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1.
Nature ; 626(8001): 1073-1083, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38355792

RESUMO

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1, which involve human-specific mechanisms2-5 that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors6. Single-cell transcriptomics and comparison to independent neural stem cells7 showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids8. Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity.


Assuntos
Esclerose Lateral Amiotrófica , Proteína C-Reativa , Proteínas de Ligação a DNA , Degeneração Lobar Frontotemporal , Rede Nervosa , Proteínas do Tecido Nervoso , Neurônios , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteína C-Reativa/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Degeneração Lobar Frontotemporal/patologia , Rede Nervosa/metabolismo , Rede Nervosa/patologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neurônios/citologia , Neurônios/metabolismo , Reprodutibilidade dos Testes
2.
EMBO J ; 42(17): e111719, 2023 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-37431963

RESUMO

Aggregation of the RNA-binding protein TAR DNA-binding protein 43 (TDP-43) is the key neuropathological feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In physiological conditions, TDP-43 is predominantly nuclear, forms oligomers, and is contained in biomolecular condensates assembled by liquid-liquid phase separation (LLPS). In disease, TDP-43 forms cytoplasmic or intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Using a variety of cellular systems to express structure-based TDP-43 variants, including human neurons and cell lines with near-physiological expression levels, we show that oligomerization and RNA binding govern TDP-43 stability, splicing functionality, LLPS, and subcellular localization. Importantly, our data reveal that TDP-43 oligomerization is modulated by RNA binding. By mimicking the impaired proteasomal activity observed in ALS/FTLD patients, we found that monomeric TDP-43 forms inclusions in the cytoplasm, whereas its RNA binding-deficient counterpart aggregated in the nucleus. These differentially localized aggregates emerged via distinct pathways: LLPS-driven aggregation in the nucleus and aggresome-dependent inclusion formation in the cytoplasm. Therefore, our work unravels the origins of heterogeneous pathological species reminiscent of those occurring in TDP-43 proteinopathy patients.


Assuntos
Esclerose Lateral Amiotrófica , Degeneração Lobar Frontotemporal , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neurônios/metabolismo , RNA/genética
3.
Mol Cell ; 73(3): 490-504.e6, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30581145

RESUMO

Fused in sarcoma (FUS) is an RNA binding protein involved in regulating many aspects of RNA processing and linked to several neurodegenerative diseases. Transcriptomics studies indicate that FUS binds a large variety of RNA motifs, suggesting that FUS RNA binding might be quite complex. Here, we present solution structures of FUS zinc finger (ZnF) and RNA recognition motif (RRM) domains bound to RNA. These structures show a bipartite binding mode of FUS comprising of sequence-specific recognition of a NGGU motif via the ZnF and an unusual shape recognition of a stem-loop RNA via the RRM. In addition, sequence-independent interactions via the RGG repeats significantly increase binding affinity and promote destabilization of structured RNA conformation, enabling additional binding. We further show that disruption of the RRM and ZnF domains abolishes FUS function in splicing. Altogether, our results rationalize why deciphering the RNA binding mode of FUS has been so challenging.


Assuntos
Proteína FUS de Ligação a RNA/química , RNA/química , Sítios de Ligação , Células HeLa , Humanos , Modelos Moleculares , Motivos de Nucleotídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA/genética , RNA/metabolismo , Motivo de Reconhecimento de RNA , Splicing de RNA , Estabilidade de RNA , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Relação Estrutura-Atividade , Dedos de Zinco
4.
EMBO J ; 41(11): e111425, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35578810

RESUMO

From the management of microtubules to the production of pathological species: liquid-liquid phase separation may tune the behavior of the protein tau in health and neurodegenerative disease. In this issue of The EMBO Journal, Hochmair et al (2022) demystify important aspects of tau condensate compilation.


