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OBJECTIVE: There are compelling ethical and practical reasons for patient engagement in research (PEIR), however, evidence for best practices remains limited. We investigated PEIR as implemented in CAPTURE ALS, a longitudinal observational study, from study inception through the first 2.5 years of operations. METHODS: Data were drawn from three engagement initiatives: a community-led letter-writing campaign; consultation with patient and caregiver focus groups; and a study-embedded 'participant partner advisory council' (PPAC). Data were derived retrospectively from study documentation. We used the International Association of Public Participation (IAP2) participation spectrum as a framework for investigation. RESULTS: 2401 letters from community members to the Canadian government affirmed study objectives and advocated for funding. Feedback from focus group consultation influenced study design and supported the study's data-sharing plan. PPAC collaboration shaped all aspects of the study. Contributions included: co-creation of governance documents, input on study protocols and public-facing communication, and development of engagement webinars for study participants and feedback surveys. Effective communication practices fostered collaboration and helped avoid tokenistic engagement. CAPTURE ALS encompassed all IAP2 participation levels. CONCLUSIONS: CAPTURE ALS was shaped by meaningful engagement initiatives over the course of the study. Lessons learned included: begin early and embed PEIR within research; build relationships and foster mutual learning; be flexible, open to adaptation, and seek diversity. Primary challenges included funding for early implementation, time needed to maintain relationships, and attrition due to disease progression. All IAP2 participation levels contributed to meaningful PEIR. 'Empowerment' was demonstrated through advocacy.
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Esclerosis Amiotrófica Lateral , Participación del Paciente , Humanos , Esclerosis Amiotrófica Lateral/psicología , Estudios Longitudinales , Participación del Paciente/psicología , Participación del Paciente/estadística & datos numéricos , Canadá/epidemiología , Masculino , Investigación Biomédica , FemeninoRESUMEN
Oligodendrocyte (OL) injury and subsequent loss is a pathologic hallmark of multiple sclerosis (MS). Stress granules (SGs) are membrane-less organelles containing mRNAs stalled in translation and considered as participants of the cellular response to stress. Here we show SGs in OLs in active and inactive areas of MS lesions as well as in normal-appearing white matter. In cultures of primary human adult brain derived OLs, metabolic stress conditions induce transient SG formation in these cells. Combining pro-inflammatory cytokines, which alone do not induce SG formation, with metabolic stress results in persistence of SGs. Unlike sodium arsenite, metabolic stress induced SG formation is not blocked by the integrated stress response inhibitor. Glycolytic inhibition also induces persistent SGs indicating the dependence of SG formation and disassembly on the energetic glycolytic properties of human OLs. We conclude that SG persistence in OLs in MS reflects their response to a combination of metabolic stress and pro-inflammatory conditions.
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Gránulos Citoplasmáticos , Esclerosis Múltiple , Humanos , Gránulos Citoplasmáticos/metabolismo , Gránulos de Estrés , Oligodendroglía , Citocinas/metabolismo , Estrés Fisiológico , Esclerosis Múltiple/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a complex, neurodegenerative disorder in which alterations in structural, physiological, and metabolic parameters act synergistically. Over the last decade there has been a considerable focus on developing drugs to slow the progression of the disease. Despite this, only four disease-modifying therapies are approved in North America. Although additional research is required for a thorough understanding of ALS, we have accumulated a large amount of knowledge that could be better integrated into future clinical trials to accelerate drug development and provide patients with improved treatment options. It is likely that future, successful ALS treatments will take a multi-pronged therapeutic approach, targeting different pathways, akin to personalized medicine in oncology. In this review, we discuss the link between ALS pathophysiology and treatments, looking at the therapeutic failures as learning opportunities that can help us refine and optimize drug development.
