RESUMO
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), 2 incurable neurodegenerative disorders, share the same pathological hallmark named TDP43 (TAR DNA binding protein 43) proteinopathy. This event is characterized by a consistent cytoplasmic mislocalization and aggregation of the protein TDP43, which loses its physiological properties, leading neurons to death. Antibody-based approaches are now emerging interventions in the field of neurodegenerative disorders. Here, we tested the target specificity, in vivo distribution, and therapeutic efficacy of a monoclonal full-length antibody, named E6, in TDP43-related conditions. We observed that the antibody recognizes specifically the cytoplasmic fraction of TDP43. We demonstrated its ability in targeting large neurons in the spinal cord of mice and in reducing TDP43 mislocalization and NF-κB activation. We also recognized the proteasome as well as the lysosome machineries as possible mechanisms used by the antibody to reduce TDP43 proteinopathy. To our knowledge, this is the first report showing the therapeutic efficacy and feasibility of a full-length antibody against TDP43 in reducing TDP43 proteinopathy in spinal neurons of an ALS/FTLD mouse model.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Anticorpos Monoclonais/farmacologia , Proteínas de Ligação a DNA/imunologia , Neurônios/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Proteinopatias TDP-43/tratamento farmacológico , Idoso , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Neurônios/imunologia , Neurônios/patologia , Medula Espinal/imunologia , Medula Espinal/patologia , Proteinopatias TDP-43/imunologia , Proteinopatias TDP-43/metabolismo , Proteinopatias TDP-43/patologiaRESUMO
BACKGROUND: Frontotemporal dementia (FTD) is the second leading cause of early onset dementia following Alzheimer's disease. It involves atrophy of the frontal and temporal regions of the brain affecting language, memory, and behavior. Transactive response DNA-binding protein 43 (TDP-43) pathology is found in most FTD and ALS cases. It plays a role in transcription, translation and serves as a shuttle between the nucleus and cytoplasm. Prior to its aggregation, TDP-43 exists as polyubiquitinated, hyperphosphorylated C-terminal fragments that correlate well with FTD disease progression. Because of the importance of TDP-43 in these diseases, reagents that can selectively recognize specific toxic TDP variants associated with onset and progression of FTD can be effective diagnostic and therapeutic tools. RESULTS: We utilized a novel atomic force microscopy (AFM) based biopanning protocol to isolate single chain variable fragments (scFvs) from a phage display library that selectively bind TDP variants present in human FTD but not cognitively normal age matched brain tissue. We then used the scFvs (FTD-TDP1 through 5) to probe post-mortem brain tissue and sera samples for the presence of FTD related TDP variants. The scFvs readily selected the FTD tissue and sera samples over age matched controls. The scFvs were used in immunohistochemical analysis of FTD and control brain slices where the reagents showed strong staining with TDP in FTD brain tissue slice. FTD-TDP1, FTD-TDP2, FTD-TDP4 and FTD-TDP5 all protected neuronal cells against FTD TDP induced toxicity suggesting potential therapeutic value. CONCLUSIONS: These results show existence of different disease specific TDP variants in FTD individuals. We have identified a panel of scFvs capable of recognizing these disease specific TDP variants in postmortem FTD tissue and sera samples over age matched controls and can thus serve as a biomarker tool.
Assuntos
Proteínas de Ligação a DNA/genética , Demência Frontotemporal/genética , Fragmentos de Imunoglobulinas/isolamento & purificação , Proteinopatias TDP-43/diagnóstico , Proteinopatias TDP-43/genética , Especificidade de Anticorpos , Biomarcadores , Biotinilação , Encéfalo/imunologia , Proteínas de Ligação a DNA/química , Demência Frontotemporal/diagnóstico , Demência Frontotemporal/imunologia , Variação Genética , Humanos , Fragmentos de Imunoglobulinas/química , Imuno-Histoquímica , Microscopia de Força Atômica , Sensibilidade e Especificidade , Proteinopatias TDP-43/imunologiaRESUMO
Frontotemporal lobar degeneration with neuronal inclusions of the TAR DNA-binding protein 43 (FTLD-TDP) represents the most common pathological subtype of FTLD. We established the international FTLD-TDP whole-genome sequencing consortium to thoroughly characterize the known genetic causes of FTLD-TDP and identify novel genetic risk factors. Through the study of 1131 unrelated Caucasian patients, we estimated that C9orf72 repeat expansions and GRN loss-of-function mutations account for 25.5% and 13.9% of FTLD-TDP patients, respectively. Mutations in TBK1 (1.5%) and other known FTLD genes (1.4%) were rare, and the disease in 57.7% of FTLD-TDP patients was unexplained by the known FTLD genes. To unravel the contribution of common genetic factors to the FTLD-TDP etiology in these patients, we conducted a two-stage association study comprising the analysis of whole-genome sequencing data from 517 FTLD-TDP patients and 838 controls, followed by targeted genotyping of the most associated genomic loci in 119 additional FTLD-TDP patients and 1653 controls. We identified three genome-wide significant FTLD-TDP risk loci: one new locus at chromosome 7q36 within the DPP6 gene led by rs118113626 (p value = 4.82e - 08, OR = 2.12), and two known loci: UNC13A, led by rs1297319 (p value = 1.27e - 08, OR = 1.50) and HLA-DQA2 led by rs17219281 (p value = 3.22e - 08, OR = 1.98). While HLA represents a locus previously implicated in clinical FTLD and related neurodegenerative disorders, the association signal in our study is independent from previously reported associations. Through inspection of our whole-genome sequence data for genes with an excess of rare loss-of-function variants in FTLD-TDP patients (n ≥ 3) as compared to controls (n = 0), we further discovered a possible role for genes functioning within the TBK1-related immune pathway (e.g., DHX58, TRIM21, IRF7) in the genetic etiology of FTLD-TDP. Together, our study based on the largest cohort of unrelated FTLD-TDP patients assembled to date provides a comprehensive view of the genetic landscape of FTLD-TDP, nominates novel FTLD-TDP risk loci, and strongly implicates the immune pathway in FTLD-TDP pathogenesis.
