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1.
Cell ; 186(22): 4898-4919.e25, 2023 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-37827155

RESUMO

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.


Assuntos
Proteína de Replicação A , Expansão das Repetições de Trinucleotídeos , Animais , Humanos , Camundongos , DNA/genética , Reparo de Erro de Pareamento de DNA , Doença de Huntington/genética , Proteínas/genética , Ataxias Espinocerebelares/genética , Proteína de Replicação A/metabolismo
2.
EMBO J ; 43(18): 3968-3999, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39103492

RESUMO

Senescence of nondividing neurons remains an immature concept, with especially the regulatory molecular mechanisms of senescence-like phenotypes and the role of proteins associated with neurodegenerative diseases in triggering neuronal senescence remaining poorly explored. In this study, we reveal that the nucleolar polyglutamine binding protein 3 (PQBP3; also termed NOL7), which has been linked to polyQ neurodegenerative diseases, regulates senescence as a gatekeeper of cytoplasmic DNA leakage. PQBP3 directly binds PSME3 (proteasome activator complex subunit 3), a subunit of the 11S proteasome regulator complex, decreasing PSME3 interaction with Lamin B1 and thereby preventing Lamin B1 degradation and senescence. Depletion of endogenous PQBP3 causes nuclear membrane instability and release of genomic DNA from the nucleus to the cytosol. Among multiple tested polyQ proteins, ataxin-1 (ATXN1) partially sequesters PQBP3 to inclusion bodies, reducing nucleolar PQBP3 levels. Consistently, knock-in mice expressing mutant Atxn1 exhibit decreased nuclear PQBP3 and a senescence phenotype in Purkinje cells of the cerebellum. Collectively, these results suggest homologous roles of the nucleolar protein PQBP3 in cellular senescence and neurodegeneration.


Assuntos
Senescência Celular , Lamina Tipo B , Complexo de Endopeptidases do Proteassoma , Animais , Humanos , Camundongos , Ataxina-1/metabolismo , Ataxina-1/genética , Células HEK293 , Lamina Tipo B/metabolismo , Lamina Tipo B/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Células de Purkinje/metabolismo
3.
Biochem Biophys Res Commun ; 736: 150453, 2024 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-39126896

RESUMO

PQBP3 is a protein binding to polyglutamine tract sequences that are expanded in a group of neurodegenerative diseases called polyglutamine diseases. The function of PQBP3 was revealed recently as an inhibitor protein of proteasome-dependent degradation of Lamin B1 that is shifted from nucleolus to peripheral region of nucleus to keep nuclear membrane stability. Here, we address whether PQBP3 is an intrinsically disordered protein (IDP) like other polyglutamine binding proteins including PQBP1, PQBP5 and VCP. Multiple bioinformatics analyses predict that N-terminal region of PQBP3 is unstructured. High-speed atomic force microscopy (HS-AFM) reveals that N-terminal region of PQBP3 is dynamically changed in the structure consistently with the predictions of the bioinformatics analyses. These data support that PQBP3 is also an IDP.

4.
Int J Mol Sci ; 25(7)2024 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-38612448

RESUMO

The mechanisms of neuronal cell death in neurodegenerative disease remain incompletely understood, although recent studies have made significant advances. Apoptosis was previously considered to be the only mechanism of neuronal cell death in neurodegenerative diseases. However, recent findings have challenged this dogma, identifying new subtypes of necrotic neuronal cell death. The present review provides an updated summary of necrosis subtypes and discusses their potential roles in neurodegenerative cell death. Among numerous necrosis subtypes, including necroptosis, paraptosis, ferroptosis, and pyroptosis, transcriptional repression-induced atypical cell death (TRIAD) has been identified as a potential mechanism of neuronal cell death. TRIAD is induced by functional deficiency of TEAD-YAP and self-amplifies via the release of HMGB1. TRIAD is a feasible potential mechanism of neuronal cell death in Alzheimer's disease and other neurodegenerative diseases. In addition to induction of cell death, HMGB1 released during TRIAD activates brain inflammatory responses, which is a potential link between neurodegeneration and neuroinflammation.


Assuntos
Proteína HMGB1 , Doenças Neurodegenerativas , Humanos , Doenças Neuroinflamatórias , Necrose , Morte Celular
5.
Brain Behav Immun ; 111: 32-45, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37004758