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Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismoRESUMEN
Multiple sclerosis is a chronic neuroinflammatory disorder characterized by demyelination, oligodendrocyte damage/loss and neuroaxonal injury in the context of immune cell infiltration in the CNS. No neuroprotective therapy is available to promote the survival of oligodendrocytes and protect their myelin processes in immune-mediated demyelinating diseases. Pro-inflammatory CD4 Th17 cells can interact with oligodendrocytes in multiple sclerosis and its animal model, causing injury to myelinating processes and cell death through direct contact. However, the molecular mechanisms underlying the close contact and subsequent detrimental interaction of Th17 cells with oligodendrocytes remain unclear. In this study we used single cell RNA sequencing, flow cytometry and immunofluorescence studies on CNS tissue from multiple sclerosis subjects, its animal model and controls to characterize the expression of cell adhesion molecules by mature oligodendrocytes. We found that a significant proportion of human and murine mature oligodendrocytes express melanoma cell adhesion molecule (MCAM) and activated leukocyte cell adhesion molecule (ALCAM) in multiple sclerosis, in experimental autoimmune encephalomyelitis and in controls, although their regulation differs between human and mouse. We observed that exposure to pro-inflammatory cytokines or to human activated T cells are associated with a marked downregulation of the expression of MCAM but not of ALCAM at the surface of human primary oligodendrocytes. Furthermore, we used in vitro live imaging, immunofluorescence and flow cytometry to determine the contribution of these molecules to Th17-polarized cell adhesion and cytotoxicity towards human oligodendrocytes. Silencing and blocking ALCAM but not MCAM limited prolonged interactions between human primary oligodendrocytes and Th17-polarized cells, resulting in decreased adhesion of Th17-polarized cells to oligodendrocytes and conferring significant protection of oligodendrocytic processes. In conclusion, we showed that human oligodendrocytes express MCAM and ALCAM, which are differently modulated by inflammation and T cell contact. We found that ALCAM is a ligand for Th17-polarized cells, contributing to their capacity to adhere and induce damage to human oligodendrocytes, and therefore could represent a relevant target for neuroprotection in multiple sclerosis.
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Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Humanos , Ratones , Animales , Linfocitos T CD4-Positivos/metabolismo , Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Adhesión Celular , Oligodendroglía/metabolismoRESUMEN
In recent years, there has been an emphasis on the role of phase-separated biomolecular condensates, especially stress granules, in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). This is largely due to several ALS-associated mutations occurring in genes involved in stress granule assembly and observations that pathological inclusions detected in ALS patient neurons contain stress granule proteins, including the ALS-linked proteins TDP-43 and FUS. However, protein components of stress granules are also found in numerous other phase-separated biomolecular condensates under physiological conditions which are inadequately discussed in the context of ALS. In this review, we look beyond stress granules and describe the roles of TDP-43 and FUS in physiological condensates occurring in the nucleus and neurites, such as the nucleolus, Cajal bodies, paraspeckles and neuronal RNA transport granules. We also discuss the consequences of ALS-linked mutations in TDP-43 and FUS on their ability to phase separate into these stress-independent biomolecular condensates and perform their respective functions. Importantly, biomolecular condensates sequester multiple overlapping protein and RNA components, and their dysregulation could contribute to the observed pleiotropic effects of both sporadic and familial ALS on RNA metabolism.
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Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Condensados Biomoleculares , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Mutación/genética , ARNRESUMEN
In this issue of Neuron, Wang et al.1 show that the RNA-binding protein G3BP2 interacts with Tau in human neurons and in brains of patients with Alzheimer's disease (AD), suggesting a new role for G3BP2 with implications for therapeutic sequestration of Tau in neurodegenerative diseases.
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Enfermedad de Alzheimer , Humanos , Encéfalo , NeuronasRESUMEN
The absence of disease modifying treatments for amyotrophic lateral sclerosis (ALS) is in large part a consequence of its complexity and heterogeneity. Deep clinical and biological phenotyping of people living with ALS would assist in the development of effective treatments and target specific biomarkers to monitor disease progression and inform on treatment efficacy. The objective of this paper is to present the Comprehensive Analysis Platform To Understand Remedy and Eliminate ALS (CAPTURE ALS), an open and translational platform for the scientific community currently in development. CAPTURE ALS is a Canadian-based platform designed to include participants' voices in its development and through execution. Standardized methods will be used to longitudinally characterize ALS patients and healthy controls through deep clinical phenotyping, neuroimaging, neurocognitive and speech assessments, genotyping and multisource biospecimen collection. This effort plugs into complementary Canadian and international initiatives to share common resources. Here, we describe in detail the infrastructure, operating procedures, and long-term vision of CAPTURE ALS to facilitate and accelerate translational ALS research in Canada and beyond.
