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1.
J Biol Chem ; 292(37): 15277-15286, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28760828

RESUMO

The aberrant accumulation of tau protein is a pathological hallmark of a class of neurodegenerative diseases known as tauopathies, including Alzheimer's disease and related dementias. On the basis of previous observations that tau is a direct substrate of histone deacetylase 6 (HDAC6), we sought to map all HDAC6-responsive sites in tau and determine how acetylation in a site-specific manner affects tau's biophysical properties in vitro Our findings indicate that several acetylation sites in tau are responsive to HDAC6 and that acetylation on Lys-321 (within a KCGS motif) is both essential for acetylation-mediated inhibition of tau aggregation in vitro and a molecular tactic for preventing phosphorylation on the downstream Ser-324 residue. To determine the functional consequence of this HDAC6-regulated phosphorylation event, we examined tau's ability to promote microtubule assembly and found that phosphorylation of Ser-324 interferes with the normal microtubule-stabilizing function of tau. Tau phosphorylation of Ser-324 (pSer-324) has not previously been evaluated in the context of tauopathy, and here we observed increased deposition of pSer-324-positive tau both in mouse models of tauopathy and in patients with Alzheimer's disease. These findings uncover a novel acetylation-phosphorylation switch at Lys-321/Ser-324 that coordinately regulates tau polymerization and function. Because the disease relevance of this finding is evident, additional studies are needed to examine the role of pSer-324 in tau pathobiology and to determine whether therapeutically modulating this acetylation-phosphorylation switch affects disease progression in vivo.


Assuntos
Doença de Alzheimer/metabolismo , Histona Desacetilases/metabolismo , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional , Tauopatias/metabolismo , Proteínas tau/metabolismo , Acetilação/efeitos dos fármacos , Idoso , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Substituição de Aminoácidos , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Feminino , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Humanos , Lisina/metabolismo , Masculino , Camundongos Transgênicos , Mutação , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Serina/metabolismo , Tauopatias/tratamento farmacológico , Tauopatias/patologia , Bancos de Tecidos , Proteínas tau/química , Proteínas tau/genética
2.
Cell Rep ; 42(8): 112822, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37471224

RESUMO

C9orf72 repeat expansions are the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Poly(GR) proteins are toxic to neurons by forming cytoplasmic inclusions that sequester RNA-binding proteins including stress granule (SG) proteins. However, little is known of the factors governing poly(GR) inclusion formation. Here, we show that poly(GR) infiltrates a finely tuned network of protein-RNA interactions underpinning SG formation. It interacts with G3BP1, the key driver of SG assembly and a protein we found is critical for poly(GR) inclusion formation. Moreover, we discovered that N6-methyladenosine (m6A)-modified mRNAs and m6A-binding YTHDF proteins not only co-localize with poly(GR) inclusions in brains of c9FTD/ALS mouse models and patients with c9FTD, they promote poly(GR) inclusion formation via the incorporation of RNA into the inclusions. Our findings thus suggest that interrupting interactions between poly(GR) and G3BP1 or YTHDF1 proteins or decreasing poly(GR) altogether represent promising therapeutic strategies to combat c9FTD/ALS pathogenesis.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Animais , Camundongos , Humanos , Esclerose Lateral Amiotrófica/patologia , DNA Helicases/metabolismo , Grânulos de Estresse , Expansão das Repetições de DNA , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Demência Frontotemporal/metabolismo , Corpos de Inclusão/metabolismo , Proteínas de Choque Térmico/metabolismo , RNA/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo
3.
Front Cell Dev Biol ; 11: 1251551, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37614226

