RESUMEN
TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein found within ribonucleoprotein granules tethered to lysosomes via annexin A11. TDP-43 protein forms inclusions in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and limbic predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Annexin A11 is also known to form aggregates in ALS cases with pathogenic variants in ANXA11. Annexin A11 aggregation has not been described in sporadic ALS, FTLD-TDP or LATE-NC cases. To explore the relationship between TDP-43 and annexin A11, genetic analysis of 822 autopsy cases was performed to identify rare ANXA11 variants. In addition, an immunohistochemical study of 368 autopsy cases was performed to identify annexin A11 aggregates. Insoluble annexin A11 aggregates which colocalize with TDP-43 inclusions were present in all FTLD-TDP Type C cases. Annexin A11 inclusions were also seen in a small proportion (3-6%) of sporadic and genetic forms of FTLD-TDP types A and B, ALS, and LATE-NC. In addition, we confirm the comingling of annexin A11 and TDP-43 aggregates in an ALS case with the pathogenic ANXA11 p.G38R variant. Finally, we found abundant annexin A11 inclusions as the primary pathologic finding in a case of progressive supranuclear palsy-like frontotemporal dementia with prominent striatal vacuolization due to a novel variant, ANXA11 p.P75S. By immunoblot, FTLD-TDP with annexinopathy and ANXA11 variant cases show accumulation of insoluble ANXA11 including a truncated fragment. These results indicate that annexin A11 forms a diverse and heterogeneous range of aggregates in both sporadic and genetic forms of TDP-43 proteinopathies. In addition, the finding of a primary vacuolar annexinopathy due to ANXA11 p.P75S suggests that annexin A11 aggregation is sufficient to cause neurodegeneration.
Asunto(s)
Anexinas , Proteínas de Unión al ADN , Degeneración Lobar Frontotemporal , Humanos , Anciano , Anexinas/genética , Anexinas/metabolismo , Femenino , Masculino , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Degeneración Lobar Frontotemporal/genética , Degeneración Lobar Frontotemporal/patología , Degeneración Lobar Frontotemporal/metabolismo , Persona de Mediana Edad , Anciano de 80 o más Años , Proteinopatías TDP-43/patología , Proteinopatías TDP-43/genética , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/metabolismo , Cuerpos de Inclusión/patología , Cuerpos de Inclusión/metabolismo , Encéfalo/patología , Encéfalo/metabolismo , Agregación Patológica de Proteínas/patología , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismoRESUMEN
Effective therapies are urgently needed to safely target TDP-43 pathology as it is closely associated with the onset and development of devastating diseases such as frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). In addition, TDP-43 pathology is present as a co-pathology in other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Our approach is to develop a TDP-43-specific immunotherapy that exploits Fc gamma-mediated removal mechanisms to limit neuronal damage while maintaining physiological TDP-43 function. Thus, using both in vitro mechanistic studies in conjunction with the rNLS8 and CamKIIa inoculation mouse models of TDP-43 proteinopathy, we identified the key targeting domain in TDP-43 to accomplish these therapeutic objectives. Targeting the C-terminal domain of TDP-43 but not the RNA recognition motifs (RRM) reduces TDP-43 pathology and avoids neuronal loss in vivo. We demonstrate that this rescue is dependent on Fc receptor-mediated immune complex uptake by microglia. Furthermore, monoclonal antibody (mAb) treatment enhances phagocytic capacity of ALS patient-derived microglia, providing a mechanism to restore the compromised phagocytic function in ALS and FTD patients. Importantly, these beneficial effects are achieved while preserving physiological TDP-43 activity. Our findings demonstrate that a mAb targeting the C-terminal domain of TDP-43 limits pathology and neurotoxicity, enabling clearance of misfolded TDP-43 through microglia engagement, and supporting the clinical strategy to target TDP-43 by immunotherapy. SIGNIFICANCE STATEMENT: TDP-43 pathology is associated with various devastating neurodegenerative disorders with high unmet medical needs such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Thus, safely and effectively targeting pathological TDP-43 represents a key paradigm for biotechnical research as currently there is little in clinical development. After years of research, we have determined that targeting the C-terminal domain of TDP-43 rescues multiple patho-mechanisms involved in disease progression in two animal models of FTD/ALS. In parallel, importantly, our studies establish that this approach does not alter the physiological functions of this ubiquitously expressed and indispensable protein. Together, our findings substantially contribute to the understanding of TDP-43 pathobiology and support the prioritization for clinical testing of immunotherapy approaches targeting TDP-43.