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Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Canadá , Biomarcadores , Progresión de la Enfermedad , NeuroimagenRESUMEN
Responding effectively to external stress is crucial for neurons. Defective stress granule dynamics has been hypothesized as one of the pathways that renders motor neurons in amyotrophic lateral sclerosis (ALS) more prone to early death. Specifically, it is thought that stress granules seed the cytoplasmic TDP-43 inclusions that are observed in the neurons of most ALS patients, as well as ~50% of all frontotemporal dementia (FTD) patients. In this study, we tested this hypothesis in an intact mammalian nervous system. We established an in vivo heat stress paradigm in mice that effectively triggers the eIF2α pathway and the formation of stress granules in the CNS. In non-transgenic mice, we report an age-dependent decline in the formation of heat-induced stress granules, with 18-month-old animals showing a significant impairment. Furthermore, although neuronal stress granules were robustly observed in non-transgenic mice and SOD1G93A mice, they were largely absent in age-matched TDP-43M337V animals. The observed defect in stress granule formation in TDP-43M337V mice correlated with deficits in expression of key protein components typically required for phase separation. Lastly, while TDP-43 was not localized to stress granules, we observed complete nuclear depletion of TDP-43 in a subset of neurons, with the highest proportion being in the TDP-43M337V mice. Overall, our results indicate that mutant TDP-43 expression is associated with defective stress granule assembly and increased TDP-43 nuclear depletion in the mammalian nervous system, which could be relevant to ALS/FTD pathogenesis.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Ratones , Animales , Esclerosis Amiotrófica Lateral/metabolismo , Demencia Frontotemporal/patología , Gránulos de Estrés , Neuronas Motoras/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Mamíferos/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease primarily impacting motor neurons. Mutations in superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS. Several of these mutations lead to misfolding or toxic gain of function in the SOD1 protein. Recently, we reported that misfolded SOD1 interacts with TNF receptor-associated factor 6 (TRAF6) in the SOD1G93A rat model of ALS. Further, we showed in cultured cells that several mutant SOD1 proteins, but not wildtype SOD1 protein, interact with TRAF6 via the MATH domain. Here, we sought to uncover the structural details of this interaction through molecular dynamics (MD) simulations of a dimeric model system, coarse grained using the AWSEM force field. We used direct MD simulations to identify buried residues, and predict binding poses by clustering frames from the trajectories. Metadynamics simulations were also used to deduce preferred binding regions on the protein surfaces from the potential of the mean force in orientation space. Well-folded SOD1 was found to bind TRAF6 via co-option of its native homodimer interface. However, if loops IV and VII of SOD1 were disordered, as typically occurs in the absence of stabilizing Zn2+ ion binding, these disordered loops now participated in novel interactions with TRAF6. On TRAF6, multiple interaction hot-spots were distributed around the equatorial region of the MATH domain beta barrel. Expression of TRAF6 variants with mutations in this region in cultured cells demonstrated that TRAF6T475 facilitates interaction with different SOD1 mutants. These findings contribute to our understanding of the disease mechanism and uncover potential targets for the development of therapeutics.
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Esclerosis Amiotrófica Lateral , Superóxido Dismutasa-1 , Factor 6 Asociado a Receptor de TNF , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Modelos Animales de Enfermedad , Mutación , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Ratas , Superóxido Dismutasa-1/química , Superóxido Dismutasa-1/genética , Factor 6 Asociado a Receptor de TNF/químicaRESUMEN
Stress granules have gained considerable exposure and interest in recent years. These micron-sized entities, composed of RNA and protein, form following a stress exposure and have been linked to several pathologies. Understanding stress granule function is paramount but has been arduous due to the membraneless nature of these organelles. Several new methodologies have recently been developed to catalogue the protein and RNA composition of stress granules. Collectively, this work has provided important insights to potential stress granule functions as well as molecular mechanisms for their assembly and disassembly. This chapter reviews the latest advancements in the understanding of stress granule dynamics and discusses the various protocols developed to study their composition.
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Gránulos Citoplasmáticos , Gránulos de Estrés , Gránulos Citoplasmáticos/metabolismo , Orgánulos/metabolismo , Estrés FisiológicoRESUMEN
RNA binding proteins (RBPs) play a key role in cellular growth, homoeostasis and survival and are tightly regulated. A deep understanding of their spatiotemporal regulation is needed to understand their contribution to physiology and pathology. Here, we have characterized the spatiotemporal expression pattern of hnRNP A1 and its splice variant hnRNP A1B in mice. We have found that hnRNP A1B expression is more restricted to the CNS compared to hnRNP A1, and that it can form an SDS-resistant dimer in the CNS. Also, hnRNP A1B expression becomes progressively restricted to motor neurons in the ventral horn of the spinal cord, compared to hnRNP A1 which is more broadly expressed. We also demonstrate that hnRNP A1B is present in neuronal processes, while hnRNP A1 is absent. This finding supports a hypothesis that hnRNP A1B may have a cytosolic function in neurons that is not shared with hnRNP A1. Our results demonstrate that both isoforms are differentially expressed across tissues and have distinct localization profiles, suggesting that the two isoforms may have specific subcellular functions that can uniquely contribute to disease progression.
