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

2021 ◽  
Author(s):  
Eric Deneault ◽  
Mathilde Chaineau ◽  
Maria Jose Castellanos-Montiel ◽  
Anna Kristyna Franco Flores ◽  
Ghazal Haghi ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Neurodegenerative Disorder ◽  
Control Line ◽  
Human Neurons ◽  
Recent Developments ◽  
Induced Pluripotent ◽  
Specific Contribution ◽  
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.

scholarly journals Immune Signaling Kinases in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD)

2021 ◽  
Vol 22 (24) ◽  
pp. 13280
Author(s):  
Raquel García-García ◽  
Laura Martín-Herrero ◽  
Laura Blanca-Pariente ◽  
Jesús Pérez-Cabello ◽  
Cintia Roodveldt
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Frontotemporal Dementia ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Neurodegenerative Disorder ◽  
Therapeutic Potential ◽  
Neuronal Loss ◽  
Immune Signaling ◽  
Health And Disease ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is the most common neurodegenerative disorder of motor neurons in adults, with a median survival of 3–5 years after appearance of symptoms, and with no curative treatment currently available. Frontotemporal dementia (FTD) is also an adult-onset neurodegenerative disease, displaying not only clinical overlap with ALS, but also significant similarities at genetic and pathologic levels. Apart from the progressive loss of neurons and the accumulation of protein inclusions in certain cells and tissues, both disorders are characterized by chronic inflammation mediated by activated microglia and astrocytes, with an early and critical impact of neurodegeneration along the disease course. Despite the progress made in the last two decades in our knowledge around these disorders, the underlying molecular mechanisms of such non-cell autonomous neuronal loss still need to be clarified. In particular, immune signaling kinases are currently thought to have a key role in determining the neuroprotective or neurodegenerative nature of the central and peripheral immune states in health and disease. This review provides a comprehensive and updated view of the proposed mechanisms, therapeutic potential, and ongoing clinical trials of immune-related kinases that have been linked to ALS and/or FTD, by covering the more established TBK1, RIPK1/3, RACK I, and EPHA4 kinases, as well as other emerging players in ALS and FTD immune signaling.

scholarly journals The Link between VAPB Loss of Function and Amyotrophic Lateral Sclerosis

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1865
Author(s):  
Nica Borgese ◽  
Nicola Iacomino ◽  
Sara Francesca Colombo ◽  
Francesca Navone
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Adaptor Proteins ◽  
Loss Of Function ◽  
Membrane Contact Sites ◽  
Physiological Processes ◽  
Main Driver ◽  
Single Allele ◽  
Induced Pluripotent ◽  
Lateral Sclerosis

The VAP proteins are integral adaptor proteins of the endoplasmic reticulum (ER) membrane that recruit a myriad of interacting partners to the ER surface. Through these interactions, the VAPs mediate a large number of processes, notably the generation of membrane contact sites between the ER and essentially all other cellular membranes. In 2004, it was discovered that a mutation (p.P56S) in the VAPB paralogue causes a rare form of dominantly inherited familial amyotrophic lateral sclerosis (ALS8). The mutant protein is aggregation-prone, non-functional and unstable, and its expression from a single allele appears to be insufficient to support toxic gain-of-function effects within motor neurons. Instead, loss-of-function of the single wild-type allele is required for pathological effects, and VAPB haploinsufficiency may be the main driver of the disease. In this article, we review the studies on the effects of VAPB deficit in cellular and animal models. Several basic cell physiological processes are affected by downregulation or complete depletion of VAPB, impinging on phosphoinositide homeostasis, Ca2+ signalling, ion transport, neurite extension, and ER stress. In the future, the distinction between the roles of the two VAP paralogues (A and B), as well as studies on motor neurons generated from induced pluripotent stem cells (iPSC) of ALS8 patients will further elucidate the pathogenic basis of p.P56S familial ALS, as well as of other more common forms of the disease.

scholarly journals The Skeletal Muscle Emerges as a New Disease Target in Amyotrophic Lateral Sclerosis

2021 ◽  
Vol 11 (7) ◽  
pp. 671
Author(s):  
Oihane Pikatza-Menoio ◽  
Amaia Elicegui ◽  
Xabier Bengoetxea ◽  
Neia Naldaiz-Gastesi ◽  
Adolfo López de Munain ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amyotrophic Lateral Sclerosis ◽  
Muscle Tissue ◽  
Motor Neurons ◽  
Neurodegenerative Disorder ◽  
Disease Onset ◽  
Fine Tuning ◽  
Current State ◽  
The Central Nervous System ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that leads to progressive degeneration of motor neurons (MNs) and severe muscle atrophy without effective treatment. Most research on ALS has been focused on the study of MNs and supporting cells of the central nervous system. Strikingly, the recent observations of pathological changes in muscle occurring before disease onset and independent from MN degeneration have bolstered the interest for the study of muscle tissue as a potential target for delivery of therapies for ALS. Skeletal muscle has just been described as a tissue with an important secretory function that is toxic to MNs in the context of ALS. Moreover, a fine-tuning balance between biosynthetic and atrophic pathways is necessary to induce myogenesis for muscle tissue repair. Compromising this response due to primary metabolic abnormalities in the muscle could trigger defective muscle regeneration and neuromuscular junction restoration, with deleterious consequences for MNs and thereby hastening the development of ALS. However, it remains puzzling how backward signaling from the muscle could impinge on MN death. This review provides a comprehensive analysis on the current state-of-the-art of the role of the skeletal muscle in ALS, highlighting its contribution to the neurodegeneration in ALS through backward-signaling processes as a newly uncovered mechanism for a peripheral etiopathogenesis of the disease.