RESUMO

Treatments for neurodegenerative disease, including Frontotemporal dementia (FTD) and Amyotrophic lateral sclerosis (ALS), remain rather limited, underscoring the need for greater mechanistic insight and disease-relevant models. Our ability to develop novel disease models of genetic risk factors, disease modifiers, and other FTD/ALS-relevant targets is impeded by the significant amount of time and capital required to develop conventional knockout and transgenic mice. To overcome these limitations, we have generated a novel CRISPRi interference (CRISPRi) knockin mouse. CRISPRi uses a catalytically dead form of Cas9, fused to a transcriptional repressor to knockdown protein expression, following the introduction of single guide RNA against the gene of interest. To validate the utility of this model we have selected the TAR DNA binding protein (TDP-43) splicing target, stathmin-2 (STMN2). STMN2 RNA is downregulated in FTD/ALS due to loss of TDP-43 activity and STMN2 loss is suggested to play a role in ALS pathogenesis. The involvement of STMN2 loss of function in FTD has yet to be determined. We find that STMN2 protein levels in familial FTD cases are significantly reduced compared to controls, supporting that STMN2 depletion may be involved in the pathogenesis of FTD. Here, we provide proof-of-concept that we can simultaneously knock down Stmn2 and express the expanded repeat in the Chromosome 9 open reading frame 72 (C9ORF72) gene, successfully replicating features of C9-associated pathology. Of interest, depletion of Stmn2 had no effect on expression or deposition of dipeptide repeat proteins (DPRs), but significantly decreased the number of phosphorylated Tdp-43 (pTdp-43) inclusions. We submit that our novel CRISPRi mouse provides a versatile and rapid method to silence gene expression in vivo and propose this model will be useful to understand gene function in isolation or in the context of other neurodegenerative disease models.

4.
Proc Natl Acad Sci U S A ; 106(18): 7607-12, 2009 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-19383787

RESUMO

Inclusions of TAR DNA-binding protein-43 (TDP-43), a nuclear protein that regulates transcription and RNA splicing, are the defining histopathological feature of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-Us) and sporadic and familial forms of amyotrophic lateral sclerosis (ALS). In ALS and FTLD-U, aggregated, ubiquitinated, and N-terminally truncated TDP-43 can be isolated from brain tissue rich in neuronal and glial cytoplasmic inclusions. The loss of TDP-43 function resulting from inappropriate cleavage, translocation from the nucleus, or its sequestration into inclusions could play important roles in neurodegeneration. However, it is not known whether TDP-43 fragments directly mediate toxicity and, more specifically, whether their abnormal aggregation is a cause or consequence of pathogenesis. We report that the ectopic expression of a approximately 25-kDa TDP-43 fragment corresponding to the C-terminal truncation product of caspase-cleaved TDP-43 leads to the formation of toxic, insoluble, and ubiquitin- and phospho-positive cytoplasmic inclusions within cells. The 25-kDa C-terminal fragment is more prone to phosphorylation at S409/S410 than full-length TDP-43, but phosphorylation at these sites is not required for inclusion formation or toxicity. Although this fragment shows no biological activity, its exogenous expression neither inhibits the function nor causes the sequestration of full-length nuclear TDP-43, suggesting that the 25-kDa fragment can induce cell death through a toxic gain-of-function. Finally, by generating a conformation-dependent antibody that detects C-terminal fragments, we show that this toxic cleavage product is specific for pathologic inclusions in human TDP-43 proteinopathies.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Demência/metabolismo , Corpos de Inclusão/metabolismo , Apoptose , Caspases/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Demência/patologia , Humanos , Corpos de Inclusão/patologia , Fosforilação , Estrutura Terciária de Proteína , Ubiquitina/metabolismo
5.
Front Cell Dev Biol ; 10: 863089, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35386195

RESUMO

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited cerebellar ataxia caused by the expansion of a polyglutamine (polyQ) repeat in the gene encoding ATXN3. The polyQ expansion induces protein inclusion formation in the neurons of patients and results in neuronal degeneration in the cerebellum and other brain regions. We used adeno-associated virus (AAV) technology to develop a new mouse model of SCA3 that recapitulates several features of the human disease, including locomotor defects, cerebellar-specific neuronal loss, polyQ-expanded ATXN3 inclusions, and TDP-43 pathology. We also found that neurofilament light is elevated in the cerebrospinal fluid (CSF) of the SCA3 animals, and the expanded polyQ-ATXN3 protein can be detected in the plasma. Interestingly, the levels of polyQ-ATXN3 in plasma correlated with measures of cerebellar degeneration and locomotor deficits in 6-month-old SCA3 mice, supporting the hypothesis that this factor could act as a biomarker for SCA3.