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A recent study by Gwon et al. identified context-specific ubiquitination of G3BP1 as critical for stress granule disassembly via VCP and the adaptor FAF2. This study provides new insights into stress granule dynamics, with potential implications for neurodegenerative disease.
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Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , ADN Helicasas/metabolismo , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARNRESUMEN
TDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death. Ras-GAP SH3-domain-binding protein 1 (G3BP1) is a critical stress granule assembly factor. Here, we define that TDP-43 stabilizes G3BP1 transcripts via direct binding of a highly conserved cis regulatory element within the 3' untranslated region. Moreover, we show in vitro and in vivo that nuclear TDP-43 depletion is sufficient to reduce G3BP1 protein levels. Finally, we establish that G3BP1 transcripts are reduced in ALS/FTD patient neurons bearing TDP-43 cytoplasmic inclusions/nuclear depletion. Thus, our data indicate that, in ALS/FTD, there is a compromised stress granule response in disease-affected neurons due to impaired G3BP1 mRNA stability caused by TDP-43 nuclear depletion. These data implicate TDP-43 and G3BP1 loss of function as contributors to disease.
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Esclerosis Amiotrófica Lateral/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/metabolismo , Neuronas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Esclerosis Amiotrófica Lateral/patología , Células Cultivadas , Demencia Frontotemporal/patología , Humanos , Neuronas/patología , ARN MensajeroRESUMEN
Cytoplasmic stress granules (SGs) are dynamic foci containing translationally arrested mRNA and RNA-binding proteins (RBPs) that form in response to a variety of cellular stressors. It has been debated that SGs may evolve into cytoplasmic inclusions observed in many neurodegenerative diseases. Recent studies have examined the SG proteome by interrogating the interactome of G3BP1. However, it is widely accepted that multiple baits are required to capture the full SG proteome. To gain further insight into the SG proteome, we employed immunoprecipitation coupled with mass spectrometry of endogenous Caprin-1, an RBP implicated in mRNP granules. Overall, we identified 1543 proteins that interact with Caprin-1. Interactors under stressed conditions were primarily annotated to the ribosome, spliceosome, and RNA transport pathways. We validated four Caprin-1 interactors that localized to arsenite-induced SGs: ANKHD1, TALIN-1, GEMIN5, and SNRNP200. We also validated these stress-induced interactions in SH-SY5Y cells and further determined that SNRNP200 also associated with osmotic- and thermal-induced SGs. Finally, we identified SNRNP200 in cytoplasmic aggregates in amyotrophic lateral sclerosis (ALS) spinal cord and motor cortex. Collectively, our findings provide the first description of the Caprin-1 protein interactome, identify novel cytoplasmic SG components, and reveal a SG protein in cytoplasmic aggregates in ALS patient neurons. Proteomic data collected in this study are available via ProteomeXchange with identifier PXD023271.
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Gránulos Citoplasmáticos , ADN Helicasas , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Proteómica , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN , Proteínas de Unión al ARN/genéticaRESUMEN
Ras-GTPase-activating protein (GAP)-binding protein 1 (G3BP1) is a multi-functional protein that is best known for its role in the assembly and dynamics of stress granules. Recent studies have highlighted that G3BP1 also has other functions related to RNA metabolism. In the context of disease, G3BP1 has been therapeutically targeted in cancers because its over-expression is correlated with proliferation of cancerous cells and metastasis. However, evidence suggests that G3BP1 is essential for neuronal development and possibly neuronal maintenance. In this review, we will examine the many functions that are carried out by G3BP1 in the context of neurons and speculate how these functions are critical to the progression of neurodegenerative diseases. Additionally, we will highlight the similarities and differences between G3BP1 and the closely related protein G3BP2, which is frequently overlooked. Although G3BP1 and G3BP2 have both been deemed important for stress granule assembly, their roles may differ in other cellular pathways, some of which are specific to the CNS, and presents an opportunity for further exploration.
