we analysed motor neurons (MNs) from an human ALS induced-pluripotent stem cells (iPSC) 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 eviden...

Nature communications, May 22, 2018

Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed r... more 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 ...

Asymmetric localization of mRNAs is a mechanism that constrains protein synthesis to subcellular ... more Asymmetric localization of mRNAs is a mechanism that constrains protein synthesis to subcellular compartments. In neurons, mRNA transcripts are transported to both dendrites and axons where they are rapidly translated in response to extracellular stimuli. To characterize the 3′ UTR isoforms localized in axons and cell bodies of sympathetic neurons we performed 3′ end-RNA sequencing. We discovered that isoforms transported to axons had significantly longer 3′ UTRs compared to cell bodies. Moreover, more than 100 short 3′ UTR isoforms were uniquely expressed in axons. Analysis of the long 3′ UTR of IMPA1 indicated that a multiprotein complex including Upf1, HuD and Ago2 mediated 3′ UTR cleavage. This event enhanced IMPA1 translation and was necessary for maintaining axon integrity. 3′ UTR cleavage took place in axons and was not limited to IMPA1 but extended to other transcripts with similar expression patterns. We conclude that the 3′ UTR of neuronal transcripts undergo post-transcri...

Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate regulators of RNA-proces... more Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate regulators of RNA-processing that are ubiquitously expressed throughout development. To understand the molecular impact of ALS-causing mutations on early neuronal development and disease, we performed transcriptomic analysis of differentiated human control and VCP-mutant induced pluripotent stem cells (iPSCs) during motor neurogenesis. We identify intron retention (IR) as the predominant splicing change affecting early stages of wild-type neural differentiation, targeting key genes involved in the splicing machinery. Importantly, IR occurs prematurely in VCP-mutant cultures compared with control counterparts; these events are also observed in independent RNAseq datasets from SOD1- and FUS-mutant motor neurons (MNs). Together with related effects on 3'UTR length variation, these findings implicate alternative RNA-processing in regulating distinct stages of lineage restriction from iPSCs to MNs, and reveal a ...

Cell reports, May 30, 2017

Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic latera... more Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral 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...

Epigenetic mechanisms have an important role in human disease, and may also be involved in the ph... more Epigenetic mechanisms have an important role in human disease, and may also be involved in the pharmacodynamic and adverse effects associated with many drugs and toxicants. The development of powerful epigenomic profiling technologies and the construction of relevant bioinformatic frameworks for epigenome analysis and integration provide unique opportunities for identifying mechanisms and biomarkers of drug-mediated toxicity during both the preclinical and clinical phases of drug development. Non-genotoxic carcinogenesis (NGC) is a common drug-induced toxicity finding in rodent models for which no well-validated short-term assays exist. Here we have used genome-wide epigenomic and transcriptomic profiling, coupled to molecular histopathology, to investigate the temporal sequence of events and identify early-mechanisms and biomarkers of NGC in vivo in a well-characterized rodent model for liver tumor promotion. Exposure of mice to a tumor-promoting dose of phenobarbital (PB) led to t...

Microelectronic Engineering, 2010

ABSTRACT In this paper, we propose a method to create a natively hydrophilic microfluidic structu... more ABSTRACT In this paper, we propose a method to create a natively hydrophilic microfluidic structure in a fast and simple way using an UV-curable polymer (NOA 63 from Norland Optics). This polymer can provide a substitute to PDMS microchannels offering high reproducibility, elevated fabrication throughput and native hydrophilicity, allowing easier filling of the microchannels with aqueous solutions. We demonstrate the manufacturing of a complete microfluidic system, composed by a NOA 63 body sealed by a NOA 63 membrane, and we study the hydrophilicity and the optical characteristics of the fabricated cartridge.