Assuntos
Doenças Neurodegenerativas , Humanos , Microtúbulos/metabolismo , Doenças Neurodegenerativas/metabolismo , Proteínas tau/metabolismo
5.
EMBO J ; 41(23): e112338, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36254605

RESUMO

A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting cellular host-factors that can modify prion propagation. We exposed prion-infected cells in high-density microplates to 35,364 ternary pools of 52,746 siRNAs targeting 17,582 genes representing the majority of the mouse protein-coding transcriptome. We identified 1,191 modulators of prion propagation. While 1,151 modified the expression of both the pathological prion protein, PrPSc , and its cellular counterpart, PrPC , 40 genes selectively affected PrPSc . Of the latter 40 genes, 20 augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrPC . Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Further, the unexpected identification of a prion-controlling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.


Assuntos
Doenças Priônicas , Príons , Camundongos , Animais , Ovinos/genética , Príons/genética , Príons/metabolismo , Drosophila melanogaster/genética , Ribonucleoproteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Doenças Priônicas/genética , Doenças Priônicas/patologia , Mamíferos/genética
6.
Cell ; 147(3): 498-508, 2011 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-22036560

RESUMO

Misfolded proteins accumulating in several neurodegenerative diseases (including Alzheimer, Parkinson, and Huntington diseases) can cause aggregation of their native counterparts through a mechanism similar to the infectious prion protein's induction of a pathogenic conformation onto its cellular isoform. Evidence for such a prion-like mechanism has now spread to the main misfolded proteins, SOD1 and TDP-43, implicated in amyotrophic lateral sclerosis (ALS). The major neurodegenerative diseases may therefore have mechanistic parallels for non-cell-autonomous spread of disease within the nervous system.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Príons/metabolismo , Animais , Humanos , Doenças Neurodegenerativas/metabolismo , Dobramento de Proteína
7.
Proc Natl Acad Sci U S A ; 119(49): e2123487119, 2022 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-36454749

RESUMO

Hexanucleotide G4C2 repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged C9orf72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.


Assuntos
Genes Reguladores , Poli A , Animais , Humanos , Camundongos , Complexo Antígeno-Anticorpo , Proteína C9orf72/genética , Dipeptídeos , Modelos Animais de Doenças
8.
Alzheimers Dement ; 20(2): 1156-1165, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37908186

RESUMO

INTRODUCTION: We assessed TAR DNA-binding protein 43 (TDP-43) seeding activity and aggregates detection in olfactory mucosa of patients with frontotemporal lobar degeneration with TDP-43-immunoreactive pathology (FTLD-TDP) by TDP-43 seeding amplification assay (TDP43-SAA) and immunocytochemical analysis. METHODS: The TDP43-SAA was optimized using frontal cortex samples from 16 post mortem cases with FTLD-TDP, FTLD with tau inclusions, and controls. Subsequently, olfactory mucosa samples were collected from 17 patients with FTLD-TDP, 15 healthy controls, and three patients carrying MAPT variants. RESULTS: TDP43-SAA discriminated with 100% accuracy post mortem cases presenting or lacking TDP-43 neuropathology. TDP-43 seeding activity was detectable in the olfactory mucosa, and 82.4% of patients with FTLD-TDP tested positive, whereas 86.7% of controls tested negative (P < 0.001). Two out of three patients with MAPT mutations tested negative. In TDP43-SAA positive samples, cytoplasmatic deposits of phosphorylated TDP-43 in the olfactory neural cells were detected. DISCUSSION: TDP-43 aggregates can be detectable in olfactory mucosa, suggesting that TDP43-SAA might be useful for identifying and monitoring FTLD-TDP in living patients.