6.
Science ; 378(6615): 94-99, 2022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36201573

RESUMO

Frontotemporal dementia and amyotrophic lateral sclerosis (FTD-ALS) are associated with both a repeat expansion in the C9orf72 gene and mutations in the TANK-binding kinase 1 (TBK1) gene. We found that TBK1 is phosphorylated in response to C9orf72 poly(Gly-Ala) [poly(GA)] aggregation and sequestered into inclusions, which leads to a loss of TBK1 activity and contributes to neurodegeneration. When we reduced TBK1 activity using a TBK1-R228H (Arg228→His) mutation in mice, poly(GA)-induced phenotypes were exacerbated. These phenotypes included an increase in TAR DNA binding protein 43 (TDP-43) pathology and the accumulation of defective endosomes in poly(GA)-positive neurons. Inhibiting the endosomal pathway induced TDP-43 aggregation, which highlights the importance of this pathway and TBK1 activity in pathogenesis. This interplay between C9orf72, TBK1, and TDP-43 connects three different facets of FTD-ALS into one coherent pathway.


Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Proteínas de Ligação a DNA , Demência Frontotemporal , Proteínas Serina-Treonina Quinases , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Expansão das Repetições de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endossomos/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Camundongos , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
7.
J Neurosci ; 30(32): 10851-9, 2010 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-20702714

RESUMO

Transactivation response DNA-binding protein 43 (TDP-43) is a principal component of ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). Mutations in TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS, yet multiple neurodegenerative diseases exhibit TDP-43 pathology without known TARDBP mutations. While TDP-43 has been ascribed a number of roles in normal biology, including mRNA splicing and transcription regulation, elucidating disease mechanisms associated with this protein is hindered by the lack of models to dissect such functions. We have generated transgenic (TDP-43PrP) mice expressing full-length human TDP-43 (hTDP-43) driven by the mouse prion promoter to provide a tool to analyze the role of wild-type hTDP-43 in the brain and spinal cord. Expression of hTDP-43 caused a dose-dependent downregulation of mouse TDP-43 RNA and protein. Moderate overexpression of hTDP-43 resulted in TDP-43 truncation, increased cytoplasmic and nuclear ubiquitin levels, and intranuclear and cytoplasmic aggregates that were immunopositive for phosphorylated TDP-43. Of note, abnormal juxtanuclear aggregates of mitochondria were observed, accompanied by enhanced levels of Fis1 and phosphorylated DLP1, key components of the mitochondrial fission machinery. Conversely, a marked reduction in mitofusin 1 expression, which plays an essential role in mitochondrial fusion, was observed in TDP-43PrP mice. Finally, TDP-43PrP mice showed reactive gliosis, axonal and myelin degeneration, gait abnormalities, and early lethality. This TDP-43 transgenic line provides a valuable tool for identifying potential roles of wild-type TDP-43 within the CNS and for studying TDP-43-associated neurotoxicity.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/metabolismo , Transtornos dos Movimentos , Análise de Variância , Animais , Peso Corporal/genética , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Dinaminas , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Neurônios Motores/ultraestrutura , Transtornos dos Movimentos/genética , Transtornos dos Movimentos/metabolismo , Transtornos dos Movimentos/mortalidade , Mutação/genética , Degeneração Neural/genética , Degeneração Neural/mortalidade , Degeneração Neural/patologia , Fosforilação/genética , Príons/genética , Príons/metabolismo , Coloração pela Prata/métodos , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/ultraestrutura
8.
Cell Rep ; 36(8): 109581, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34433069

RESUMO

Loss-of-function mutations in the progranulin gene (GRN), which encodes progranulin (PGRN), are a major cause of frontotemporal dementia (FTD). GRN-associated FTD is characterized by TDP-43 inclusions and neuroinflammation, but how PGRN loss causes disease remains elusive. We show that Grn knockout (KO) mice have increased microgliosis in white matter and an accumulation of myelin debris in microglial lysosomes in the same regions. Accumulation of myelin debris is also observed in white matter of patients with GRN-associated FTD. In addition, our findings also suggest that PGRN insufficiency in microglia leads to impaired lysosomal-mediated clearance of myelin debris. Finally, Grn KO mice that are deficient in cathepsin D (Ctsd), a key lysosomal enzyme, have augmented myelin debris and increased neuronal TDP-43 pathology. Together, our data strongly imply that PGRN loss affects microglial activation and lysosomal function, resulting in the accumulation of myelin debris and contributing to TDP-43 pathology.


