Biology subjects

Deneault, E.

Publications and source records attributed to Deneault, E..

11 recordsLinked to original sources

bioRxiv2025

LRRK2 Kinase Mediates Increased GCase Activity in Microglia in Response to Proinflammatory Stimuli

Variants in the LRRK2 and GBA1 genes are among the most common risk factors associated with Parkinsons disease (PD). Both patients carrying PD-associated variants in GBA1, encoding lysosomal enzyme glucocerebrosidase (GCase), and a subset of non-carrier patients have been shown to have reduced GCase enzymatic activity, suggesting that reduced GCase activity may be a feature of both genetic and a subset of sporadic PD. However, the effect of PD-associated variants in LRRK2, encoding a serine/threonine kinase, on GCase activity remains controversial, with conflicting results in various tissues and cell types. Moreover, rare patients carrying both GBA1 and LRRK2 risk alleles seem to have a more benign disease course than carriers of GBA1 variants alone, suggesting a complex interplay between these two genes in PD. Here we evaluate the effect of LRRK2 kinase activity on GCase activity in human induced pluripotent stem cell (iPSC)-derived microglia (iMGs), a PD-relevant brain cell type expressing high levels of LRRK2. Using CRISPR editing, isogenic control iPSC lines were generated to match PD patient-derived iPSC lines harbouring the LRRK2 p.G2019S, p.M1646T, or p.N551K-p.R1398H protective haplotype variants. Whereas iMGs harbouring the p.M1646T variant, and the protective haplotype, respectively increased and decreased phosphorylation of canonical LRRK2 substrate, Rab10, GCase protein levels and activity were not altered in any of the LRRK2 variant lines. Additionally, whereas pharmacological inhibition of LRRK2 kinase activity had no impact on GCase activity in iMGs under basal conditions, it attenuated the increase in GCase activity elicited in response to interferon {gamma} (IFN{gamma}) treatment. Moreover, GCase activity induced by IFN{gamma} was reduced in PD risk LRRK2 p.M1646T iMGs and increased in p.N551K-p.R1398H protective haplotype iMGs compared to their isogenic corrected controls, congruent with their respective effects on LRRK2 kinase activity and PD risk. Thus, our data suggests a role for LRRK2 kinase activity in regulation of GCase activity in response to neuroinflammation.

neuroscience↗

bioRxiv2025

Glucosylceramide induced ectosomes propagate pathogenic α-synuclein in Parkinson's disease

Intercellular transmission of -synuclein contributes to Parkinsons disease pathology. Yet, the mechanisms of -synuclein spread are not fully understood. Here, we used live-cell microscopy to examine the impact of Parkinsons disease associated lipid alterations on -synuclein release. We discovered that increased glucosylceramides induce ectosome shedding from primary neurons, and from dopaminergic neurons derived from Parkinsons disease patient iPSCs harboring mutations in GBA1 (N370S, L444P and W378G) and LRRK2 (G2019S and R1441H) compared to their isogenic control. We show that elevated glucosylceramide similarly increases vesicle release and uptake by other neurons in living mouse brains using 2-photon microscopy. Finally, we show that ectosomes are loaded with pathogenic -synuclein and lead to the transmission of -synuclein pathology to neighbouring neurons. These data reveal ectosomes as the predominant route for -synuclein transmission that can only be appreciated by live-cell imaging technologies.

cell biology↗

bioRxiv2025

Mutations in SOD1 induce ALS-related phenotypes in 3D iPSC-derived motor neuron (MN) spheroids

A significant challenge in ALS research is the heterogeneity of the disease. Even mutations within the same gene can lead to different disease prognosis. For instance, in silico protein modeling predicts distinct properties for distinct SOD1 mutations. With this in mind, in this study, we generated and characterized 3D iPSC-derived MN spheroids carrying homozygous knock-in SOD1 mutations (D90A and G93A), as well as a double mutation (D90A/G93A), to evaluate potential synergistic effects. An isogenic control line with the same genetic background was used for phenotypic comparisons with the knock-in variants. Mutant SOD1 MN spheroids exhibited multiple ALS-related phenotypes including altered SOD1 expression, reduced cell viability, downregulation of neurofilament (NF) subunit expression, hypoactivity, and altered burst activity. Our results highlight the advantages of using 3D MN spheroids as a disease model and stress the importance of considering phenotype variability at the genetic level in ALS.

neuroscience↗

bioRxiv2023

A deep learning convolutional neural network distinguishes neuronal models of Parkinson's disease from matched controls

