RESUMEN
The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.
Asunto(s)
Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN/genética , Degeneración Nerviosa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Axones/metabolismo , Proteína C9orf72/genética , Muerte Celular , Células Cultivadas , Corteza Cerebral/patología , Cromatina/metabolismo , Daño del ADN , Modelos Animales de Enfermedad , Drosophila , Ratones Endogámicos C57BL , Degeneración Nerviosa/patología , Estabilidad Proteica , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Regiones no Traducidas 3'/genética , Secuencia de Bases , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Mutación/genética , Motivos de Nucleótidos/genética , Transición de Fase , Mutación Puntual/genética , Poli A/metabolismo , Unión Proteica , Multimerización de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Eliminación de SecuenciaRESUMEN
Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Nucleotidiltransferasas/metabolismo , Alarminas/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/metabolismo , Interferón Tipo I/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Degeneración Nerviosa/patología , Fosfotransferasas (Aceptor de Grupo Alcohol) , Subunidades de Proteína/metabolismo , Transducción de SeñalRESUMEN
RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-ß2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-ß1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-ß2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-ß2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.
Asunto(s)
Transporte Activo de Núcleo Celular , Priones/química , Proteínas de Unión al ARN/química , Receptores Citoplasmáticos y Nucleares/química , Adulto , Anciano , Animales , Citoplasma/química , Proteínas de Unión al ADN/química , Drosophila melanogaster , Femenino , Proteínas Fluorescentes Verdes/química , Células HEK293 , Células HeLa , Homeostasis , Humanos , Carioferinas/química , Masculino , Persona de Mediana Edad , Chaperonas Moleculares/química , Mutación , Enfermedades Neurodegenerativas/patología , Dominios Proteicos , Proteína EWS de Unión a ARN/química , Factores Asociados con la Proteína de Unión a TATA/química , beta Carioferinas/químicaRESUMEN
Over 40% of the human genome is composed of retrotransposons, DNA species that hold the potential to replicate via an RNA intermediate and are evolutionarily related to retroviruses. Retrotransposons are most studied for their ability to jump within a genome, which can cause DNA damage and novel insertional mutations. Retrotransposon-encoded products, including viral-like proteins, double-stranded RNAs, and extrachromosomal circular DNAs, can also be potent activators of the innate immune system. A growing body of evidence suggests that retrotransposons are activated in age-related neurodegenerative disorders and that such activation causally contributes to neurotoxicity. Here we provide an overview of retrotransposon biology and outline evidence of retrotransposon activation in age-related neurodegenerative disorders, with an emphasis on those involving TAR-DNA binding protein-43 (TDP-43) and tau. Studies to date provide the basis for ongoing clinical trials and hold promise for innovative strategies to ameliorate the adverse effects of retrotransposon dysregulation in neurodegenerative disorders.
Asunto(s)
Envejecimiento , Retrovirus Endógenos , Enfermedades Neurodegenerativas , Retroelementos , Humanos , Enfermedades Neurodegenerativas/genética , Retroelementos/genética , Retrovirus Endógenos/genética , Animales , Envejecimiento/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
RNA methylation at adenosine N6 (m6A) is one of the most common RNA modifications, impacting RNA stability, transport, and translation. Previous studies uncovered RNA destabilization in amyotrophic lateral sclerosis (ALS) models in association with accumulation of the RNA-binding protein TDP43. Here, we show that TDP43 recognizes m6A RNA and that RNA methylation is critical for both TDP43 binding and autoregulation. We also observed extensive RNA hypermethylation in ALS spinal cord, corresponding to methylated TDP43 substrates. Emphasizing the importance of m6A for TDP43 binding and function, we identified several m6A factors that enhance or suppress TDP43-mediated toxicity via single-cell CRISPR-Cas9 in primary neurons. The most promising modifier-the canonical m6A reader YTHDF2-accumulated within ALS spinal neurons, and its knockdown prolonged the survival of human neurons carrying ALS-associated mutations. Collectively, these data show that m6A modifications modulate RNA binding by TDP43 and that m6A is pivotal for TDP43-related neurodegeneration in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Esclerosis Amiotrófica Lateral/patología , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Metilación , Neuronas/metabolismo , ARN/genética , ARN/metabolismoRESUMEN
Nuclear exclusion of the RNA- and DNA-binding protein TDP-43 can induce neurodegeneration in different diseases. Diverse processes have been implicated to influence TDP-43 mislocalization, including disrupted nucleocytoplasmic transport (NCT); however, the physiological pathways that normally ensure TDP-43 nuclear localization are unclear. The six-transmembrane enzyme glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) cleaves the glycosylphosphatidylinositol (GPI) anchor that tethers some proteins to the membrane. Here we show that GDE2 maintains TDP-43 nuclear localization by regulating the dynamics of canonical Wnt signaling. Ablation of GDE2 causes aberrantly sustained Wnt activation in adult neurons, which is sufficient to cause NCT deficits, nuclear pore abnormalities, and TDP-43 nuclear exclusion. Disruption of GDE2 coincides with TDP-43 abnormalities in postmortem tissue from patients with amyotrophic lateral sclerosis (ALS). Further, GDE2 deficits are evident in human neural cell models of ALS, which display erroneous Wnt activation that, when inhibited, increases mRNA levels of genes regulated by TDP-43. Our study identifies GDE2 as a critical physiological regulator of Wnt signaling in adult neurons and highlights Wnt pathway activation as an unappreciated mechanism contributing to nucleocytoplasmic transport and TDP-43 abnormalities in disease.