Asunto(s)
Enfermedad de Alzheimer , Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Enfermedad de Pick , Ratones , Animales , Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/genética , Enfermedad de Alzheimer/genética , Neuroprotección , Proteínas de Unión al ADN/metabolismo , InmunoterapiaRESUMEN
Limbic-predominant age-related TDP-43 encephalopathy (LATE) is characterized by the accumulation of TAR-DNA-binding protein 43 (TDP-43) aggregates in older adults. LATE coexists with Lewy body disease (LBD) as well as other neuropathological changes including Alzheimer's disease (AD). We aimed to identify the pathological, clinical, and genetic characteristics of LATE in LBD (LATE-LBD) by comparing it with LATE in AD (LATE-AD), LATE with mixed pathology of LBD and AD (LATE-LBD + AD), and LATE alone (Pure LATE). We analyzed four cohorts of autopsy-confirmed LBD (n = 313), AD (n = 282), LBD + AD (n = 355), and aging (n = 111). We assessed the association of LATE with patient profiles including LBD subtype and AD neuropathologic change (ADNC). We studied the morphological and distributional differences between LATE-LBD and LATE-AD. By frequency analysis, we staged LATE-LBD and examined the association with cognitive impairment and genetic risk factors. Demographic analysis showed LATE associated with age in all four cohorts and the frequency of LATE was the highest in LBD + AD followed by AD, LBD, and Aging. LBD subtype and ADNC associated with LATE in LBD or AD but not in LBD + AD. Pathological analysis revealed that the hippocampal distribution of LATE was different between LATE-LBD and LATE-AD: neuronal cytoplasmic inclusions were more frequent in cornu ammonis 3 (CA3) in LATE-LBD compared to LATE-AD and abundant fine neurites composed of C-terminal truncated TDP-43 were found mainly in CA2 to subiculum in LATE-LBD, which were not as numerous in LATE-AD. Some of these fine neurites colocalized with phosphorylated α-synuclein. LATE-LBD staging showed LATE neuropathological changes spread in the dentate gyrus and brainstem earlier than in LATE-AD. The presence and prevalence of LATE in LBD associated with cognitive impairment independent of either LBD subtype or ADNC; LATE-LBD stage also associated with the genetic risk variants of TMEM106B rs1990622 and GRN rs5848. These data highlight clinicopathological and genetic features of LATE-LBD.
Asunto(s)
Envejecimiento/patología , Encéfalo/patología , Enfermedad por Cuerpos de Lewy/patología , Proteinopatías TDP-43/patología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Femenino , Humanos , Enfermedad por Cuerpos de Lewy/complicaciones , Enfermedad por Cuerpos de Lewy/genética , Masculino , Persona de Mediana Edad , Proteinopatías TDP-43/complicaciones , Proteinopatías TDP-43/genéticaRESUMEN
AIM: The heterogeneity in the distribution and morphological features of TAR DNA-binding protein-43 (TDP-43) pathology in the brains of frontotemporal lobar degeneration (FTLD-TDP) patients and their different clinical manifestations suggest that distinct pathological TDP-43 strains could play a role in this heterogeneity between different FTLD-TDP subtypes (A-E). Our aim was to evaluate the existence of distinct TDP-43 strains in the brains of different FTLD-TDP subtypes and characterise their specific seeding properties in vitro and in vivo. METHODS AND RESULTS: We used an inducible stable cell line expressing a mutant cytoplasmic TDP-43 (iGFP-NLSm) to evaluate the seeding properties of distinct pathological TDP-43 strains. Brain-derived TDP-43 protein extracts from FTLD-TDP types A (n = 6) and B (n = 3) cases induced the formation of round/spherical phosphorylated TDP-43 aggregates that morphologically differed from the linear and wavy wisps and bigger heterogeneous filamentous (skein-like) aggregates induced by type E (n = 3) cases. These morphological differences correlated with distinct biochemical banding patterns of sarkosyl-insoluble TDP-43 protein recovered from the transduced cells. Moreover, brain-derived TDP-43 extracts from type E cases showed higher susceptibility to PK digestion of full-length TDP-43 and the most abundant C-terminal fragments that characterise type E extracts. Finally, we showed that intracerebral injections of different TDP-43 strains induced a distinctive morphological and subcellular distribution of TDP-43 pathology and different spreading patterns in the brains of CamKIIa-hTDP-43NLSm Tg mice. CONCLUSIONS: We show the existence of distinct TDP-43 strains in the brain of different FTLD-TDP subtypes with distinctive seeding and spreading properties in the brains of experimental animal models.
