RESUMEN
The human genome expresses thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability or translation of their overlapping genes1,2. Here we describe MAPT-AS1, a brain-enriched NAT that is conserved in primates and contains an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing for ribosomal RNA pairing with the MAPT mRNA internal ribosome entry site3. MAPT encodes tau, a neuronal intrinsically disordered protein (IDP) that stabilizes axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies4. Mutations in MAPT cause familial frontotemporal dementia, and common variations forming the MAPT H1 haplotype are a significant risk factor in many tauopathies5 and Parkinson's disease. Notably, expression of MAPT-AS1 or minimal essential sequences from MAPT-AS1 (including MIR) reduces-whereas silencing MAPT-AS1 expression increases-neuronal tau levels, and correlate with tau pathology in human brain. Moreover, we identified many additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. These results demonstrate a key role for MAPT-AS1 in tauopathies and reveal a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs6, with particular relevance for proteostasis in neurodegeneration.
Asunto(s)
Biosíntesis de Proteínas/genética , Proteostasis/genética , ARN sin Sentido/genética , Tauopatías/genética , Tauopatías/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Anciano , Animales , Sitios de Unión , Encéfalo/metabolismo , Encéfalo/patología , Estudios de Casos y Controles , Diferenciación Celular , Progresión de la Enfermedad , Femenino , Humanos , Sitios Internos de Entrada al Ribosoma/genética , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Neuronas/metabolismo , Neuronas/patología , Ribosomas/metabolismo , Proteínas tau/biosíntesisRESUMEN
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. Here we show that IR is a physiological process that is spatiotemporally regulated during MN lineage restriction and that IRTs in the cytoplasm are detected in as many as 13% (n = 2297) of the genes expressed during this process. We identify a major class of cytoplasmic IRTs that are not associated with reduced expression of their own genes but instead show a high capacity for RNA-binding protein and miRNA occupancy. Finally, we show that ALS-causing VCP mutations lead to a selective increase in cytoplasmic abundance of this particular class of IRTs, which in turn temporally coincides with an increase in the nuclear expression level of predicted miRNA target genes. Altogether, our study identifies a previously unrecognized class of cytoplasmic intronic sequences with potential regulatory function beyond gene expression.
Asunto(s)
MicroARNs , Neuronas Motoras , Humanos , Intrones , Citoplasma/genética , Citoplasma/metabolismo , Neurogénesis/genética , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
Telomere shortening is a well-established hallmark of cellular aging. Telomerase reverse transcriptase (TERT) plays a crucial role in maintaining the length of telomeres, which are specialised protective caps at the end of chromosomes. The lack of in vitro aging models, particularly for the central nervous system (CNS), has impeded progress in understanding aging and age-associated neurodegenerative diseases. In this study, we aimed to explore the possibility of inducing aging-associated features in cell types of the CNS using hiPSC (human induced pluripotent stem cell) technology. To achieve this, we utilised CRISPR/Cas9 to generate hiPSCs with a loss of telomerase function and shortened telomeres. Through directed differentiation, we generated motor neurons and astrocytes to investigate whether telomere shortening could lead to age-associated phenotypes. Our findings revealed that shortened telomeres induced age-associated characteristics in both motor neurons and astrocytes including increased cellular senescence, heightened inflammation, and elevated DNA damage. We also observed cell-type specific age-related morphology changes. Additionally, our study highlighted the fundamental role of TERT and telomere shortening in neural progenitor cell (NPC) proliferation and neuronal differentiation. This study serves as a proof of concept that telomere shortening can effectively induce aging-associated phenotypes, thereby providing a valuable tool to investigate age-related decline and neurodegenerative diseases.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Telomerasa , Humanos , Acortamiento del Telómero , Células Madre Pluripotentes Inducidas/metabolismo , Astrocitos/metabolismo , Telomerasa/genética , Telómero , Neuronas Motoras/metabolismo , FenotipoRESUMEN