Asunto(s)
Proteínas de Unión al ADN , Neuronas , Hidrolasas Diéster Fosfóricas , Vía de Señalización Wnt , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Animales , Neuronas/metabolismo , Ratones , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismoRESUMEN
Despite the prominent role of TDP-43 in neurodegeneration, its physiological and pathological functions are not fully understood. Here, we report an unexpected role of TDP-43 in the formation of dynamic, reversible, liquid droplet-like nuclear bodies (NBs) in response to stress. Formation of NBs alleviates TDP-43-mediated cytotoxicity in mammalian cells and fly neurons. Super-resolution microscopy reveals distinct functions of the two RRMs in TDP-43 NB formation. TDP-43 NBs are partially colocalized with nuclear paraspeckles, whose scaffolding lncRNA NEAT1 is dramatically upregulated in stressed neurons. Moreover, increase of NEAT1 promotes TDP-43 liquid-liquid phase separation (LLPS) in vitro. Finally, we discover that the ALS-associated mutation D169G impairs the NEAT1-mediated TDP-43 LLPS and NB assembly, causing excessive cytoplasmic translocation of TDP-43 to form stress granules, which become phosphorylated TDP-43 cytoplasmic foci upon prolonged stress. Together, our findings suggest a stress-mitigating role and mechanism of TDP-43 NBs, whose dysfunction may be involved in ALS pathogenesis.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Neuronas/metabolismo , ARN Largo no Codificante/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Arsenitos/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/ultraestructura , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Drosophila melanogaster , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Cuerpos de Inclusión Intranucleares/efectos de los fármacos , Cuerpos de Inclusión Intranucleares/ultraestructura , Ratones , Mutación , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Cultivo Primario de Células , Transporte de Proteínas/efectos de los fármacos , ARN Largo no Codificante/metabolismo , Transducción de Señal , Estrés FisiológicoRESUMEN
Aggregation of the RNA-binding protein TAR DNA-binding protein 43 (TDP-43) is the key neuropathological feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In physiological conditions, TDP-43 is predominantly nuclear, forms oligomers, and is contained in biomolecular condensates assembled by liquid-liquid phase separation (LLPS). In disease, TDP-43 forms cytoplasmic or intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Using a variety of cellular systems to express structure-based TDP-43 variants, including human neurons and cell lines with near-physiological expression levels, we show that oligomerization and RNA binding govern TDP-43 stability, splicing functionality, LLPS, and subcellular localization. Importantly, our data reveal that TDP-43 oligomerization is modulated by RNA binding. By mimicking the impaired proteasomal activity observed in ALS/FTLD patients, we found that monomeric TDP-43 forms inclusions in the cytoplasm, whereas its RNA binding-deficient counterpart aggregated in the nucleus. These differentially localized aggregates emerged via distinct pathways: LLPS-driven aggregation in the nucleus and aggresome-dependent inclusion formation in the cytoplasm. Therefore, our work unravels the origins of heterogeneous pathological species reminiscent of those occurring in TDP-43 proteinopathy patients.