scholarly journals Spatiotemporal Dynamics of Molecular Pathology in Amyotrophic Lateral Sclerosis

2018 ◽  
Author(s):  
Silas Maniatis ◽  
Tarmo Äijö ◽  
Sanja Vickovic ◽  
Catherine Braine ◽  
Kristy Kang ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Early Time ◽  
Spatiotemporal Dynamics ◽  
Course Of Disease ◽  
Molecular Events ◽  
Als Patients ◽  
Lateral Sclerosis

AbstractParalysis occurring in amyotrophic lateral sclerosis (ALS) results from denervation of skeletal muscle as a consequence of motor neuron degeneration. Interactions between motor neurons and glia contribute to motor neuron loss, but the spatiotemporal ordering of molecular events that drive these processes in intact spinal tissue remains poorly understood. Here, we use spatial transcriptomics to obtain gene expression measurements of mouse spinal cords over the course of disease, as well as of postmortem tissue from ALS patients, to characterize the underlying molecular mechanisms in ALS. We identify novel pathway dynamics, regional differences between microglia and astrocyte populations at early time-points, and discern perturbations in several transcriptional pathways shared between murine models of ALS and human postmortem spinal cords.One Sentence SummaryAnalysis of the ALS spinal cord using Spatial Transcriptomics reveals spatiotemporal dynamics of disease driven gene regulation.

scholarly journals Pulmonary function in patients with Amyotrophic Lateral Sclerosis at disease onset

2015 ◽  
Vol 77 (3-4) ◽  
Author(s):  
B. Chandrasoma ◽  
D. Balfe ◽  
T. Naik ◽  
A. Elsayegh ◽  
M. Lewis ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Pulmonary Function ◽  
Muscle Weakness ◽  
Motor Neurons ◽  
Neurodegenerative Disorder ◽  
Pulmonary Function Tests ◽  
Disease Onset ◽  
Group Versus ◽  
Combination Methods ◽  
Lateral Sclerosis

Background. Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder affecting both the upper and lower motor neurons. Deteriorating pulmonary function as a reflection of progressive respiratory muscle weakness is a common feature, accounting for the majority of deaths. The aim of the study was to describe a trend in initial pulmonary function tests (PFT) of Amyotrophic Lateral Sclerosis (ALS) patients, in addition, differentiating between the types of disease onset, bulbar, limb muscle, and a combination. Methods. Initial PFT were gathered from 32 consecutive patients in our clinic with the diagnosis of ALS, they were categorized by the type of disease onset. Values obtained were referenced to the 95% confidence limits for normality. Results. There was evidence of significant reductions in both the FEV1 (64.7% predicted) and FVC (61.2%), with preservation of the FEV1/FVC (81.7%). The MVV was significantly reduced(43%). Total lung capacity was 93.2%, the residual volumes was increased at 145.7%. Subgroup analysis failed to show significant differences between types of disease onset. In the bulbar onset group (versus the limb group) there was a trend for the MVV to be further reduced (p=0.15) and the RV to be higher (157.4% versus 135.9%, P=0.24). Conclusions. ALS is a devastating disease that invariably leads to respiratory failure. Abnormal spirometric variables such as the FVC and MVV, likely reflect inspiratory muscle weakness and increased RV likely reflect expiratory muscle weakness. The type of disease onset did not result in a different pattern of PFT abnormalities.

scholarly journals Impact of traumatic brain injury on amyotrophic lateral sclerosis: from bedside to bench