Assuntos
Demência Frontotemporal , Degeneração Lobar Frontotemporal , Humanos , Demência Frontotemporal/genética , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/patologia , Proteínas tau/genética , Proteínas tau/metabolismo , Lobo Frontal/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo
9.
Trends Immunol ; 41(9): 755-757, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32800706

RESUMO

C9ORF72 mutations are the most common genetic cause of ALS and FTD, leading to neurodegeneration via complex mechanisms. Mutations also lead to loss of C9ORF72 function and inflammatory diseases in patients and knockout mice. Burberry et al. now show that C9orf72-associated inflammation and premature death in mice are directly modified by the gut microbiome.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Esclerose Lateral Amiotrófica/genética , Animais , Bactérias , Proteína C9orf72 , Humanos , Inflamação , Camundongos
10.
EMBO Rep ; 22(12): e53877, 2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34806807

RESUMO

Morphologically distinct TDP-43 aggregates occur in clinically different FTLD-TDP subtypes, yet the mechanism of their emergence and contribution to clinical heterogeneity are poorly understood. Several lines of evidence suggest that pathological TDP-43 follows a prion-like cascade, but the molecular determinants of this process remain unknown. We use advanced microscopy techniques to compare the seeding properties of pathological FTLD-TDP-A and FTLD-TDP-C aggregates. Upon inoculation of patient-derived aggregates in cells, FTLD-TDP-A seeds amplify in a template-dependent fashion, triggering neoaggregation more efficiently than those extracted from FTLD-TDP-C patients, correlating with the respective disease progression rates. Neoaggregates are sequentially phosphorylated with N-to-C directionality and with subtype-specific timelines. The resulting FTLD-TDP-A neoaggregates are large and contain densely packed fibrils, reminiscent of the pure compacted fibrils present within cytoplasmic inclusions in postmortem brains. In contrast, FTLD-TDP-C dystrophic neurites show less dense fibrils mixed with cellular components, and their respective neoaggregates are small, amorphous protein accumulations. These cellular seeding models replicate aspects of the patient pathological diversity and will be a useful tool in the quest for subtype-specific therapeutics.


Assuntos
Demência Frontotemporal , Príons , Encéfalo/metabolismo , Demência Frontotemporal/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Príons/metabolismo
11.
Chimia (Aarau) ; 73(6): 380-390, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31118120

RESUMO

Altered cellular localization and pathologic aggregation of RNA binding proteins (RPBs) containing low complexity regions (LCRs) is a hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Given the importance of RBPs in maintaining a healthy RNA homeostasis, a common mechanism in disease progression is the loss of RNA-related cellular functions. In this review, we summarize and discuss the knowledge gained in the recent years on the molecular mechanisms of TDP-43 proteinopathies that comprise a set of neurodegenerative diseases characterized by the mislocalization and aggregation of the RNA-binding protein TDP-43. Based on biophysical, biochemical and in vivo data, we highlight pathways that are misregulated early in disease and contribute to its progression, thereby representing attractive therapeutic targets.


Assuntos
Doenças Neurodegenerativas , Humanos , Proteinopatias TDP-43
12.
J Proteome Res ; 17(12): 4072-4084, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30137990

RESUMO

The Biology/Disease-driven (B/D) working groups of the Human Proteome Project are alliances of research groups aimed at developing or improving proteomic tools to support specific biological or disease-related research areas. Here, we describe the activities and progress to date of the B/D working group focused on protein aggregation diseases (PADs). PADs are characterized by the intra- or extracellular accumulation of aggregated proteins and include devastating diseases such as Parkinson's and Alzheimer's disease and systemic amyloidosis. The PAD B/D working group aims for the development of proteomic assays for the quantification of aggregation-prone proteins involved in PADs to support basic and clinical research on PADs. Because the proteins in PADs undergo aberrant conformational changes, a goal is to quantitatively resolve altered protein structures and aggregation states in complex biological specimens. We have developed protein-extraction protocols and a set of mass spectrometric (MS) methods that enable the detection and quantification of proteins involved in the systemic and localized amyloidosis and the probing of aberrant protein conformational transitions in cell and tissue extracts. In several studies, we have demonstrated the potential of MS-based proteomics approaches for specific and sensitive clinical diagnoses and for the subtyping of PADs. The developed methods have been detailed in both protocol papers and manuscripts describing applications to facilitate implementation by nonspecialized laboratories, and assay coordinates are shared through public repositories and databases. Clinicians actively involved in the PAD working group support the transfer to clinical practice of the developed methods, such as assays to quantify specific disease-related proteins and their fragments in biofluids and multiplexed MS-based methods for the diagnosis and typing of systemic amyloidosis. We believe that the increasing availability of tools to precisely measure proteins involved in PADs will positively impact research on the molecular bases of these diseases and support early disease diagnosis and a more-confident subtyping.