Asunto(s)
Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Unión al ADN/metabolismo , Animales , Proteínas de Unión al ADN/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Degeneración Lobar Frontotemporal/patología , Humanos , Ratones TransgénicosRESUMEN
The neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TAR DNA-binding protein-43 (TDP-43) inclusions (FTLD-TDP) share the neuropathological hallmark of aggregates of TDP-43. However, factors governing the severity and regional distribution of TDP-43 pathology, which may account for the divergent clinical presentations of ALS and FTLD-TDP, are not well understood. Here, we investigated the influence of genotypes at TMEM106B, a locus associated with risk for FTLD-TDP, and hexanucleotide repeat expansions in C9orf72, a known genetic cause for both ALS and FTLD-TDP, on global TDP-43 pathology and regional distribution of TDP-43 pathology in 899 postmortem cases from a spectrum of neurodegenerative diseases. We found that, among the 110 ALS cases, minor (C)-allele homozygotes at the TMEM106B locus sentinel SNP rs1990622 had more TDP-43 pathology globally, as well as in select brain regions. C9orf72 expansions similarly associated with greater TDP-43 pathology in ALS. However, adjusting for C9orf72 expansion status did not affect the relationship between TMEM106B genotype and TDP-43 pathology. To elucidate the direction of causality for this association, we directly manipulated TMEM106B levels in an inducible cell system that expresses mislocalized TDP-43 protein. We found that partial knockdown of TMEM106B, to levels similar to what would be expected in rs1990622 C allele carriers, led to development of more TDP-43 cytoplasmic aggregates, which were more insoluble, in this system. Taken together, our results support a causal role for TMEM106B in modifying the development of TDP-43 proteinopathy.
Asunto(s)
Enfermedad de Alzheimer/etiología , Proteína C9orf72/fisiología , Proteínas de Unión al ADN/fisiología , Enfermedad por Cuerpos de Lewy/etiología , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteinopatías TDP-43/etiología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Estudios de Cohortes , Femenino , Humanos , Enfermedad por Cuerpos de Lewy/patología , Masculino , Persona de Mediana Edad , Proteinopatías TDP-43/patologíaRESUMEN
TAR-DNA binding protein-43 (TDP-43) proteinopathy is seen in multiple brain diseases. A standardized terminology was recommended recently for common age-related TDP-43 proteinopathy: limbic-predominant, age-related TDP-43 encephalopathy (LATE) and the underlying neuropathological changes, LATE-NC. LATE-NC may be co-morbid with Alzheimer's disease neuropathological changes (ADNC). However, there currently are ill-defined diagnostic classification issues among LATE-NC, ADNC, and frontotemporal lobar degeneration with TDP-43 (FTLD-TDP). A practical challenge is that different autopsy cohorts are composed of disparate groups of research volunteers: hospital- and clinic-based cohorts are enriched for FTLD-TDP cases, whereas community-based cohorts have more LATE-NC cases. Neuropathological methods also differ across laboratories. Here, we combined both cases and neuropathologists' diagnoses from two research centres-University of Pennsylvania and University of Kentucky. The study was designed to compare neuropathological findings between FTLD-TDP and pathologically severe LATE-NC. First, cases were selected from the University of Pennsylvania with pathological diagnoses of either FTLD-TDP (n = 33) or severe LATE-NC (mostly stage 3) with co-morbid ADNC (n = 30). Sections from these University of Pennsylvania cases were cut from amygdala, anterior cingulate, superior/mid-temporal, and middle frontal gyrus. These sections were stained for phospho-TDP-43 immunohistochemically and evaluated independently by two University of Kentucky neuropathologists blinded to case data. A simple set of criteria hypothesized to differentiate FTLD-TDP from LATE-NC was generated based on density of TDP-43 immunoreactive neuronal cytoplasmic inclusions in the neocortical regions. Criteria-based sensitivity and specificity of differentiating severe LATE-NC from FTLD-TDP cases with blind evaluation was â¼90%. Another proposed neuropathological feature related to TDP-43 proteinopathy in aged individuals is 'Alpha' versus 'Beta' in amygdala. Alpha and Beta status was diagnosed by neuropathologists from both universities (n = 5 raters). There was poor inter-rater reliability of Alpha/Beta classification (mean κ = 0.31). We next tested a separate cohort of cases from University of Kentucky with either FTLD-TDP (n = 8) or with relatively 'pure' severe LATE-NC (lacking intermediate or severe ADNC; n = 14). The simple criteria were applied by neuropathologists blinded to the prior diagnoses at University of Pennsylvania. Again, the criteria for differentiating LATE-NC from FTLD-TDP was effective, with sensitivity and specificity â¼90%. If more representative cases from each cohort (including less severe TDP-43 proteinopathy) had been included, the overall accuracy for identifying LATE-NC was estimated at >98% for both cohorts. Also across both cohorts, cases with FTLD-TDP died younger than those with LATE-NC (P < 0.0001). We conclude that in most cases, severe LATE-NC and FTLD-TDP can be differentiated by applying simple neuropathological criteria.