Parkinsons disease (PD) is a neurodegenerative disorder that results in the loss of dopaminergic neurons in the substantia nigra pars compacta. Despite advances in understanding PD, there is a critical need for novel therapeutics that can slow or halt its progression. Induced pluripotent stem cell (iPSC)-derived dopaminergic neurons have been used to model PD but measuring differences between PD and control cells in a robust, reproducible, and scalable manner remains a challenge. In this study, we developed a binary classifier convolutional neural network (CNN) to accurately classify microscopy images of PD models and matched control cells. We acquired images of iPSC-derived neural precursor cells (NPCs) and dopaminergic (DANs) and trained multiple CNN models comparing control cells to genetic and chemical models of PD. Our CNN accurately predicted whether control NPC cells were treated with the PD-inducing pesticide rotenone with 97.60% accuracy. We also compared control to a genetic model of PD (deletion of the Parkin gene) and found a predictive accuracy of 86.77% and 95.47% for NPC and DAN CNNs, respectively. Our cells were stained for nuclei, mitochondria, and plasma membrane, and we compared the contribution of each to the CNNs accuracy. Using all three features together produced the best accuracy, but nuclear staining alone produced a highly predictive CNN. Our study demonstrates the power of deep learning and computer vision for analyzing complex PD-related phenotypes in DANs and suggests that these tools hold promise for identifying new targets for therapy and improving our understanding of PD.

neuroscience↗

bioRxiv2023

Transcriptional dysregulation and impaired neuronal activity in FMR1 knock-out and Fragile X patients iPSC-derived models.

The lack of fragile X mental retardation protein (FMRP) protein, due to a repression of the FMR1 gene, causes Fragile X syndrome (FXS), one of the most prevalent forms of syndromic autisms. The FMR1 gene codes for an RNA binding protein involved in the regulation of gene expression through RNA processing, control of local translation, and protein-protein interactions; processes that are crucial for proper brain development. Taking advantage of induced pluripotent stem cells (iPSCs) and CRISPR-Cas9 genome editing technologies, we generated iPSC-derived cortical neural progenitors and cortical neurons from an FMR1 knock-out and patient cell line with the aim of identifying common phenotypes between the two cellular models. Using RNA sequencing, quantitative PCR and multielectrode array approaches, we assessed how the absence of the functional FMR1 gene affects the transcriptional profiles and the activities of iPSC-derived cortical neuronal progenitor cells (NPCs) and neurons with both models. We observed that FMR1 KO and FXS patient cells have a decrease in their mean firing rate; a cellular activity that can also be blocked by tetrodotoxin (TTX) application in wild-type active neurons. Relative to wild-type neurons, in FMR1 KO neurons, increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and Autism Related Genes found amongst downregulated genes. This implies that an absence of functional FMRP affects transcriptional profiles at the NPC stage, resulting in impaired activity and differentiation of the progenitors into mature neurons over time. These findings from the FMR1 KO lines were also shared with FXS patients iPSC-derived cells that also present with an impairment in activity and neuronal differentiation, illustrating the critical role of FMRP protein in neuronal development.

neuroscience↗

bioRxiv2023

Homozygous ALS-linked mutations in TARDBP/TDP-43 lead to progressive synaptic dysfunction in human iPSC-derived motor neurons

Cytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 is a pathological hallmark of the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS). Furthermore, while mutations in the TARDBP gene (encoding TDP-43) have been associated with ALS, the pathogenic consequences of these mutations remain poorly understood. Using CRISPR/Cas9, we engineered two homozygous knock-in iPSC lines carrying mutations in TARDBP encoding TDP-43A382T and TDP-43G348C, two common yet understudied ALS TDP-43 variants. MNs differentiated from knock-in iPSCs had normal viability and displayed no significant changes in TDP-43 subcellular localization, phosphorylation, solubility, or aggregation compared with isogenic control MNs. However, our results highlight synaptic impairments in both TDP-43A382T and TDP-43G348C MN cultures, as reflected in synapse abnormalities and alterations in spontaneous neuronal activity. Collectively, our findings suggest that MN dysfunction may precede the occurrence of TDP-43 pathology and neurodegeneration in ALS and further implicate synaptic and excitability defects in the pathobiology of this disease.

neuroscience↗

bioRxiv2022

Neuron-specific protein network mapping of autism risk genes identifies shared biological mechanisms and disease relevant pathologies

Manuscript summaryThere are hundreds of risk genes associated with autism spectrum disorder (ASD), but signaling networks at the protein level remain unexplored. We use neuron-specific proximity-labeling proteomics (BioID) to identify protein-protein interaction (PPI) networks for 41 ASD-risk genes. Neuron-specific PPI networks, including synaptic transmission proteins, are disrupted by de novo missense variants. The PPI network map reveals convergent pathways, including mitochondrial/metabolic processes, Wnt signaling, and MAPK signaling. CRISPR knockout reveal an association between mitochondrial activity and ASD-risk genes. The PPI network shows an enrichment of 112 additional ASD-risk genes and differentially expressed genes from post-mortem ASD patients. Clustering of risk genes based on PPI networks identifies gene groups corresponding to clinical behavior score severity. Our data reveal that cell type-specific PPI networks can identify individual and convergent ASD signaling networks, provide a method to assess patient variants, and reveal biological insight into disease mechanisms and sub-cohorts of ASD.