Assuntos
Demência Frontotemporal/metabolismo , Lisossomos/metabolismo , Microglia/metabolismo , Progranulinas/metabolismo , Substância Branca/metabolismo , Animais , Feminino , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Humanos , Lisossomos/patologia , Masculino , Camundongos , Camundongos Knockout , Microglia/patologia , Progranulinas/genética , Substância Branca/patologia
9.
Front Cell Dev Biol ; 9: 809942, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35096836

RESUMO

The aberrant translation of a repeat expansion in chromosome 9 open reading frame 72 (C9orf72), the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), results in the accumulation of toxic dipeptide repeat (DPR) proteins in the central nervous system We have found that, among the sense DPR proteins, HDAC6 specifically interacts with the poly (GA) and co-localizes with inclusions in both patient tissue and a mouse model of this disease (c9FTD/ALS). Overexpression of HDAC6 increased poly (GA) levels in cultured cells independently of HDAC6 deacetylase activity, suggesting that HDAC6 can modulate poly (GA) pathology through a mechanism that depends upon their physical interaction. Moreover, decreasing HDAC6 expression by stereotaxic injection of antisense oligonucleotides significantly reduced the number of poly (GA) inclusions in c9FTD/ALS mice. These findings suggest that pharmacologically reducing HDAC6 levels could be of therapeutic value in c9FTD/ALS.

10.
Sci Transl Med ; 12(559)2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32878979

RESUMO

TAR DNA-binding protein 43 (TDP-43) inclusions are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), including cases caused by G4C2 repeat expansions in the C9orf72 gene (c9FTD/ALS). Providing mechanistic insight into the link between C9orf72 mutations and TDP-43 pathology, we demonstrated that a glycine-arginine repeat protein [poly(GR)] translated from expanded G4C2 repeats was sufficient to promote aggregation of endogenous TDP-43. In particular, toxic poly(GR) proteins mediated sequestration of full-length TDP-43 in an RNA-independent manner to induce cytoplasmic TDP-43 inclusion formation. Moreover, in GFP-(GR)200 mice, poly(GR) caused the mislocalization of nucleocytoplasmic transport factors and nuclear pore complex proteins. These mislocalization events resulted in the aberrant accumulation of endogenous TDP-43 in the cytoplasm where it co-aggregated with poly(GR). Last, we demonstrated that treating G4C2 repeat-expressing mice with repeat-targeting antisense oligonucleotides lowered poly(GR) burden, which was accompanied by reduced TDP-43 pathology and neurodegeneration, including lowering of plasma neurofilament light (NFL) concentration. These results contribute to clarification of the mechanism by which poly(GR) drives TDP-43 proteinopathy, confirm that G4C2-targeted therapeutics reduce TDP-43 pathology in vivo, and demonstrate that alterations in plasma NFL provide insight into the therapeutic efficacy of disease-modifying treatments.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Proteinopatias TDP-43 , Esclerose Lateral Amiotrófica/genética , Animais , Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/genética , Camundongos
11.
Cell Rep ; 31(5): 107616, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32375043

RESUMO

A G4C2 hexanucleotide repeat expansion in an intron of C9orf72 is the most common cause of frontal temporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). A remarkably similar intronic TG3C2 repeat expansion is associated with spinocerebellar ataxia 36 (SCA36). Both expansions are widely expressed, form RNA foci, and can undergo repeat-associated non-ATG (RAN) translation to form similar dipeptide repeat proteins (DPRs). Yet, these diseases result in the degeneration of distinct subsets of neurons. We show that the expression of these repeat expansions in mice is sufficient to recapitulate the unique features of each disease, including this selective neuronal vulnerability. Furthermore, only the G4C2 repeat induces the formation of aberrant stress granules and pTDP-43 inclusions. Overall, our results demonstrate that the pathomechanisms responsible for each disease are intrinsic to the individual repeat sequence, highlighting the importance of sequence-specific RNA-mediated toxicity in each disorder.