RESUMO

The molecular pathological mechanisms underlying schizophrenia remain unclear; however, genomic analysis has identified genes encoding important risk molecules. One such molecule is neurexin 1α (NRXN1α), a presynaptic cell adhesion molecule. In addition, novel autoantibodies that target the nervous system have been found in patients with encephalitis and neurological disorders. Some of these autoantibodies inhibit synaptic antigen molecules. Studies have examined the association between schizophrenia and autoimmunity; however, the pathological data remain unclear. Here, we identified a novel autoantibody against NRXN1α in patients with schizophrenia (n = 2.1%) in a Japanese cohort (n = 387). None of the healthy control participants (n = 362) were positive for anti-NRXN1α autoantibodies. Anti-NRXN1α autoantibodies isolated from patients with schizophrenia inhibited the molecular interaction between NRXN1α and Neuroligin 1 (NLGN1) and between NRXN1α and Neuroligin 2 (NLGN2). Additionally, these autoantibodies reduced the frequency of the miniature excitatory postsynaptic current in the frontal cortex of mice. Administration of anti-NRXN1α autoantibodies from patients with schizophrenia into the cerebrospinal fluid of mice reduced the number of spines/synapses in the frontal cortex and induced schizophrenia-related behaviors such as reduced cognition, impaired pre-pulse inhibition, and reduced social novelty preference. These changes were improved through the removal of anti-NRXN1α autoantibodies from the IgG fraction of patients with schizophrenia. These findings demonstrate that anti-NRXN1α autoantibodies transferred from patients with schizophrenia cause schizophrenia-related pathology in mice. Removal of anti-NRXN1α autoantibodies may be a therapeutic target for a subgroup of patients who are positive for these autoantibodies.


Assuntos
Esquizofrenia , Camundongos , Animais , Esquizofrenia/genética , Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , Autoanticorpos/metabolismo , Fenótipo
6.
Int J Mol Sci ; 23(11)2022 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-35682906

RESUMO

The idea that a common pathology underlies various neurodegenerative diseases and dementias has attracted considerable attention in the basic and medical sciences. Polyglutamine binding protein-1 (PQBP1) was identified in 1998 after a molecule was predicted to bind to polyglutamine tract amino acid sequences, which are associated with a family of neurodegenerative disorders called polyglutamine diseases. Hereditary gene mutations of PQBP1 cause intellectual disability, whereas acquired loss of function of PQBP1 contributes to dementia pathology. PQBP1 functions in innate immune cells as an intracellular receptor that recognizes pathogens and neurodegenerative proteins. It is an intrinsically disordered protein that generates intracellular foci, similar to other neurodegenerative disease proteins such as TDP43, FUS, and hnRNPs. The knowledge accumulated over more than 20 years has given rise to a new concept that shifts in the equilibrium between physiological and pathological processes have their basis in the dysregulation of common protein structure-linked molecular mechanisms.


Assuntos
Deficiência Intelectual , Doenças Neurodegenerativas , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Imunidade Inata , Deficiência Intelectual/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Proteínas Nucleares/genética
7.
Neuropathology ; 41(2): 93-98, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33876503

RESUMO

Using a new marker of necrosis, pSer46-MARCKS, which was identified by comprehensive phosphoproteome analysis as a phosphoprotein changed before appearance of extracellular amyloid aggregation, we discovered that neuronal necrosis occurs much earlier in Alzheimer's disease pathology than previously expected. The necrosis is induced by intracellular amyloid accumulation that deprives a critical effector molecule, Yes-associated protein (YAP), in the Hippo signaling pathway that is essential for cell survival, similarly to TRIAD necrosis observed in transcriptional repression and in other neurodegenerative diseases such as Huntington's disease. The initial TRIAD necrosis due to the intracellular amyloid releases HMGB1 into extracellular space and induces cluster of secondary necrosis around the primary necrotic neurons. Finally, the cluster grows into extracellular amyloid plaque. Inhibition of HMGB1 by anti-HMGB1 antibody prevents expansion of neurodegeneration. Administration even after onset significantly ameliorates the cognitive decline of Alzheimer's disease model mice. Our results present a new theory of Alzheimer's disease pathology, which can be referred to as the "intracellular amyloid hypothesis".


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Disfunção Cognitiva/fisiopatologia , Placa Amiloide/patologia , Doença de Alzheimer/metabolismo , Animais , Disfunção Cognitiva/metabolismo , Modelos Animais de Doenças , Humanos , Neurônios/patologia , Placa Amiloide/metabolismo
8.
J Neurosci ; 39(4): 678-691, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30504273