neuroscience↗

bioRxiv2021

A streamlined CRISPR workflow to introduce mutations and generate isogenic iPSCs for modeling amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) represents a complex neurodegenerative disorder with significant genetic heterogeneity. To date, both the genetic etiology and the underlying molecular mechanisms driving this disease remain poorly understood, although in recent years a number of studies have highlighted a number of genetic mutations causative for ALS. With these mutations pointing to potential pathways that may be affected within individuals with ALS, having the ability to generate human neurons and other disease relevant cells containing these mutations becomes even more critical if new therapies are to emerge. Recent developments with the advent of induced pluripotent stem cells (iPSCs) and clustered regularly interspaced short palindromic repeats (CRISPR) gene editing fields gave us the tools to introduce or correct a specific mutation at any site within the genome of an iPSC, and thus model the specific contribution of risk mutations. In this study we describe a rapid and efficient way to either introduce a mutation into a control line, or to correct a mutation, generating an isogenic control line from patient-derived iPSCs with a given mutation. The mutations introduced were the G93A mutation into SOD1 or H517Q into FUS, and the mutation corrected was a patient iPSC line with I114T in SOD1. A combination of small molecules and growth factors were used to guide a stepwise differentiation of the edited cells into motor neurons in order to demonstrate that disease-relevant cells could be generated for downstream applications. Through a combination of iPSCs and CRISPR editing, the cells generated here will provide fundamental insights into the molecular mechanisms underlying neuron degeneration in ALS.

neuroscience↗

bioRxiv2021

Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy

SNCA, the first gene associated with Parkinsons disease, encodes the -synuclein (-syn) protein, the predominant component within pathological inclusions termed Lewy bodies (LBs). The presence of LBs is one of the classical hallmarks found in the brain of patients with Parkinsons disease, and LBs have also been observed in patients with other synucleinopathies. However, the study of -syn pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use 3D midbrain organoids (hMOs), differentiated from human induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These hMOs recapitulate key features of -syn pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication hMOs express elevated levels of -syn and exhibit an age-dependent increase in -syn aggregation, manifested by the presence of both oligomeric and phosphorylated forms of -syn. These phosphorylated -syn aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, hMOs from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinsons disease and provide a powerful system to study the pathogenesis of synucleinopathies.

neuroscience↗

bioRxiv2018

CNTN5-/+ or EHMT2-/+ iPSC-Derived Neurons from Individuals with Autism Develop Hyperactive Neuronal Networks

Induced pluripotent stem cell (iPSC)-derived cortical neurons are increasingly used as a model to study developmental aspects of Autism Spectrum Disorder (ASD), which is clinically and genetically heterogeneous. To study the complex relationship of rare (penetrant) variant(s) and common (weaker) polygenic risk variant(s) to ASD, \"isogenic\" iPSC-derived neurons from probands and family-based controls, for modeling, is critical. We developed a standardized set of procedures, designed to control for heterogeneity in reprogramming and differentiation, and generated 53 different iPSC-derived glutamatergic neuronal lines from 25 participants from 12 unrelated families with ASD (14 ASD-affected individuals, 3 unaffected siblings, 8 unaffected parents). Heterozygous de novo (7 families; 16p11.2, NRXN1, DLGAP2, CAPRIN1, VIP, ANOS1, THRA) and rare-inherited (2 families; CNTN5, AGBL4) presumed-damaging variants were characterized in ASD risk genes/loci. In three additional families, functional candidates for ASD (SET), and combinations of putative etiologic variants (GLI3/KIF21A and EHMT2/UBE2I combinations in separate families), were modeled. We used a large-scale multi-electrode array (MEA) as our primary high-throughput phenotyping assay, followed by patch clamp recordings. Our most compelling new results revealed a consistent spontaneous network hyperactivity in neurons deficient for CNTN5 or EHMT2. Our biobank of iPSC-derived neurons and accompanying genomic data are available to accelerate ASD research.

neuroscience↗

bioRxiv2018

Complete Disruption of Autism-Susceptibility Genes by Gene-Editing Predominantly Reduces Functional Connectivity of Isogenic Human Neurons

Autism Spectrum Disorder is phenotypically and genetically heterogeneous, but genomic analyses have identified candidate susceptibility genes. We present a CRISPR gene editing strategy to insert a protein tag and premature termination sites creating an induced pluripotent stem cell (iPSC) knockout resource for functional studies of 10 ASD-relevant genes (AFF2/FMR2, ANOS1, ASTN2, ATRX, CACNA1C, CHD8, DLGAP2, KCNQ2, SCN2A, TENM1). Neurogenin 2 (NEUROG2)-directed differentiation of iPSCs allowed production of cortical excitatory neurons, and mutant proteins were not detectable. RNAseq revealed convergence of several neuronal networks. Using both patch-clamp and multi-electrode array approaches, the electrophysiological deficits measured were distinct for different mutations. However, they culminated in a consistent reduction in synaptic activity, including reduced spontaneous excitatory post-synaptic current frequencies in AFF2/FMR2-, ASTN2-, ATRX-, KCNQ2- and SCN2A-null neurons. Despite ASD susceptibility genes belonging to different gene ontologies, isogenic stem cell resources can reveal common functional phenotypes, such as reduced functional connectivity.

neuroscience↗