Assuntos
Proteína C9orf72/genética , Proteínas Nucleares/genética , RNA/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Expansão das Repetições de DNA/genética , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Camundongos , Neurônios/metabolismo
12.
Acta Neuropathol Commun ; 7(1): 10, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30674342

RESUMO

Pathogenic mutations in the tau gene (microtubule associated protein tau, MAPT) are linked to the onset of tauopathy, but the A152T variant is unique in acting as a risk factor for a range of disorders including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and dementia with Lewy bodies (DLB). In order to provide insight into the mechanism by which A152T modulates disease risk, we developed a novel mouse model utilizing somatic brain transgenesis with adeno-associated virus (AAV) to drive tau expression in vivo, and validated the model by confirming the distinct biochemical features of A152T tau in postmortem brain tissue from human carriers. Specifically, TauA152T-AAV mice exhibited increased tau phosphorylation that unlike animals expressing the pathogenic P301L mutation remained localized to the soluble fraction. To investigate the possibility that the A152T variant might alter the phosphorylation state of tau on T152 or the neighboring T153 residue, we generated a novel antibody that revealed significant accumulation of soluble tau species that were hyperphosphorylated on T153 (pT153) in TauA152T-AAV mice, which were absent the soluble fraction of TauP301L-AAV mice. Providing new insight into the role of A152T in modifying risk of tauopathy, as well as validating the TauA152T-AAV model, we demonstrate that the presence of soluble pT153-positive tau species in human postmortem brain tissue differentiates A152T carriers from noncarriers, independent of disease classification. These results implicate both phosphorylation of T153 and an altered solubility profile in the mechanism by which A152T modulates disease risk.


Assuntos
Encéfalo/metabolismo , Predisposição Genética para Doença , Doenças Neurodegenerativas/metabolismo , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Gliose/patologia , Humanos , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Fosforilação , Proteínas tau/genética
13.
Science ; 363(6428)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30765536

RESUMO

How hexanucleotide GGGGCC (G4C2) repeat expansions in C9orf72 cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is not understood. We developed a mouse model engineered to express poly(PR), a proline-arginine (PR) dipeptide repeat protein synthesized from expanded G4C2 repeats. The expression of green fluorescent protein-conjugated (PR)50 (a 50-repeat PR protein) throughout the mouse brain yielded progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis, culminating in motor and memory impairments. We found that poly(PR) bound DNA, localized to heterochromatin, and caused heterochromatin protein 1α (HP1α) liquid-phase disruptions, decreases in HP1α expression, abnormal histone methylation, and nuclear lamina invaginations. These aberrations of histone methylation, lamins, and HP1α, which regulate heterochromatin structure and gene expression, were accompanied by repetitive element expression and double-stranded RNA accumulation. Thus, we uncovered mechanisms by which poly(PR) may contribute to the pathogenesis of C9orf72-associated FTD and ALS.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/metabolismo , Dipeptídeos/metabolismo , Heterocromatina/patologia , RNA de Cadeia Dupla/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Encéfalo/metabolismo , Proteína C9orf72/genética , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Dipeptídeos/genética , Modelos Animais de Doenças , Proteínas de Fluorescência Verde , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Lâmina Nuclear/patologia , Sequências Repetitivas de Ácido Nucleico
14.
Nat Med ; 24(8): 1136-1142, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29942091