Assuntos
Objetivos , Agregação Patológica de Proteínas , Proteoma/química , Proteômica/métodos , Logro , Doença de Alzheimer , Amiloidose , Projeto Genoma Humano , Humanos , Doença de Parkinson
13.
Immunity ; 29(6): 998-1008, 2008 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-19100703

RESUMO

Prior to invading the nervous system, prions frequently colonize lymphoid organs and sites of inflammatory lymphoneogenesis, where they colocalize with Mfge8+ follicular dendritic cells (FDCs). Here, we report that soft-tissue granulomas, a frequent feature of chronic inflammation, expressed the cellular prion protein (PrPC, encoded by Prnp) and the lymphotoxin receptor (LTbetaR), even though they lacked FDCs and did not display lymphoneogenesis. After intraperitoneal prion inoculation, granulomas of Prnp(+/+) mice, but not Prnp(-/-) granulomas or unaffected Prnp(+/+) skin, accumulated prion infectivity and disease-associated prion protein. Bone-marrow transfers between Prnp(+/+) and Prnp(-/-) mice and administration of lymphotoxin signaling antagonists indicated that prion replication required radioresistant PrPC-expressing cells and LTbetaR signaling. Granulomatous PrPC was mainly expressed by stromal LTbetaR+ mesenchymal cells that were absent from unaffected subcutis. Hence, granulomas can act as clinically silent reservoirs of prion infectivity. Furthermore, lymphotoxin-dependent prion replication can occur in inflammatory stromal cells that are distinct from FDCs.


Assuntos
Células Dendríticas Foliculares/imunologia , Granuloma/imunologia , Receptor beta de Linfotoxina/imunologia , Linfotoxina-alfa/imunologia , Príons/metabolismo , Animais , Células Dendríticas Foliculares/metabolismo , Granuloma/genética , Granuloma/patologia , Receptor beta de Linfotoxina/metabolismo , Linfotoxina-alfa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Priônicas , Príons/genética , Células Estromais/imunologia , Células Estromais/metabolismo
14.
J Neurochem ; 138 Suppl 1: 163-83, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27502124

RESUMO

Frontotemporal dementia is a devastating neurodegenerative disease causing stark alterations in personality and language. Characterized by severe atrophy of the frontal and temporal brain lobes, frontotemporal dementia (FTD) shows extreme heterogeneity in clinical presentation, genetic causes, and pathological findings. Like most neurodegenerative diseases, the initial symptoms of FTD are subtle, but increase in severity over time, as the disease progresses. Clinical progression is paralleled by exacerbation of pathological findings and the involvement of broader brain regions, which currently lack mechanistic explanation. Yet, a flurry of studies indicate that protein aggregates accumulating in neurodegenerative diseases can act as propagating entities, amplifying their pathogenic conformation, in a way similar to infectious prions. In this prion-centric view, FTD can be divided into three subtypes, TDP-43 or FUS proteinopathy and tauopathy. Here, we review the current evidence that FTD-linked pathology propagates in a prion-like manner and discuss the implications of these findings for disease progression and heterogeneity. Frontotemporal dementia (FTD) is a progressive neurodegenerative disease causing severe personality dysfunctions, characterized by profound heterogeneity. Accumulation of tau, TDP-43 or FUS cytoplasmic aggregates characterize molecularly distinct and non-overlapping FTD subtypes. Here, we discuss the current evidence suggesting that prion-like propagation and cell-to-cell spread of each of these cytoplasmic aggregates may underlie disease progression and heterogeneity. This article is part of the Frontotemporal Dementia special issue.