Asunto(s)
Degeneración Lobar Frontotemporal/diagnóstico por imagen , Sistema Límbico/diagnóstico por imagen , Proteinopatías TDP-43/diagnóstico por imagen , Factores de Edad , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Degeneración Lobar Frontotemporal/fisiopatología , Humanos , Sistema Límbico/fisiopatología , Masculino , Persona de Mediana Edad , Proteinopatías TDP-43/fisiopatologíaRESUMEN
Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) can typically be categorized into one of four distinct histopathologic patterns of TDP-43 pathology, types A to D. The strength of this histopathologic classification lies in the association between FTLD-TDP subtypes and various clinical and genetic features of disease. Seven cases of FTLD-TDP were identified here which were difficult to classify based on existing pathologic criteria. Distinct features common to these cases included TDP-43 aggregates over a wide neuroanatomic distribution comprised of granulofilamentous neuronal inclusions, abundant grains, and oligodendroglial inclusions. TDP-43 aggregates were phosphorylated and associated with loss of normal nuclear TDP-43 protein (nuclear clearance) but were negative for ubiquitin. Biochemical analysis confirmed the presence of insoluble and phosphorylated TDP-43 and also revealed a distinct pattern of TDP-43 C-terminal fragments relative to other FTLD-TDP subtypes. Finally, these cases were uniformly associated with a very rapid clinical course culminating in death within ~3 years of disease onset. We suggest that these cases may represent a unique clinicopathologic subtype of FTLD-TDP which we provisionally call "type E." The immature appearance of TDP-43 aggregates, widespread distribution, uniform biochemical profile and rapid clinical course highlights the clinical and pathologic variability within FTLD-TDP, and raises the possibility that type E neuropathology is the sequelae of a particularly virulent strain of TDP-43 proteinopathy.
Asunto(s)
Degeneración Lobar Frontotemporal/clasificación , Edad de Inicio , Anciano , Anciano de 80 o más Años , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente , Lóbulo Frontal/metabolismo , Lóbulo Frontal/patología , Degeneración Lobar Frontotemporal/genética , Degeneración Lobar Frontotemporal/metabolismo , Degeneración Lobar Frontotemporal/patología , Humanos , Persona de Mediana Edad , Fenotipo , Análisis de RegresiónRESUMEN
Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches.
Asunto(s)
Enfermedad de Huntington/etiología , Proteínas del Tejido Nervioso/genética , ARN Mensajero , Exones , Silenciador del Gen , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/patología , Péptidos/genética , Péptidos/metabolismo , ARN Mensajero/metabolismo , Expansión de Repetición de TrinucleótidoRESUMEN
Adenosine A2A receptor (A2AR) is a G-protein coupled receptor that stimulates adenylyl cyclase activity. In the brain, A2ARs are found highly enriched in striatal GABAergic medium spiny neurons, related to the control of voluntary movement. Pharmacological modulation of A2ARs is particularly useful in Parkinson's disease (PD) due to their property of antagonizing dopamine D2 receptor activity. Increases in A2AR levels have been described in PD patients showing an important loss of dopaminergic denervation markers, but no data have been reported about A2AR levels in incidental PD brains. In the present report, we show that increased A2ARs protein levels were also detected in the putamen of incidental PD cases (Braak PD stages 1-2) with respect to age-matched controls. By contrast, A2ARs mRNA levels remained unchanged, suggesting that posttranslational mechanisms could be involved in the regulation of A2ARs. It has been described how miR-34b/c downregulation is an early event in PD cases. We found that miR-34b levels are also significantly reduced in the putamen of incidental PD cases and along disease progression. Given that 3'UTR of A2AR contains a predicted target site for miR-34b, the potential role of this miRNA in protein A2AR levels was assessed. In vitro studies revealed that endogenous A2AR protein levels increased when miR-34b function was blocked using a specific anti-miR-34b. Moreover, using a luciferase reporter assay with point mutations in a miR-34b predicted binding site within the 3'UTR region of A2AR mRNA abolished the effect of the miRNA using a miR-34b mimic. In addition, we showed a reduced percentage of DNA methylation in the 5'UTR region of ADORA2A in advanced PD cases. Overall, these findings reveal that increased A2AR protein levels occur in asymptomatic PD patients and provide new insights into the molecular mechanisms underlying A2AR expression levels along the progression of this neurodegenerative disease.