Asunto(s)
Esclerosis Amiotrófica Lateral , Degeneración Lobar Frontotemporal , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , Proteínas de Unión al ADN/metabolismo , Neuronas/metabolismo , ARN/genéticaRESUMEN
Oxidative stress, which can be activated by a variety of environmental risk factors, has been implicated as an important pathogenic factor for inflammatory bowel disease (IBD). However, how oxidative stress drives IBD onset remains elusive. Here, we found that oxidative stress was strongly activated in inflamed tissues from both ulcerative colitis patients and Crohn's disease patients, and it caused nuclear-to-cytosolic TDP-43 transport and a reduction in the TDP-43 protein level. To investigate the function of TDP-43 in IBD, we inducibly deleted exons 2 to 3 of Tardbp (encoding Tdp-43) in mouse intestinal epithelium, which disrupted its nuclear localization and RNA-processing function. The deletion gave rise to spontaneous intestinal inflammation by inducing epithelial cell necroptosis. Suppression of the necroptotic pathway with deletion of Mlkl or the RIP1 inhibitor Nec-1 rescued colitis phenotypes. Mechanistically, disruption of nuclear TDP-43 caused excessive R-loop accumulation, which triggered DNA damage and genome instability and thereby induced PARP1 hyperactivation, leading to subsequent NAD+ depletion and ATP loss, consequently activating mitochondrion-dependent necroptosis in intestinal epithelial cells. Importantly, restoration of cellular NAD+ levels with NAD+ or NMN supplementation, as well as suppression of ALKBH7, an α-ketoglutarate dioxygenase in mitochondria, rescued TDP-43 deficiency-induced cell death and intestinal inflammation. Furthermore, TDP-43 protein levels were significantly inversely correlated with γ-H2A.X and p-MLKL levels in clinical IBD samples, suggesting the clinical relevance of TDP-43 deficiency-induced mitochondrion-dependent necroptosis. Taken together, these findings identify a unique pathogenic mechanism that links oxidative stress to intestinal inflammation and provide a potent and valid strategy for IBD intervention.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Necroptosis , Humanos , Animales , Ratones , NAD/metabolismo , Estructuras R-Loop , Enfermedades Inflamatorias del Intestino/metabolismo , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Inflamación/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Mitocondrias/metabolismoRESUMEN
Cytoplasmic mislocalization and aggregation of TDP-43 protein are hallmarks of amyotrophic lateral sclerosis (ALS) and are observed in the vast majority of both familial and sporadic cases. How these two interconnected processes are regulated on a molecular level, however, remains enigmatic. Genome-wide screens for modifiers of the ALS-associated genes TDP-43 and FUS have identified the phospholipase D (Pld) pathway as a key regulator of ALS-related phenotypes in the fruit fly Drosophila melanogaster [M. W. Kankel et al., Genetics 215, 747-766 (2020)]. Here, we report the results of our search for downstream targets of the enzymatic product of Pld, phosphatidic acid. We identify two conserved negative regulators of the cAMP/PKA signaling pathway, the phosphodiesterase dunce and the inhibitory subunit PKA-R2, as modifiers of pathogenic phenotypes resulting from overexpression of the Drosophila TDP-43 ortholog TBPH. We show that knockdown of either of these genes results in a mitigation of both TBPH aggregation and mislocalization in larval motor neuron cell bodies, as well as an amelioration of adult-onset motor defects and shortened lifespan induced by TBPH. We determine that PKA kinase activity is downstream of both TBPH and Pld and that overexpression of the PKA target CrebA can rescue TBPH mislocalization. These findings suggest a model whereby increasing cAMP/PKA signaling can ameliorate the molecular and functional effects of pathological TDP-43.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico , AMP Cíclico , Proteínas de Unión al ADN , Proteínas de Drosophila , Drosophila melanogaster , Transducción de Señal , Animales , AMP Cíclico/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Humanos , Neuronas Motoras/metabolismoRESUMEN
RNA-binding proteins (RBPs) are critical players in RNA expression and metabolism, thus, the proper regulation of this class of proteins is critical for cellular health. Regulation of RBPs often occurs through post-translational modifications (PTMs), which allow the cell to quickly and efficiently respond to cellular and environmental stimuli. PTMs have recently emerged as important regulators of RBPs implicated in neurodegenerative disorders, in particular amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we summarize how disease-associated PTMs influence the biophysical properties, molecular interactions, subcellular localization, and function of ALS/FTD-linked RBPs, such as FUS and TDP-43. We will discuss how PTMs are believed to play pathological, protective, or ambiguous roles in these neurodegenerative disorders.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Humanos , Procesamiento Proteico-Postraduccional , Proteína FUS de Unión a ARN/genética , Proteínas de Unión al ARN/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.