AIMS: Although morphological attributes of cells and their substructures are recognised readouts of physiological or pathophysiological states, these have been relatively understudied in amyotrophic lateral sclerosis (ALS) research. METHODS: In this study, we integrate multichannel fluorescence high-content microscopy data with deep learning imaging methods to reveal-directly from unsegmented images-novel neurite-associated morphological perturbations associated with (ALS-causing) VCP-mutant human motor neurons (MNs). RESULTS: Surprisingly, we reveal that previously unrecognised disease-relevant information is withheld in broadly used and often considered 'generic' biological markers of nuclei (DAPI) and neurons ( ß III-tubulin). Additionally, we identify changes within the information content of ALS-related RNA binding protein (RBP) immunofluorescence imaging that is captured in VCP-mutant MN cultures. Furthermore, by analysing MN cultures exposed to different extrinsic stressors, we show that heat stress recapitulates key aspects of ALS. CONCLUSIONS: Our study therefore reveals disease-relevant information contained in a range of both generic and more specific fluorescent markers and establishes the use of image-based deep learning methods for rapid, automated and unbiased identification of biological hypotheses.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Aprendizaje Profundo , Neuronas Motoras/metabolismo , Neuritas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , FenotipoRESUMEN
RNA-binding proteins (RBPs) have been shown to play a key role in the pathogenesis of a variety of neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS) is an exemplar neurodegenerative disease characterised by rapid progression and relatively selective motor neuron loss. Nuclear-to-cytoplasmic mislocalisation and accumulation of RBPs have been identified as a pathological hallmark of the disease, yet the spatiotemporal responses of RBPs to different extrinsic stressors in human neurons remain incompletely understood. Here, we used healthy induced pluripotent stem cell (iPSC)-derived motor neurons to model how different types of cellular stress affect the nucleocytoplasmic localisation of key ALS-linked RBPs. We found that osmotic stress robustly induced nuclear loss of TDP-43, SPFQ, FUS, hnRNPA1 and hnRNPK, with characteristic changes in nucleocytoplasmic localisation in an RBP-dependent manner. Interestingly, we found that RBPs displayed stress-dependent characteristics, with unique responses to both heat and oxidative stress. Alongside nucleocytoplasmic protein distribution changes, we identified the formation of stress- and RBP-specific nuclear and cytoplasmic foci. Furthermore, the kinetics of nuclear relocalisation upon recovery from extrinsic stressors was also found to be both stress- and RBP-specific. Importantly, these experiments specifically highlight TDP-43 and FUS, two of the most recognised RBPs in ALS pathogenesis, as exhibiting delayed nuclear relocalisation following stress in healthy human motor neurons as compared to SFPQ, hnRNPA1 and hnRNPK. Notably, ALS-causing valosin containing protein (VCP) mutations did not disrupt the relocalisation dynamics of TDP-43 or FUS in human motor neurons following stress. An increased duration of TDP-43 and FUS within the cytoplasm after stress may render the environment more aggregation-prone, which may be poorly tolerated in the context of ALS and related neurodegenerative disorders. In summary, our study addresses stress-specific spatiotemporal responses of neurodegeneration-related RBPs in human motor neurons. The insights into the nucleocytoplasmic dynamics of RBPs provided here may be informative for future studies examining both disease mechanisms and therapeutic strategy.
Asunto(s)
Neuronas Motoras/metabolismo , Transporte de Proteínas/fisiología , Proteínas de Unión al ARN/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/fisiología , Mutación , Enfermedades Neurodegenerativas/metabolismo , Estrés Oxidativo/fisiología , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Análisis Espacio-TemporalAsunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Núcleo Celular/metabolismo , Neuronas Motoras/metabolismo , Células-Madre Neurales/metabolismo , Neuritas/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Proteína que Contiene Valosina/genética , Proteína que Contiene Valosina/metabolismo , Citoplasma/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , MutaciónRESUMEN
Neuronal differentiation requires building a complex intracellular architecture, and therefore the coordinated regulation of defined sets of genes. RNA-binding proteins (RBPs) play a key role in this regulation. However, while their action on individual mRNAs has been explored in depth, the mechanisms used to coordinate gene expression programs shaping neuronal morphology are poorly understood. To address this, we studied how the paradigmatic RBP IMP1 (IGF2BP1), an essential developmental factor, selects and regulates its RNA targets during the human neuronal differentiation. We perform a combination of system-wide and molecular analyses, revealing that IMP1 developmentally transitions to and directly regulates the expression of mRNAs encoding essential regulators of the microtubule network, a key component of neuronal morphology. Furthermore, we show that m6A methylation drives the selection of specific IMP1 mRNA targets and their protein expression during the developmental transition from neural precursors to neurons, providing a molecular principle for the onset of target selectivity.