Asunto(s)
MicroARNs/metabolismo , Enfermedad de Parkinson/fisiopatología , Putamen/fisiopatología , Receptor de Adenosina A2A/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Metilación de ADN , Progresión de la Enfermedad , Femenino , Humanos , Masculino , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Persona de Mediana Edad , ARN Mensajero/metabolismo , Receptor de Adenosina A2A/genética , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
People with Down syndrome (DS) exhibit abnormal brain structure. Alterations affecting neurotransmission and signalling pathways that govern brain function are also evident. A large number of genes are simultaneously expressed at abnormal levels in DS; therefore, it is a challenge to determine which gene(s) contribute to specific abnormalities, and then identify the key molecular pathways involved. We generated RCAN1-TG mice to study the consequences of RCAN1 over-expression and investigate the contribution of RCAN1 to the brain phenotype of DS. RCAN1-TG mice exhibit structural brain abnormalities in those areas affected in DS. The volume and number of neurons within the hippocampus is reduced and this correlates with a defect in adult neurogenesis. The density of dendritic spines on RCAN1-TG hippocampal pyramidal neurons is also reduced. Deficits in hippocampal-dependent learning and short- and long-term memory are accompanied by a failure to maintain long-term potentiation (LTP) in hippocampal slices. In response to LTP induction, we observed diminished calcium transients and decreased phosphorylation of CaMKII and ERK1/2-proteins that are essential for the maintenance of LTP and formation of memory. Our data strongly suggest that RCAN1 plays an important role in normal brain development and function and its up-regulation likely contributes to the neural deficits associated with DS.
Asunto(s)
Hipocampo/patología , Hipocampo/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Aprendizaje por Laberinto , Memoria a Corto Plazo , Proteínas Musculares/metabolismo , Animales , Proteínas de Unión al Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Espinas Dendríticas , Síndrome de Down/genética , Síndrome de Down/patología , Síndrome de Down/fisiopatología , Fenómenos Electrofisiológicos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Potenciación a Largo Plazo , Masculino , Memoria a Largo Plazo , Ratones , Ratones Transgénicos , Proteínas Musculares/genética , Neuronas/patologíaRESUMEN
MicroRNAs (miRNAs) are post-transcriptional gene expression regulators, playing key roles in neuronal development, plasticity and disease. Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by the presence of protein inclusions or Lewy bodies and a progressive loss of dopaminergic neurons in the midbrain. Here, we have evaluated miRNA expression deregulation in PD brain samples. MiRNA expression profiling revealed decreased expression of miR-34b and miR-34c in brain areas with variable neuropathological affectation at clinical (motor) stages (Braak stages 4 and 5) of the disease, including the amygdala, frontal cortex, substantia nigra and cerebellum. Furthermore, misregulation of miR-34b/c was detected in pre-motor stages (stages 1-3) of the disease, and thus in cases that did not receive any PD-related treatment during life. Depletion of miR-34b or miR-34c in differentiated SH-SY5Y dopaminergic neuronal cells resulted in a moderate reduction in cell viability that was accompanied by altered mitochondrial function and dynamics, oxidative stress and reduction in total cellular adenosin triphosphate content. MiR-34b/c downregulation was coupled to a decrease in the expression of DJ1 and Parkin, two proteins associated to familial forms of PD that also have a role in idiopathic cases. Accordingly, DJ1 and Parkin expression was reduced in PD brain samples displaying strong miR-34b/c downregulation. We propose that early deregulation of miR-34b/c in PD triggers downstream transcriptome alterations underlying mitochondrial dysfunction and oxidative stress, which ultimately compromise cell viability. A better understanding of the cellular pathways controlling and/or controlled by miR-34b/c should allow identification of targets for development of therapeutic approaches.