Asunto(s)
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
Progranulin is an evolutionarily conserved protein that has been implicated in human neurodevelopmental and neurodegenerative diseases. Human progranulin is comprised of multiple cysteine-rich, biologically active granulin peptides. Granulin peptides accumulate with age and stress, however their functional contributions relative to full-length progranulin remain unclear. To address this, we generated C. elegans strains that produced quantifiable levels of both full-length progranulin/PGRN-1 protein and cleaved granulin peptide. Using these strains, we demonstrated that even in the presence of intact PGRN-1, granulin peptides suppressed the activity of the lysosomal aspartyl protease activity, ASP-3/CTSD. Granulin peptides were also dominant over PGRN-1 in compromising animal fitness as measured by progress through development and stress response. Finally, the degradation of human TDP-43 was impaired when the granulin to PGRN-1 ratio was increased, representing a disease-relevant downstream impact of impaired lysosomal function. In summary, these studies suggest that not only absolute progranulin levels, but also the balance between full-length progranulin and its cleavage products, is important in regulating lysosomal biology. Given its relevance in human disease, this suggests that the processing of progranulin into granulins should be considered as part of disease pathobiology and may represent a site of therapeutic intervention.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Granulinas , Progranulinas , Animales , Humanos , Caenorhabditis elegans/fisiología , Granulinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Enfermedades Neurodegenerativas , Progranulinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismoRESUMEN
Post-translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA-binding protein TAR DNA-binding protein (TDP-43), is hyperphosphorylated in disease on several C-terminal serine residues, a process generally believed to promote TDP-43 aggregation. Here, we however find that Casein kinase 1δ-mediated TDP-43 hyperphosphorylation or C-terminal phosphomimetic mutations reduce TDP-43 phase separation and aggregation, and instead render TDP-43 condensates more liquid-like and dynamic. Multi-scale molecular dynamics simulations reveal reduced homotypic interactions of TDP-43 low-complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP-43, but suppress accumulation of TDP-43 in membrane-less organelles and promote its solubility in neurons. We speculate that TDP-43 hyperphosphorylation may be a protective cellular response to counteract TDP-43 aggregation.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Agregado de Proteínas , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Despite the efficacy of highly active antiretroviral therapy in controlling the incidence and mortality of AIDS, effective interventions for HIV-1-induced neurological damage and cognitive impairment remain elusive. In this study, we found that HIV-1 infection can induce proteolytic cleavage and aberrant aggregation of TAR DNA-binding protein 43 (TDP-43), a pathological protein associated with various severe neurological disorders. The HIV-1 accessory protein Vpu was found to be responsible for the cleavage of TDP-43, as ectopic expression of Vpu alone was sufficient to induce TDP-43 cleavage, whereas HIV-1 lacking Vpu failed to cleave TDP-43. Mechanistically, the cleavage of TDP-43 at Asp89 by HIV-1 relies on Vpu-mediated activation of Caspase 3, and pharmacological inhibition of Caspase 3 activity effectively suppressed the HIV-1-induced aggregation and neurotoxicity of TDP-43. Overall, these results suggest that TDP-43 is a conserved host target of HIV-1 Vpu and provide evidence for the involvement of TDP-43 dysregulation in the neural pathogenesis of HIV-1.
RESUMEN
In amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD), cytoplasmic aggregates of hyperphosphorylated TDP-43 accumulate and colocalize with some stress granule components, but how pathological TDP-43 aggregation is nucleated remains unknown. In Drosophila, we establish that downregulation of tankyrase, a poly(ADP-ribose) (PAR) polymerase, reduces TDP-43 accumulation in the cytoplasm and potently mitigates neurodegeneration. We establish that TDP-43 non-covalently binds to PAR via PAR-binding motifs embedded within its nuclear localization sequence. PAR binding promotes liquid-liquid phase separation of TDP-43 in vitro and is required for TDP-43 accumulation in stress granules in mammalian cells and neurons. Stress granule localization initially protects TDP-43 from disease-associated phosphorylation, but upon long-term stress, stress granules resolve, leaving behind aggregates of phosphorylated TDP-43. Finally, small-molecule inhibition of Tankyrase-1/2 in mammalian cells inhibits formation of cytoplasmic TDP-43 foci without affecting stress granule assembly. Thus, Tankyrase inhibition antagonizes TDP-43-associated pathology and neurodegeneration and could have therapeutic utility for ALS and FTD.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Células COS , Línea Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Drosophila , Femenino , Degeneración Lobar Frontotemporal/metabolismo , Masculino , Mamíferos/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Fosforilación/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
An evolutionarily conserved region of the TDP-43 low-complexity domain (LCD) twenty residues in length can adopt either an α-helical or ß-strand conformation. When in the latter conformation, TDP-43 self-associates via the formation of a labile, cross-ß structure. Self-association can be monitored via the formation of phase-separated protein droplets. Exposure of droplets to hydrogen peroxide leads to oxidation of conserved methionine residues distributed throughout the LCD. Oxidation disassembles the cross-ß structure, thus eliminating both self-association and phase separation. Here, we demonstrate that this process reciprocally enables formation of α-helical structure in precisely the same region formerly functioning to facilitate ß-strand-mediated self-association. We further observe that the α-helical conformation allows interaction with a lipid-like detergent and that exposure to lipids enhances the ß-to-α conformational switch. We hypothesize that regulation of this oxidative switch will prove to be important to the control of localized translation within vertebrate cells. The experimental observations reported herein were heavily reliant on studies of 1,6-hexanediol, a chemical agent that selectively dissolves labile structures formed via the self-association of protein domains of low sequence complexity. This aliphatic alcohol is shown to exert its dissociative activity primarily via hydrogen-bonding interactions with carbonyl oxygen atoms of the polypeptide backbone. Such observations underscore the central importance of backbone-mediated protein:protein interactions that facilitate the self-association and phase separation of LCDs.