Asunto(s)
Diferenciación Celular , Microtúbulos , Neuronas , ARN Mensajero , Proteínas de Unión al ARN , Humanos , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Microtúbulos/metabolismo , Neuronas/metabolismo , Neuronas/citología , Diferenciación Celular/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Metilación , Neurogénesis/genética , Adenosina/metabolismo , Adenosina/análogos & derivados , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are now known as parts of a disease spectrum with common pathological features and genetic causes. However, as both conditions are clinically heterogeneous, patient groups may be phenotypically similar but pathogenically and genetically variable. Despite numerous clinical trials, there remains no effective therapy for these conditions, which, in part, may be due to challenges of therapy development in a heterogeneous patient population. Disruption to protein homeostasis is a key feature of different forms of ALS and FTD. Targeting the endogenous protein chaperone system, the heat shock response (HSR) may, therefore, be a potential therapeutic approach. We conducted a preclinical study of a known pharmacological amplifier of the HSR, called arimoclomol, in mice with a mutation in valosin-containing protein (VCP) which causes both ALS and FTD in patients. We demonstrate that amplification of the HSR ameliorates the ALS/FTD-like phenotype in the spinal cord and brain of mutant VCP mice and prevents neuronal loss, replicating our earlier findings in the SOD1 mouse model of ALS. Moreover, in human cell models, we demonstrate improvements in pathology upon arimoclomol treatment in mutant VCP patient fibroblasts and iPSC-derived motor neurons. Our findings suggest that targeting of the HSR may have therapeutic potential, not only in non-SOD1 ALS, but also for the treatment of FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Animales , Ratones , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Demencia Frontotemporal/tratamiento farmacológico , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Hidroxilaminas/uso terapéutico , Respuesta al Choque Térmico , Mutación/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is characterized by nucleocytoplasmic mislocalization of the RNA-binding protein (RBP) TDP-43. However, emerging evidence suggests more widespread mRNA and protein mislocalization. Here, we employed nucleocytoplasmic fractionation, RNA sequencing, and mass spectrometry to investigate the localization of mRNA and protein in induced pluripotent stem cell-derived motor neurons (iPSMNs) from ALS patients with TARDBP and VCP mutations. ALS mutant iPSMNs exhibited extensive nucleocytoplasmic mRNA redistribution, RBP mislocalization, and splicing alterations. Mislocalized proteins exhibited a greater affinity for redistributed transcripts, suggesting a link between RBP mislocalization and mRNA redistribution. Notably, treatment with ML240, a VCP ATPase inhibitor, partially restored mRNA and protein localization in ALS mutant iPSMNs. ML240 induced changes in the VCP interactome and lysosomal localization and reduced oxidative stress and DNA damage. These findings emphasize the link between RBP mislocalization and mRNA redistribution in ALS motor neurons and highlight the therapeutic potential of VCP inhibition.
Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , ARN Mensajero/metabolismo , Neuronas Motoras/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteína que Contiene Valosina/genéticaRESUMEN
In the process of generating organoids, basement membrane extracts or Matrigel are often used to encapsulate cells but they are poorly defined and contribute to reproducibility issues. While defined hydrogels are increasingly used for organoid culture, the effects of replacing Matrigel with a defined hydrogel on neural progenitor growth, neural differentiation, and maturation within organoids are not well-explored. In this study, the use of alginate hydrogels as a Matrigel substitute in spinal cord organoid generation is explored. It is found that alginate encapsulation reduces organoid size variability by preventing organoid aggregation. Importantly, alginate supports neurogenesis and gliogenesis of the spinal cord organoids at a similar efficiency to Matrigel, with mature myelinated neurons observed by day 120. Furthermore, using alginate leads to lower expression of non-spinal markers such as FOXA2, suggesting better control over neural fate specification. To demonstrate the feasibility of using alginate-based organoid cultures as disease models, an isogenic pair of induced pluripotent stem cells discordant for the ALS-causing mutation TDP43G298S is used, where increased TDP43 mislocalization in the mutant organoids is observed. This study shows that alginate is an ideal substitute for Matrigel for spinal cord organoid derivation, especially when a xeno-free and fully defined 3D culture condition is desired.
Asunto(s)
Hidrogeles , Enfermedades de la Médula Espinal , Humanos , Hidrogeles/farmacología , Hidrogeles/metabolismo , Alginatos/farmacología , Reproducibilidad de los Resultados , Organoides , Enfermedades de la Médula Espinal/metabolismoRESUMEN
RNA binding proteins fulfil a wide number of roles in gene expression. Multiple mechanisms of RNA binding protein dysregulation have been implicated in the pathomechanisms of several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Oxidative stress and mitochondrial dysfunction also play important roles in these diseases. In this review, we highlight the mechanistic interplay between RNA binding protein dysregulation, oxidative stress and mitochondrial dysfunction in ALS. We also discuss different potential therapeutic strategies targeting these pathways.
RESUMEN
RNA binding proteins have been shown to play a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Mutations in valosin-containing protein (VCP/p97) cause ALS and exhibit the hallmark nuclear-to-cytoplasmic mislocalization of RNA binding proteins (RBPs). However, the mechanism by which mutations in VCP lead to this mislocalization of RBPs remains incompletely resolved. To address this, we used human-induced pluripotent stem cell-derived motor neurons carrying VCP mutations. We first demonstrate reduced nuclear-to-cytoplasmic ratios of transactive response DNA-binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS) and splicing factor proline and glutamine rich (SFPQ) in VCP mutant motor neurons. Upon closer analysis, we also find these RBPs are mislocalized to motor neuron neurites themselves. To address the hypothesis that altered function of the D2 ATPase domain of VCP causes RBP mislocalization, we used pharmacological inhibition of this domain in control motor neurons and found this does not recapitulate RBP mislocalization phenotypes. However, D2 domain inhibition in VCP mutant motor neurons was able to robustly reverse mislocalization of both TDP-43 and FUS, in addition to partially relocalizing SFPQ from the neurites. Together these results argue for a gain-of-function of D2 ATPase in VCP mutant human motor neurons driving the mislocalization of TDP-43 and FUS. Our data raise the intriguing possibility of harnessing VCP D2 ATPase inhibitors in the treatment of VCP-related ALS.
RESUMEN
The discovery of mutations within genes associated with autosomal recessive Parkinson's disease allowed for the identification of PINK1/Parkin regulated mitophagy as an important pathway for the removal of damaged mitochondria. While recent studies suggest that AKT-dependent signalling regulates Parkin recruitment to depolarised mitochondria, little is known as to whether this can also regulate PINK1 mitochondrial accumulation and downstream mitophagy. Here, we demonstrate that inhibition of AKT signalling decreases endogenous PINK1 accumulation in response to mitochondria depolarisation, subsequent Parkin recruitment, phosphorylation of ubiquitin, and ultimately mitophagy. Conversely, we show that upon stimulation of AKT signalling via insulin, the mitophagy pathway is increased in SHSY5Y cells. These data suggest that AKT signalling is an upstream regulator of PINK1 accumulation on damaged mitochondria. Importantly, we show that the AKT pathway also regulates endogenous PINK1-dependent mitophagy in human iPSC-derived neurons.
Asunto(s)
Mitocondrias/metabolismo , Mitofagia , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/fisiología , Línea Celular Tumoral , Humanos , Células Madre Pluripotentes Inducidas , Neuronas/citología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
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 and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.