RESUMO

The major genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a C9orf72 G4C2 repeat expansion1,2. Proposed mechanisms by which the expansion causes c9FTD/ALS include toxicity from repeat-containing RNA and from dipeptide repeat proteins translated from these transcripts. To investigate the contribution of poly(GR) dipeptide repeat proteins to c9FTD/ALS pathogenesis in a mammalian in vivo model, we generated mice that expressed GFP-(GR)100 in the brain. GFP-(GR)100 mice developed age-dependent neurodegeneration, brain atrophy, and motor and memory deficits through the accumulation of diffuse, cytoplasmic poly(GR). Poly(GR) co-localized with ribosomal subunits and the translation initiation factor eIF3η in GFP-(GR)100 mice and, of importance, in c9FTD/ALS patients. Combined with the differential expression of ribosome-associated genes in GFP-(GR)100 mice, these findings demonstrate poly(GR)-mediated ribosomal distress. Indeed, poly(GR) inhibited canonical and non-canonical protein translation in HEK293T cells, and also induced the formation of stress granules and delayed their disassembly. These data suggest that poly(GR) contributes to c9FTD/ALS by impairing protein translation and stress granule dynamics, consequently causing chronic cellular stress and preventing cells from mounting an effective stress response. Decreasing poly(GR) and/or interrupting interactions between poly(GR) and ribosomal and stress granule-associated proteins may thus represent potential therapeutic strategies to restore homeostasis.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/metabolismo , Grânulos Citoplasmáticos/metabolismo , Dipeptídeos/farmacologia , Demência Frontotemporal/metabolismo , Biossíntese de Proteínas , Estresse Fisiológico , Animais , Comportamento Animal , Análise por Conglomerados , Grânulos Citoplasmáticos/efeitos dos fármacos , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Ribossômicas/metabolismo , Estresse Fisiológico/efeitos dos fármacos
15.
Sci Transl Med ; 9(383)2017 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-28356511

RESUMO

There is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. However, discovery of a G4C2 repeat expansion in the C9ORF72 gene as the most common genetic cause of ALS has opened up new avenues for therapeutic intervention for this form of ALS. G4C2 repeat expansion RNAs and proteins of repeating dipeptides synthesized from these transcripts are believed to play a key role in C9ORF72-associated ALS (c9ALS). Therapeutics that target G4C2 RNA, such as antisense oligonucleotides (ASOs) and small molecules, are thus being actively investigated. A limitation in moving such treatments from bench to bedside is a lack of pharmacodynamic markers for use in clinical trials. We explored whether poly(GP) proteins translated from G4C2 RNA could serve such a purpose. Poly(GP) proteins were detected in cerebrospinal fluid (CSF) and in peripheral blood mononuclear cells from c9ALS patients and, notably, from asymptomatic C9ORF72 mutation carriers. Moreover, CSF poly(GP) proteins remained relatively constant over time, boding well for their use in gauging biochemical responses to potential treatments. Treating c9ALS patient cells or a mouse model of c9ALS with ASOs that target G4C2 RNA resulted in decreased intracellular and extracellular poly(GP) proteins. This decrease paralleled reductions in G4C2 RNA and downstream G4C2 RNA-mediated events. These findings indicate that tracking poly(GP) proteins in CSF could provide a means to assess target engagement of G4C2 RNA-based therapies in symptomatic C9ORF72 repeat expansion carriers and presymptomatic individuals who are expected to benefit from early therapeutic intervention.


Assuntos
Esclerose Lateral Amiotrófica/genética , Biomarcadores/metabolismo , Proteína C9orf72/genética , Repetições de Dinucleotídeos/genética , Adulto , Idoso , Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Esclerose Lateral Amiotrófica/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Estudos Longitudinais , Camundongos , Pessoa de Meia-Idade , Neurônios/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Prognóstico , RNA/genética
16.
Brain Res ; 1647: 57-64, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27155453

RESUMO

Inclusions of Tar DNA- binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP). Pathological TDP-43 exhibits the disease-specific biochemical signatures, which include its ubiquitination, phosphorylation and truncation. Recently, we demonstrated that the extreme N-terminus of TDP-43 regulates formation of abnormal cytoplasmic TDP-43 aggregation in cultured cells and primary neurons. However, it remained unclear whether this N-terminal domain mediates TDP-43 aggregation and the associated toxicity in vivo. To investigate this, we expressed a GFP-tagged TDP-43 with a nuclear localization signal mutation (GFP-TDP-43NLSm) and a truncated form without the extreme N-terminus (GFP-TDP-4310-414-NLSm) by adeno-associated viral (AAV) vectors in mouse primary cortical neurons and murine central nervous system. Compared to neurons containing GFP alone, expression of GFP-TDP-43NLSm resulted in the formation of ubiquitin-positive cytoplasmic inclusions and activation of caspase-3, an indicator of cell death. Moreover, mice expressing GFP-TDP-43NLSm proteins show reactive gliosis and develop neurological abnormalities. However, by deletion of TDP-43's extreme N-terminus, these pathological alterations can be abrogated. Together, our study provides further evidence confirming the critical role of the extreme N-terminus of TDP-43 in regulating protein structure as well as mediating toxicity associated with its aggregation. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease.