Assuntos
Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Doenças Priônicas/genética , Doenças Priônicas/patologia , Príons/genética , Encéfalo/patologia , Demência Frontotemporal/etiologia , Humanos
15.
Proc Natl Acad Sci U S A ; 110(8): E736-45, 2013 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-23382207

RESUMO

Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43(Q331K) and TDP-43(M337V)), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43-dependent alternative splicing events conferred by both human wild-type and mutant TDP-43(Q331K), but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43(Q331K) enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.


Assuntos
Esclerose Lateral Amiotrófica/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Mutação , Splicing de RNA , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Proteínas de Ligação a DNA/metabolismo , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Ubiquitinação
16.
Proc Natl Acad Sci U S A ; 110(47): E4530-9, 2013 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-24170860

RESUMO

Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions.


Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Expansão das Repetições de DNA/genética , Degeneração Lobar Frontotemporal/tratamento farmacológico , Terapia Genética/métodos , Oligonucleotídeos Antissenso/farmacologia , Proteínas/genética , Esclerose Lateral Amiotrófica/genética , Animais , Southern Blotting , Proteína C9orf72 , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Primers do DNA/genética , Fibroblastos/metabolismo , Degeneração Lobar Frontotemporal/genética , Genótipo , Hibridização in Situ Fluorescente , Camundongos , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA
17.
EMBO Mol Med ; 16(9): 2024-2042, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39080493

RESUMO

Extracellularly released molecular inflammasome assemblies -ASC specks- cross-seed Aß amyloid in Alzheimer's disease. Here we show that ASC governs the extent of inflammation-induced amyloid A (AA) amyloidosis, a systemic disease caused by the aggregation and peripheral deposition of the acute-phase reactant serum amyloid A (SAA) in chronic inflammatory conditions. Using super-resolution microscopy, we found that ASC colocalized tightly with SAA in human AA amyloidosis. Recombinant ASC specks accelerated SAA fibril formation and mass spectrometry after limited proteolysis showed that ASC interacts with SAA via its pyrin domain (PYD). In a murine model of inflammatory AA amyloidosis, splenic amyloid load was conspicuously decreased in Pycard-/- mice which lack ASC. Treatment with anti-ASCPYD antibodies decreased amyloid loads in wild-type mice suffering from AA amyloidosis. The prevalence of natural anti-ASC IgG (-logEC50 ≥ 2) in 19,334 hospital patients was <0.01%, suggesting that anti-ASC antibody treatment modalities would not be confounded by natural autoimmunity. These findings expand the role played by ASC and IL-1 independent inflammasome employments to extraneural proteinopathies and suggest that anti-ASC immunotherapy may contribute to resolving such diseases.


Assuntos
Amiloidose , Proteínas Adaptadoras de Sinalização CARD , Inflamassomos , Proteína Amiloide A Sérica , Animais , Proteína Amiloide A Sérica/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Amiloidose/metabolismo , Amiloidose/patologia , Humanos , Inflamassomos/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais de Doenças , Inflamação/metabolismo , Inflamação/patologia , Agregados Proteicos , Camundongos Endogâmicos C57BL
18.
Cells ; 12(4)2023 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-36831264