Asunto(s)
MicroARNs/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Western Blotting , Línea Celular Tumoral , Humanos , Microscopía Electrónica de Transmisión , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/ultraestructura , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
MicroRNAs (miRNAs) and other small non-coding RNAs (sncRNAs) are post-transcriptional regulators of gene expression, playing key roles in neuronal development, plasticity, and disease. Transcriptome deregulation caused by miRNA dysfunction has been associated to neurodegenerative diseases. Parkinson disease (PD) is the second most common neurodegenerative disease showing deregulation of the coding and small non-coding transcriptome. On profiling sncRNA in PD brain areas differently affected, we found that upregulation of a small vault RNA (svtRNA2-1a) is widespread in PD brains, occurring early in the course of the disease (at pre-motor stages). SvtRNA2-1a biogenesis was dependent on Dicer activity on its precursor (vtRNA2-1) but independent of Drosha endonuclease, unlike the canonical miRNAs. Although endogenous svtRNA2-1a was enriched in Ago-2 immunoprecipitates in differentiated SH-SY5Y neuronal cells, overexpression of svtRNA2-1a induced subtle transcriptomic changes, suggesting that gene expression regulation may involve other mechanisms than mRNA decay only. Function enrichment analysis of the genes deregulated by svtRNA2-1a overexpression or svtRNA2-1a predicted targets identified pathways related to nervous system development and cell type specification. The expression pattern of svtRNA2-1a during development and aging of the human brain and the detrimental consequences of a svtRNA2-1a mimic overexpression in neuronal cells further indicate that low svtRNA2-1a levels may be important for the maintenance of neurons. Our results suggest that early svtRNA2-1a upregulation in PD may contribute to perturbations of gene expression networks, underlying metabolic impairment and cell dysfunction. A better understanding of the pathways regulated by svtRNA2-a, and also the mechanisms regulating its expression should facilitate the identification of new targets for therapeutic approaches in PD.
Asunto(s)
Regulación de la Expresión Génica , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , ARN Pequeño no Traducido/genética , Proteínas Argonautas/metabolismo , Secuencia de Bases , Encéfalo/metabolismo , Diferenciación Celular/genética , Línea Celular , Expresión Génica , Humanos , Datos de Secuencia Molecular , Neuronas/citología , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , Enfermedad de Parkinson/tratamiento farmacológico , ARN Pequeño no Traducido/química , Reproducibilidad de los Resultados , Ribonucleasa III/metabolismo , Alineación de Secuencia , Transcriptoma , Regulación hacia ArribaRESUMEN
We introduce a new automated machine learning analysis pipeline to precisely classify cellular structures captured through single molecule localization microscopy, which we call ECLiPSE (Enhanced Classification of Localized Pointclouds by Shape Extraction). ECLiPSE leverages 67 comprehensive shape descriptors encompassing geometric, boundary, skeleton and other properties, the majority of which are directly extracted from the localizations to accurately characterize individual structures. We validate ECLiPSE through unsupervised and supervised classification on a dataset featuring five distinct cellular structures, achieving exceptionally high classification accuracies nearing 100%. Moreover, we demonstrate the versatility of our approach by applying it to two novel biological applications: quantifying the clearance of tau protein aggregates, a critical marker for neurodegenerative diseases, and differentiating between two distinct morphological features (morphotypes) of TAR DNA-binding protein 43 proteinopathy, potentially associated to different TDP-43 strains, each exhibiting unique seeding and spreading properties. We anticipate that this versatile approach will significantly enhance the way we study cellular structures across various biological contexts, elucidating their roles in disease development and progression.