Assuntos
Córtex Cerebral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neurônios/metabolismo , Animais , Caspase 3/metabolismo , Morte Celular , Células Cultivadas , Córtex Cerebral/patologia , Proteínas de Ligação a DNA/genética , Gliose/metabolismo , Corpos de Inclusão/metabolismo , Camundongos , Atividade Motora , Neurônios/patologia
17.
Nat Neurosci ; 19(5): 668-677, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26998601

RESUMO

Neuronal inclusions of poly(GA), a protein unconventionally translated from G4C2 repeat expansions in C9ORF72, are abundant in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) caused by this mutation. To investigate poly(GA) toxicity, we generated mice that exhibit poly(GA) pathology, neurodegeneration and behavioral abnormalities reminiscent of FTD and ALS. These phenotypes occurred in the absence of TDP-43 pathology and required poly(GA) aggregation. HR23 proteins involved in proteasomal degradation and proteins involved in nucleocytoplasmic transport were sequestered by poly(GA) in these mice. HR23A and HR23B similarly colocalized to poly(GA) inclusions in C9ORF72 expansion carriers. Sequestration was accompanied by an accumulation of ubiquitinated proteins and decreased xeroderma pigmentosum C (XPC) levels in mice, indicative of HR23A and HR23B dysfunction. Restoring HR23B levels attenuated poly(GA) aggregation and rescued poly(GA)-induced toxicity in neuronal cultures. These data demonstrate that sequestration and impairment of nuclear HR23 and nucleocytoplasmic transport proteins is an outcome of, and a contributor to, poly(GA) pathology.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neurônios/patologia , Proteínas/toxicidade , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Atrofia/patologia , Comportamento Animal , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Proteína C9orf72 , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Expressão Gênica/genética , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/ultraestrutura , Camundongos , Mutação , Degeneração Neural/patologia , Neurônios/metabolismo , Cultura Primária de Células , Proteínas/genética , Proteínas/metabolismo , Proteínas Ubiquitinadas/metabolismo
18.
Neurobiol Aging ; 35(7): 1769-77, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24503275

RESUMO

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is a neurodegenerative tauopathy caused by mutations in the tau gene (MAPT). Individuals with FTDP-17 have deficits in learning, memory, and language, in addition to personality and behavioral changes that are often characterized by a lack of social inhibition. Several transgenic mouse models expressing tau mutations have been tested extensively for memory or motor impairments, though reports of amygdala-dependent behaviors are lacking. To this end, we tested the rTg4510 mouse model on a behavioral battery that included amygdala-dependent tasks of exploration. As expected, rTg4510 mice exhibit profound impairments in hippocampal-dependent learning and memory tests, including contextual fear conditioning. However, rTg4510 mice also display an abnormal hyperexploratory phenotype in the open-field assay, elevated plus maze, light-dark exploration, and cued fear conditioning, indicative of amygdala dysfunction. Furthermore, significant tau burden is detected in the amygdala of both rTg4510 mice and human FTDP-17 patients, suggesting that the rTg4510 mouse model recapitulates the behavioral disturbances and neurodegeneration of the amygdala characteristic of FTDP-17.