RESUMO

TDP-43 is the primary or secondary pathological hallmark of neurodegenerative diseases, such as amyotrophic lateral sclerosis, half of frontotemporal dementia cases, and limbic age-related TDP-43 encephalopathy, which clinically resembles Alzheimer's dementia. In such diseases, a biomarker that can detect TDP-43 proteinopathy in life would help to stratify patients according to their definite diagnosis of pathology, rather than in clinical subgroups of uncertain pathology. For therapies developed to target pathological proteins that cause the disease a biomarker to detect and track the underlying pathology would greatly enhance such undertakings. This article reviews the latest developments and outlooks of deriving TDP-43-specific biomarkers from the pathophysiological processes involved in the development of TDP-43 proteinopathy and studies using biosamples from clinical entities associated with TDP-43 pathology to investigate biomarker candidates.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Proteinopatias TDP-43 , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Demência Frontotemporal/patologia , Biomarcadores , Proteínas de Ligação a DNA/metabolismo
19.
J Biol Chem ; 286(14): 12149-56, 2011 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-21324909

RESUMO

Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrPSc, which is enriched in the ß-sheet structure. Although the high resolution structure of the normal PrP was determined, the structure of the converted form of PrP remains inaccessible to high resolution techniques. To map the PrP conversion process we introduced disulfide bridges into different positions within the globular domain of PrP, tethering selected secondary structure elements. The majority of tethered PrP mutants exhibited increased thermodynamic stability, nevertheless, they converted efficiently. Only the disulfides that tether subdomain B1-H1-B2 to subdomain H2-H3 prevented PrP conversion in vitro and in prion-infected cell cultures. Reduction of disulfides recovered the ability of these mutants to convert, demonstrating that the separation of subdomains is an essential step in conversion. Formation of disulfide-linked proteinase K-resistant dimers in fibrils composed of a pair of single cysteine mutants supports the model based on domain-swapped dimers as the building blocks of prion fibrils. In contrast to previously proposed structural models of PrPSc suggesting conversion of large secondary structural segments, we provide evidence for the conservation of secondary structural elements of the globular domain upon PrP conversion. Previous studies already showed that dimerization is the rate-limiting step in PrP conversion. We show that separation and swapping of subdomains of the globular domain is necessary for conversion. Therefore, we propose that the domain-swapped dimer of PrP precedes amyloid formation and represents a potential target for therapeutic intervention.


Assuntos
Príons/química , Príons/metabolismo , Animais , Linhagem Celular , Dicroísmo Circular , Dissulfetos/síntese química , Dissulfetos/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Mutação , Príons/genética , Príons/ultraestrutura
20.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 11): 1501-12, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23090399

RESUMO

Prion diseases are neurodegenerative diseases characterized by the conversion of the cellular prion protein PrP(c) into a pathogenic isoform PrP(sc). Passive immunization with antiprion monoclonal antibodies can arrest the progression of prion diseases. Here, the crystal structure of the Fab fragment of an antiprion monoclonal antibody, POM1, in complex with human prion protein (huPrP(c)) has been determined to 2.4 Å resolution. The prion epitope of POM1 is in close proximity to the epitope recognized by the purportedly therapeutic antibody fragment ICSM18 Fab in complex with huPrP(c). POM1 Fab forms a 1:1 complex with huPrP(c) and the measured K(d) of 4.5 × 10(-7) M reveals moderately strong binding between them. Structural comparisons have been made among three prion-antibody complexes: POM1 Fab-huPrP(c), ICSM18 Fab-huPrP(c) and VRQ14 Fab-ovPrP(c). The prion epitopes recognized by ICSM18 Fab and VRQ14 Fab are adjacent to a prion glycosylation site, indicating possible steric hindrance and/or an altered binding mode to the glycosylated prion protein in vivo. However, both of the glycosylation sites on huPrP(c) are positioned away from the POM1 Fab binding epitope; thus, the binding mode observed in this crystal structure and the binding affinity measured for this antibody are most likely to be the same as those for the native prion protein in vivo.


Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/química , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas PrPC/química , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Sítios de Ligação de Anticorpos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Proteínas PrPC/imunologia , Doenças Priônicas/imunologia , Dobramento de Proteína , Estrutura Terciária de Proteína
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