2019 ◽  
Vol 122 (3) ◽  
pp. 1174-1185 ◽  
Author(s):  
Colin K. Franz ◽  
Divya Joshi ◽  
Elizabeth L. Daley ◽  
Rogan A. Grant ◽  
Kyriakos Dalamagkas ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Amyotrophic Lateral Sclerosis ◽  
Brain Injury ◽  
Motor Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Epidemiological Studies ◽  
History Of ◽  
Induced Pluripotent ◽  
Progressive Weakness ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of upper and lower motor neurons, which manifests clinically as progressive weakness. Although several epidemiological studies have found an association between traumatic brain injury (TBI) and ALS, there is not a consensus on whether TBI is an ALS risk factor. It may be that it can cause ALS in a subset of susceptible patients, based on a history of repetitive mild TBI and genetic predisposition. This cannot be determined based on clinical observational studies alone. Better preclinical models are necessary to evaluate the effects of TBI on ALS onset and progression. To date, only a small number of preclinical studies have been performed, mainly in the superoxide dismutase 1 transgenic rodents, which, taken together, have mixed results and notable methodological limitations. The more recent incorporation of additional animal models such as Drosophila flies, as well as patient-induced pluripotent stem cell-derived neurons, should facilitate a better understanding of a potential functional interaction between TBI and ALS.

scholarly journals Spatiotemporal dynamics of molecular pathology in amyotrophic lateral sclerosis

Science ◽  
2019 ◽  
Vol 364 (6435) ◽  
pp. 89-93 ◽  
Author(s):  
Silas Maniatis ◽  
Tarmo Äijö ◽  
Sanja Vickovic ◽  
Catherine Braine ◽  
Kristy Kang ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Early Time ◽  
Course Of Disease ◽  
Molecular Events ◽  
Motor Neuron Loss ◽  
Als Patients ◽  
Lateral Sclerosis

Paralysis occurring in amyotrophic lateral sclerosis (ALS) results from denervation of skeletal muscle as a consequence of motor neuron degeneration. Interactions between motor neurons and glia contribute to motor neuron loss, but the spatiotemporal ordering of molecular events that drive these processes in intact spinal tissue remains poorly understood. Here, we use spatial transcriptomics to obtain gene expression measurements of mouse spinal cords over the course of disease, as well as of postmortem tissue from ALS patients, to characterize the underlying molecular mechanisms in ALS. We identify pathway dynamics, distinguish regional differences between microglia and astrocyte populations at early time points, and discern perturbations in several transcriptional pathways shared between murine models of ALS and human postmortem spinal cords.

scholarly journals The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis

2017 ◽  
Vol 9 (391) ◽  
pp. eaaf3962 ◽  
Author(s):  
Keiko Imamura ◽  
Yuishin Izumi ◽  
Akira Watanabe ◽  
Kayoko Tsukita ◽  
Knut Woltjen ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neuron ◽  
Motor Neurons ◽  
New Drugs ◽  
Small Interfering Rnas ◽  
Altered Expression ◽  
Sporadic Als ◽  
Induced Pluripotent ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), a fatal disease causing progressive loss of motor neurons, still has no effective treatment. We developed a phenotypic screen to repurpose existing drugs using ALS motor neuron survival as readout. Motor neurons were generated from induced pluripotent stem cells (iPSCs) derived from an ALS patient with a mutation in superoxide dismutase 1 (SOD1). Results of the screen showed that more than half of the hits targeted the Src/c-Abl signaling pathway. Src/c-Abl inhibitors increased survival of ALS iPSC-derived motor neurons in vitro. Knockdown of Src or c-Abl with small interfering RNAs (siRNAs) also rescued ALS motor neuron degeneration. One of the hits, bosutinib, boosted autophagy, reduced the amount of misfolded mutant SOD1 protein, and attenuated altered expression of mitochondrial genes. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS or other forms of familial ALS caused by mutations in TAR DNA binding protein (TDP-43) or repeat expansions in C9orf72. Furthermore, bosutinib treatment modestly extended survival of a mouse model of ALS with an SOD1 mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS.

scholarly journals Pathological Roles of Wild-Type Cu, Zn-Superoxide Dismutase in Amyotrophic Lateral Sclerosis

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Yoshiaki Furukawa
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Superoxide Dismutase ◽  
Motor Neurons ◽  
Wild Type ◽  
Sporadic Als ◽  
Familial Form ◽  
Recent Developments ◽  
Lateral Sclerosis

Dominant mutations in a Cu, Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS). While it remains controversial how SOD1 mutations lead to onset and progression of the disease, manyin vitroandin vivostudies have supported a gain-of-toxicity mechanism where pathogenic mutations contribute to destabilizing a native structure of SOD1 and thus facilitate misfolding and aggregation. Indeed, abnormal accumulation of SOD1-positive inclusions in spinal motor neurons is a pathological hallmark in SOD1-related familial ALS. Furthermore, similarities in clinical phenotypes and neuropathology of ALS cases with and without mutations insod1gene have implied a disease mechanism involving SOD1 common to all ALS cases. Although pathogenic roles of wild-type SOD1 in sporadic ALS remain controversial, recent developments of novel SOD1 antibodies have made it possible to characterize wild-type SOD1 under pathological conditions of ALS. Here, I have briefly reviewed recent progress on biochemical and immunohistochemical characterization of wild-type SOD1 in sporadic ALS cases and discussed possible involvement of wild-type SOD1 in a pathomechanism of ALS.

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