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ADN Helicasas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Animales , HumanosRESUMEN
Progressive loss of neuromuscular junctions (NMJs) is an early event in amyotrophic lateral sclerosis (ALS), preceding the global degeneration of motor axons and being accompanied by new axonal sprouting within the same axonal arbor. Some aspects of ALS onset and progression seem to be affected by sex in animal models of the disease. However, whether there are sex-specific differences in the pattern or time course of NMJ loss and repair within single motor axons remains unknown. We performed further analysis of a previously published in vivo dataset, obtained from male and female SOD1G37R mice. We found that NMJ losses are as frequent in male and female motor axons but, intriguingly, axonal sprouting is more frequent in female than male mice, resulting in a net increase of axonal arborization. Interestingly, these numerous new axonal branches in female mice are associated with a slightly faster decline in grip strength, increased NMJ denervation, and reduced α-motor neuron survival. Collectively, these results suggest that excessive axonal sprouting and motor-unit (MU) expansion in female SOD1G37R mice are maladaptive during ALS progression.
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Esclerosis Amiotrófica Lateral , Esclerosis Amiotrófica Lateral/genética , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Transgénicos , Neuronas Motoras , Unión Neuromuscular , Superóxido Dismutasa/genética , Superóxido Dismutasa-1/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1G93A rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1G93A rats and conformation-restricted antibodies specific for misfolded SOD1 (B8H10 and AMF7-63), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding proteins that uniquely interacted with either AMF7-63 or B8H10-reactive SOD1 conformers as well as a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) as a SOD1 interactor, and we determined that exposure of the SOD1 functional loops facilitates this interaction. Of note, this conformational change was not universally fulfilled by all SOD1 variants and differentiated TRAF6 interacting from TRAF6 noninteracting SOD1 variants. Functionally, TRAF6 stimulated polyubiquitination and aggregation of the interacting SOD1 variants. TRAF6 E3 ubiquitin ligase activity was required for the former but was dispensable for the latter, indicating that TRAF6-mediated polyubiquitination and aggregation of the SOD1 variants are independent events. We propose that the interaction between misfolded SOD1 and TRAF6 may be relevant to the etiology of ALS.
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Esclerosis Amiotrófica Lateral/patología , Superóxido Dismutasa-1/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Anticuerpos/inmunología , Línea Celular , Modelos Animales de Enfermedad , Mitocondrias/metabolismo , Mutagénesis Sitio-Dirigida , FN-kappa B/metabolismo , Agregado de Proteínas , Pliegue de Proteína , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Transgénicas , Superóxido Dismutasa-1/química , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/inmunología , Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidores , Factor 6 Asociado a Receptor de TNF/genética , UbiquitinaciónRESUMEN
In recent years, cytoplasmic RNA granules, which are micron-sized membrane-less entities formed by phase separation, have progressively gained recognition as essential constituents of neuronal RNA metabolism. Stress granules form under adverse growth conditions in order to protect nontranslating mRNA, shift translation toward the production of prosurvival factors, as well as potentially serve as hubs for intracellular signaling. In contrast, processing bodies play a role in RNA degradation in both stressed and homeostatic conditions. Lastly, transport granules permit, as their name indicates, the transport of mRNA within neurons. All of these granule subtypes are required for proper neuronal function; thus, impairments in their regulation and/or composition are expected to be deleterious. Here, we review these cytoplasmic RNA granule subtypes and discuss how they have been implicated in some neurodegenerative diseases.
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Enfermedades Neurodegenerativas , ARN Mensajero , Gránulos Citoplasmáticos/metabolismo , Humanos , Enfermedades Neurodegenerativas/fisiopatología , ARN Mensajero/metabolismoRESUMEN
Despite being an early event in ALS, it remains unclear whether the denervation of neuromuscular junctions (NMJ) is simply the first manifestation of a globally degenerating motor neuron. Using in vivo imaging of single axons and their NMJs over a three-month period, we identify that single motor-units are dismantled asynchronously in SOD1G37R mice. We reveal that weeks prior to complete axonal degeneration, the dismantling of axonal branches is accompanied by contemporaneous new axonal sprouting resulting in synapse formation onto nearby NMJs. Denervation events tend to propagate from the first lost NMJ, consistent with a contribution of neuromuscular factors extrinsic to motor neurons, with distal branches being more susceptible. These results show that NMJ denervation in ALS is a complex and dynamic process of continuous denervation and new innervation rather than a manifestation of sudden global motor neuron degeneration.