RESUMEN
Despite the strong evidence linking the transactive response DNA-binding protein 43 (TDP-43) aggregation to the pathogenesis of frontotemporal lobar degeneration with TDP-43, amyotrophic lateral sclerosis and several neurodegenerative diseases, our knowledge of the sequence and structural determinants of its aggregation and neurotoxicity remains incomplete. Herein, we present a new method for producing recombinant full-length TDP-43 filaments that exhibit sequence and morphological features similar to those of brain-derived TDP-43 filaments. We show that TDP-43 filaments contain a ß-sheet-rich helical amyloid core that is fully buried by the flanking structured domains of the protein. We demonstrate that the proteolytic cleavage of TDP-43 filaments and exposure of this amyloid core are necessary for propagating TDP-43 pathology and enhancing the seeding of brain-derived TDP-43 aggregates. Only TDP-43 filaments with exposed amyloid core efficiently seeded the aggregation of endogenous TDP-43 in cells. These findings suggest that inhibiting the enzymes mediating cleavage of TDP-43 aggregates represents a viable disease-modifying strategy to slow the progression of amyotrophic lateral sclerosis and other TDP-43 proteinopathies.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Degeneración Lobar Frontotemporal , Proteinopatías TDP-43 , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Proteinopatías TDP-43/patología , Degeneración Lobar Frontotemporal/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismoRESUMEN
Huntington disease (HD) is a neurodegenerative disorder that predominantly affects neurons of the forebrain. We have applied the Illumina massively parallel sequencing to deeply analyze the small RNA populations of two different forebrain areas, the frontal cortex (FC) and the striatum (ST) of healthy individuals and individuals with HD. More than 80% of the small-RNAs were annotated as microRNAs (miRNAs) in all samples. Deep sequencing revealed length and sequence heterogeneity (IsomiRs) for the vast majority of miRNAs. Around 80-90% of the miRNAs presented modifications in the 3'-terminus mainly in the form of trimming and/or as nucleotide addition variants, while the 5'-terminus of the miRNAs was specially protected from changes. Expression profiling showed strong miRNA and isomiR expression deregulation in HD, most being common to both FC and ST. The analysis of the upstream regulatory regions in co-regulated miRNAs suggests a role for RE1-Silencing Transcription Factor (REST) and P53 in miRNAs downregulation in HD. The putative targets of deregulated miRNAs and seed-region IsomiRs strongly suggest that their altered expression contributes to the aberrant gene expression in HD. Our results show that miRNA variability is a ubiquitous phenomenon in the adult human brain, which may influence gene expression in physiological and pathological conditions.
Asunto(s)
Encéfalo/metabolismo , Variación Genética , Enfermedad de Huntington/genética , MicroARNs/química , Adulto , Lóbulo Frontal/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Enfermedad de Huntington/metabolismo , MicroARNs/metabolismo , Neostriado/metabolismo , ARN Pequeño no Traducido/química , Proteínas Represoras/metabolismo , Análisis de Secuencia de ARNRESUMEN
Genes located on chromosome 21, over-expressed in Down syndrome (DS) and Alzheimer's disease (AD) and which regulate vesicle trafficking, are strong candidates for involvement in AD neuropathology. Regulator of calcineurin activity 1 (RCAN1) is one such gene. We have generated mutant mice in which RCAN1 is either over-expressed (RCAN1(ox)) or ablated (Rcan1-/-) and examined whether exocytosis from chromaffin cells, a classic cellular model of neuronal exocytosis, is altered using carbon fibre amperometry. We find that Rcan1 regulates the number of vesicles undergoing exocytosis and the speed at which the vesicle fusion pore opens and closes. Cells from both Rcan1-/- and RCAN1(ox) mice display reduced levels of exocytosis. Changes in single-vesicle fusion kinetics are also evident resulting in the less catecholamine released per vesicle with increasing Rcan1 expression. Acute calcineurin inhibition did not replicate the effect of RCAN1 overexpression. These changes are not due to alterations in Ca2+ entry or the readily releasable vesicle pool size. Thus, we illustrate a novel regulator of vesicle exocytosis, Rcan1, which influences both exocytotic rate and vesicle fusion kinetics. If Rcan1 functions similarly in neurons then overexpression of this protein, as occurs in DS and AD brains, will reduce both the number of synaptic vesicles undergoing exocytosis and the amount of neurotransmitter released per fusion event. This has direct implications for the pathogenesis of these diseases as sufficient levels of neurotransmission are required for synaptic maintenance and the prevention of neurodegeneration and vesicle trafficking defects are the earliest hallmark of AD neuropathology.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Síndrome de Down/metabolismo , Exocitosis , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fusión de Membrana , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Animales , Calcineurina/metabolismo , Calcio/metabolismo , Células Cromafines/fisiología , Vesículas Citoplasmáticas/metabolismo , Proteínas de Unión al ADN , Humanos , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Cranberry (Vaccinium macrocarpon) has been used for decades to prevent urinary tract infections (UTIs) that are among the most common bacterial infections in women. As to the traditional use of cranberry and its A-type proanthocyanidins' ability to inhibit adherence of the bacterial P fimbriae in a dose-dependent manner, clinical trials have been conducted on different subpopulations. A Cochrane meta-analysis in 244 females with symptomatic UTI suggests that the effect was more pronounced in women with recurrent UTIs than elderly males and females or people requiring catheterization. A first head-to-head trial in older females has been published comparing effectiveness of a low-dose antibiotic versus cranberry in which investigators suggest that cranberry products may have a role in older females with recurrent UTI. Still with regard to antibiotic treatment in women, a recently published study investigated also the potential cranberry juice interaction with beta-lactam antibiotics supporting the hypothesis that cranberry juice in usual quantities as prophylaxis for UTI is not likely to alter the pharmacokinetics of these oral antibiotics. In addition, the effects of cranberry in pregnant female patients have been investigated. A first pilot trial has been published in which, while a possible protective effect was shown, more than one third of the females withdrew mainly for gastrointestinal upset.