Assuntos
Tonsila do Cerebelo/patologia , Tonsila do Cerebelo/fisiopatologia , Demência Frontotemporal/genética , Demência Frontotemporal/fisiopatologia , Mutação/genética , Degeneração Neural , Proteínas tau/genética , Animais , Comportamento Animal , Condicionamento Psicológico , Modelos Animais de Doenças , Comportamento Exploratório , Medo , Demência Frontotemporal/patologia , Demência Frontotemporal/psicologia , Humanos , Idioma , Aprendizagem , Memória , Camundongos , Camundongos Transgênicos , Índice de Gravidade de Doença
19.
PLoS One ; 8(7): e69864, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23922830

RESUMO

Tar DNA binding protein 43 (TDP-43) is the major component of pathological deposits in frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in amyotrophic lateral sclerosis (ALS). It has been reported that TDP-43 transgenic mouse models expressing human TDP-43 wild-type or ALS-associated mutations recapitulate certain ALS and FTLD pathological phenotypes. Of note, expression of human TDP-43 (hTDP-43) reduces the levels of mouse Tdp-43 (mTdp-43). However, it remained unclear whether the mechanisms through which TDP-43 induces ALS or FTLD-like pathologies resulted from a reduction in mTdp-43, an increase in hTDP-43, or a combination of both. In elucidating the role of mTdp-43 and hTDP-43 in hTDP-43 transgenic mice, we observed that reduction of mTdp-43 in non-transgenic mice by intraventricular brain injection of AAV1-shTardbp leads to a dramatic increase in the levels of splicing variants of mouse sortilin 1 and translin. However, the levels of these two abnormal splicing variants are not increased in hTDP-43 transgenic mice despite significant downregulation of mTdp-43 in these mice. Moreover, further downregulation of mTdp-43 in hTDP-43 hemizygous mice, which are asymptomatic, to the levels equivalent to that of mTdp-43 in hTDP-43 homozygous mice does not induce the pathological phenotypes observed in the homozygous mice. Lastly, the number of dendritic spines and the RNA levels of TDP-43 RNA targets critical for synapse formation and function are significantly decreased in symptomatic homozygous mice. Together, our findings indicate that mTdp-43 downregulation does not lead to a loss of function mechanism or account for the pathological phenotypes observed in hTDP-43 homozygous mice because hTDP-43 compensates for the reduction, and associated functions of mTdp-43. Rather, expression of hTDP-43 beyond a certain threshold leads to abnormal metabolism of TDP-43 RNA targets critical for neuronal structure and function, which might be responsible for the ALS or FTLD-like pathologies observed in homozygous hTDP-43 transgenic mice.


Assuntos
Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Degeneração Lobar Frontotemporal/patologia , Processamento Alternativo/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Ligação a DNA/genética , Espinhas Dendríticas/metabolismo , Modelos Animais de Doenças , Degeneração Lobar Frontotemporal/metabolismo , Hemizigoto , Homozigoto , Humanos , Injeções Intraventriculares , Camundongos , Camundongos Transgênicos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sinapses/metabolismo
20.
PLoS One ; 4(6): e5888, 2009 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-19517018

RESUMO

BACKGROUND: Covalent linkage of ubiquitin regulates the function and, ultimately, the degradation of many proteins by the ubiquitin-proteasome system (UPS). Given its essential role in protein regulation, even slight perturbations in UPS activity can substantially impair cellular function. METHODOLOGY/PRINCIPAL FINDINGS: We have generated and characterized a novel transgenic mouse model which expresses a previously described reporter for UPS function. This UPS reporter contains a degron sequence attached to the C-terminus of green fluorescent protein, and is predominantly expressed in neurons throughout the brain of our transgenic model. We then demonstrated that this reporter system is sensitive to UPS inhibition in vivo. CONCLUSIONS/SIGNIFICANCE: Given the obstacles associated with evaluating proteasomal function in the brain, our mouse model uniquely provides the capability to monitor UPS function in real time in individual neurons of a complex organism. Our novel mouse model now provides a useful resource with which to evaluate the impact of aging, as well as various genetic and/or pharmacological modifiers of neurodegenerative disease(s).


Assuntos
Envelhecimento , Regulação da Expressão Gênica , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/fisiologia , Ubiquitina/genética , Ubiquitina/fisiologia , Animais , Feminino , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doenças Neurodegenerativas/genética , Neurônios/citologia , Neurônios/metabolismo , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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