Assuntos
Proteínas do Tecido Nervoso/genética , Proteinopatias TDP-43/genética , Idoso , Expansão das Repetições de DNA , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Feminino , Lobo Frontal/metabolismo , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/imunologia , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Antígenos HLA-DQ/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/fisiologia , Canais de Potássio/genética , Progranulinas/genética , Progranulinas/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas/genética , Proteínas/fisiologia , RNA Mensageiro/biossíntese , Fatores de Risco , Análise de Sequência de RNA , Sociedades Científicas , Proteinopatias TDP-43/imunologia , População Branca/genéticaRESUMO
Pathological aggregation and mutation of the 43-kDa TAR DNA-binding protein (TDP-43) are strongly implicated in the pathogenesis amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 neurotoxicity has been extensively modeled in mice, zebrafish, Caenorhabditis elegans and Drosophila, where selective expression of TDP-43 in motoneurons led to paralysis and premature lethality. Through a genetic screen aimed to identify genetic modifiers of TDP-43, we found that the Drosophila dual leucine kinase Wallenda (Wnd) and its downstream kinases JNK and p38 influenced TDP-43 neurotoxicity. Reducing Wnd gene dosage or overexpressing its antagonist highwire partially rescued TDP-43-associated premature lethality. Downstream of Wnd, the JNK and p38 kinases played opposing roles in TDP-43-associated neurodegeneration. LOF alleles of the p38b gene as well as p38 inhibitors diminished TDP-43-associated premature lethality, whereas p38b GOF caused phenotypic worsening. In stark contrast, disruptive alleles of Basket (Bsk), the Drosophila homologue of JNK, exacerbated longevity shortening, whereas overexpression of Bsk extended lifespan. Among possible mechanisms, we found motoneuron-directed expression of TDP-43 elicited oxidative stress and innate immune gene activation that were exacerbated by p38 GOF and Bsk LOF, respectively. A key pathologic role for innate immunity in TDP-43-associated neurodegeneration was further supported by the finding that genetic suppression of the Toll/Dif and Imd/Relish inflammatory pathways dramatically extended lifespan of TDP-43 transgenic flies. We propose that oxidative stress and neuroinflammation are intrinsic components of TDP-43-associated neurodegeneration and that the balance between cytoprotective JNK and cytotoxic p38 signaling dictates phenotypic outcome to TDP-43 expression in Drosophila.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteinopatias TDP-43/imunologia , Proteinopatias TDP-43/patologia , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/imunologia , Genes Letais , Humanos , Imunidade Inata , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , Estresse Oxidativo , Proteinopatias TDP-43/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Proteinaceous inclusions are common hallmarks of many neurodegenerative diseases. TDP-43 proteinopathies, consisting of several neurodegenerative diseases, including frontotemporal lobar dementia (FTLD) and amyotrophic lateral sclerosis (ALS), are characterized by inclusion bodies formed by polyubiquitinated and hyperphosphorylated full-length and truncated TDP-43. The structural properties of TDP-43 aggregates and their relationship to pathogenesis are still ambiguous. Here we demonstrate that the recombinant full-length human TDP-43 forms structurally stable, spherical oligomers that share common epitopes with an anti-amyloid oligomer-specific antibody. The TDP-43 oligomers are stable, have exposed hydrophobic surfaces, exhibit reduced DNA binding capability and are neurotoxic in vitro and in vivo. Moreover, TDP-43 oligomers are capable of cross-seeding Alzheimer's amyloid-ß to form amyloid oligomers, demonstrating interconvertibility between the amyloid species. Such oligomers are present in the forebrain of transgenic TDP-43 mice and FTLD-TDP patients. Our results suggest that aside from filamentous aggregates, TDP-43 oligomers may play a role in TDP-43 pathogenesis.