Asunto(s)
Bebidas , Fitoterapia , Infecciones Urinarias/terapia , Vaccinium macrocarpon , Factores de Edad , Antibacterianos/uso terapéutico , Medicina Basada en la Evidencia , Femenino , Interacciones Alimento-Droga , Frutas , Humanos , Masculino , Recurrencia , Factores Sexuales , Resultado del Tratamiento , Infecciones Urinarias/microbiología , Salud de la MujerRESUMEN
The angiopoietin (Ang)-Tie pathway has been intensely pursued as candidate second-generation anti-angiogenic target. While much of the translational work has focused on the ligand Ang2, the clinical efficacy of Ang2-targeting drugs is limited and failed to improve patient survival. In turn, the orphan receptor Tie1 remains therapeutically unexplored, although its endothelial-specific genetic deletion has previously been shown to result in a strong reduction in metastatic growth. Here, we report a novel Tie1 function-blocking antibody (AB-Tie1-39), which suppressed postnatal retinal angiogenesis. During primary tumor growth, neoadjuvant administration of AB-Tie1-39 strongly impeded systemic metastasis. Furthermore, the administration of AB-Tie1-39 in a perioperative therapeutic window led to a significant survival advantage as compared to control-IgG-treated mice. Additional in vivo experimental metastasis and in vitro transmigration assays concurrently revealed that AB-Tie1-39 treatment suppressed tumor cell extravasation at secondary sites. Taken together, the data phenocopy previous genetic work in endothelial Tie1 KO mice and thereby validate AB-Tie1-39 as a Tie1 function-blocking antibody. The study establishes Tie1 as a therapeutic target for metastasis in a perioperative or neoadjuvant setting.
Asunto(s)
Neoplasias , Receptor TIE-1 , Angiopoyetina 1 , Angiopoyetina 2 , Animales , Eliminación de Gen , Humanos , Ratones , Neovascularización Patológica , Receptor TIE-1/genética , Receptor TIE-2RESUMEN
Neurodegeneration in Alzheimer's disease (AD) is closely associated with the accumulation of pathologic tau aggregates in the form of neurofibrillary tangles. We found that a p.Asp395Gly mutation in VCP (valosin-containing protein) was associated with dementia characterized neuropathologically by neuronal vacuoles and neurofibrillary tangles. Moreover, VCP appeared to exhibit tau disaggregase activity in vitro, which was impaired by the p.Asp395Gly mutation. Additionally, intracerebral microinjection of pathologic tau led to increased tau aggregates in mice in which p.Asp395Gly VCP mice was knocked in, as compared with injected wild-type mice. These findings suggest that p.Asp395Gly VCP is an autosomal-dominant genetic mutation associated with neurofibrillary degeneration in part owing to reduced tau disaggregation, raising the possibility that VCP may represent a therapeutic target for the treatment of AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Agregado de Proteínas , Agregación Patológica de Proteínas/genética , Proteína que Contiene Valosina/metabolismo , Proteínas tau/metabolismo , Animales , Ácido Aspártico/genética , Técnicas de Sustitución del Gen , Genes Dominantes , Glicina/genética , Humanos , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación , Ovillos Neurofibrilares/genética , Ovillos Neurofibrilares/metabolismo , Fosforilación , Proteína que Contiene Valosina/genéticaRESUMEN
The original version of this Article contained an error in the author affiliations. The affiliation of Alice Chen-Plotkin with the Department of Neurology, Perelman School of Medicine, Philadelphia, PA, 19104 USA was inadvertently omitted. This has now been corrected in both the PDF and HTML versions of the Article.