Asunto(s)
Proteínas de Unión al ADN , Péptidos , Proteínas de Unión al ADN/metabolismo , Péptidos/química , Dominios Proteicos , Metionina/metabolismo , Estrés OxidativoRESUMEN
Mixed pathologies are common in neurodegenerative disease; however, antemortem imaging rarely captures copathologic effects on brain atrophy due to a lack of validated biomarkers for non-Alzheimer's pathologies. We leveraged a dataset comprising antemortem MRI and postmortem histopathology to assess polypathologic associations with atrophy in a clinically heterogeneous sample of 125 human dementia patients (41 female, 84 male) with T1-weighted MRI ≤ 5 years before death and postmortem ordinal ratings of amyloid-[Formula: see text], tau, TDP-43, and [Formula: see text]-synuclein. Regional volumes were related to pathology using linear mixed-effects models; approximately 25% of data were held out for testing. We contrasted a polypathologic model comprising independent factors for each proteinopathy with two alternatives: a model that attributed atrophy entirely to the protein(s) associated with the patient's primary diagnosis and a protein-agnostic model based on the sum of ordinal scores for all pathology types. Model fits were evaluated using log-likelihood and correlations between observed and fitted volume scores. Additionally, we performed exploratory analyses relating atrophy to gliosis, neuronal loss, and angiopathy. The polypathologic model provided superior fits in the training and testing datasets. Tau, TDP-43, and [Formula: see text]-synuclein burden were inversely associated with regional volumes, but amyloid-[Formula: see text] was not. Gliosis and neuronal loss explained residual variance in and mediated the effects of tau, TDP-43, and [Formula: see text]-synuclein on atrophy. Regional brain atrophy reflects not only the primary molecular pathology but also co-occurring proteinopathies; inflammatory immune responses may independently contribute to degeneration. Our findings underscore the importance of antemortem biomarkers for detecting mixed pathology.
Asunto(s)
Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Humanos , Masculino , Femenino , Enfermedades Neurodegenerativas/diagnóstico por imagen , Enfermedades Neurodegenerativas/patología , Sustancia Gris/patología , Proteínas tau/metabolismo , Gliosis/patología , Atrofia/patología , Amiloide , Sinucleínas , Proteínas de Unión al ADN/metabolismo , Biomarcadores , Enfermedad de Alzheimer/patologíaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder marked by progressive motor neuron degeneration and muscle denervation. A recent transcriptomic study integrating a wide range of human ALS samples revealed that the upregulation of p53, a downstream target of inflammatory stress, is commonly detected in familial and sporadic ALS cases by a mechanism linked to a transactive response DNA-binding protein 43 (TDP-43) dysfunction. In this study, we show that prolonged interferon-gamma (IFNγ) treatment of human induced pluripotent stem cell-derived spinal motor neurons results in a severe cytoplasmic aggregation of TDP-43. TDP-43 dysfunction resulting from either IFNγ exposure or an ALS-associated TDP-43 mutation was associated with the activation of the p53 pathway. This was accompanied by the hyperactivation of neuronal firing, followed by the complete loss of their electrophysiological function. Through a comparative single-cell transcriptome analysis, we have identified significant alterations in ALS-associated genes in motor neurons exposed to IFNγ, implicating their direct involvement in ALS pathology. Interestingly, IFNγ was found to induce significant levels of programmed death-ligand 1 (PD-L1) expression in motor neurons without affecting the levels of any other immune checkpoint proteins. This finding suggests a potential role of excessive PD-L1 expression in ALS development, given that PD-L1 was recently reported to impair neuronal firing ability in mice. Our findings suggest that exposing motor neurons to IFNγ could directly derive ALS pathogenesis, even without the presence of the inherent genetic mutation or functional glia component. Furthermore, this study provides a comprehensive list of potential candidate genes for future immunotherapeutic targets with which to treat sporadic forms of ALS, which account for 90% of all reported cases.