RESUMO

The actin cytoskeleton is crucial for neuronal migration in the mammalian developing cerebral cortex. The adaptor protein Drebrin-like (Dbnl) plays important roles in reorganization of the actin cytoskeleton, dendrite formation, and endocytosis by interacting with F-actin, cobl, and dynamin. Although Dbnl is known to be expressed in the brain, the functions of this molecule during brain development are largely unknown. In this study, to examine the roles of Dbnl in the developing cerebral cortex, we conducted experiments using mice of both sexes with knockdown of Dbnl, effected by in utero electroporation, in the migrating neurons of the embryonic cortex. Time-lapse imaging of the Dbnl-knockdown neurons revealed that the presence of Dbnl is a prerequisite for appropriate formation of processes in the multipolar neurons in the multipolar cell accumulation zone or the deep part of the subventricular zone, and for neuronal polarization and entry into the cortical plate. We found that Dbnl knockdown decreased the amount of N-cadherin protein expressed on the plasma membrane of the cortical neurons. The defect in neuronal migration caused by Dbnl knockdown was rescued by moderate overexpression of N-cadherin and αN-catenin or by transfection of the phospho-mimic form (Y337E, Y347E), but not the phospho-resistant form (Y337F, Y347F), of Dbnl. These results suggest that Dbnl controls neuronal migration, neuronal multipolar morphology, and cell polarity in the developing cerebral cortex via regulating N-cadherin expression.SIGNIFICANCE STATEMENT Disruption of neuronal migration can cause neuronal disorders, such as lissencephaly and subcortical band heterotopia. During cerebral cortical development, the actin cytoskeleton plays a key role in neuronal migration; however, the mechanisms of regulation of neuronal migration by the actin cytoskeleton still remain unclear. Herein, we report that the novel protein Dbnl, an actin-binding protein, controls multiple events during neuronal migration in the developing mouse cerebral cortex. We also showed that this regulation is mediated by phosphorylation of Dbnl at tyrosine residues 337 and 347 and αN-catenin/N-cadherin, suggesting that the Dbnl-αN-catenin/N-cadherin pathway is important for neuronal migration in the developing cortex.


Assuntos
Caderinas/biossíntese , Movimento Celular/fisiologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/fisiologia , Proteínas dos Microfilamentos/fisiologia , Neurônios/fisiologia , Domínios de Homologia de src/fisiologia , Animais , Caderinas/genética , Membrana Celular/metabolismo , Córtex Cerebral/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas dos Microfilamentos/biossíntese , Proteínas dos Microfilamentos/genética , Neurônios/ultraestrutura , Gravidez , Domínios de Homologia de src/genética
9.
Biochem Biophys Res Commun ; 523(4): 894-899, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31959475

RESUMO

Polyglutamine tract-binding protein 1 (PQBP1), an intellectual disability causative gene, is involved in transcriptional and post-transcriptional regulation of gene expression in animals, and possibly also in plants. In our previous work, reduced brain size, associated with an elongated cell cycle duration in neural stem cells (NSCs), was observed in the NSCs conditional Pqbp1 gene knockout (cKO) mice, which mimic microcephaly patients. However, the physiological significance of PQBP1 in bone metabolism has not been elucidated. Here, we analyzed the bone phenotype of nestin-Cre Pqbp1-cKO mice. Surprisingly, the Pqbp1-cKO mice were significantly shorter than control mice and had a lower bone mass, shown by micro-computed tomography. Furthermore, bone histology showed impaired bone formation in the Pqbp1-cKO mice as well as a chondrocyte deficiency. Real-time PCR analysis showed reduced osteoblast- and chondrocyte-related gene expression in the Pqbp1-cKO mice, while the osteoclast-related gene expression remained unchanged. These results suggest that PQBP1 in bone marrow mesenchymal stem cells may play a crucial role in bone formation and cartilage development.


Assuntos
Desenvolvimento Ósseo/genética , Proteínas de Ligação a DNA/genética , Crescimento e Desenvolvimento/genética , Deficiência Intelectual/genética , Animais , Osso e Ossos/metabolismo , Cartilagem/embriologia , Diferenciação Celular , Feminino , Masculino , Camundongos Knockout , Tamanho do Órgão , Osteoblastos/metabolismo , Osteoclastos/metabolismo
10.
J Struct Biol ; 206(3): 305-313, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30951824

RESUMO

Polyglutamine tract-binding protein-1 (PQBP-1) is a nuclear intrinsically disordered protein playing important roles in transcriptional regulation and RNA splicing during embryonic and postembryonic development. In human, its mutations lead to severe cognitive impairment known as the Renpenning syndrome, a form of X-linked intellectual disability (XLID). Here, we report a combined biophysical study of two PQBP-1 frameshift mutants, K192Sfs*7 and R153Sfs*41. Both mutants are dimeric in solution, in contrast to the monomeric wild-type protein. These mutants contain more folded contents and have increased thermal stabilities. Using small-angle X-ray scattering data, we generated three-dimensional envelopes which revealed their overall flat shapes. We also described each mutant using an ensemble model based on a native-like initial pool with a dimeric structural core. PQBP-1 is known to repress transcription by way of interacting with the C-terminal domain of RNA polymerase II, which consists of 52 repeats of a consensus heptapeptide sequence YSPTSPS. We studied the binding of PQBP-1 variants to the labelled peptide which is phosphorylated at positions 2 and 5 (YpSPTpSPS) and found that this interaction is significantly weakened in the two mutants.


Assuntos
Paralisia Cerebral/genética , Proteínas de Ligação a DNA/genética , Deficiência Intelectual/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Transcrição Gênica , Fenômenos Biofísicos , Paralisia Cerebral/patologia , Proteínas de Ligação a DNA/química , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Mutação da Fase de Leitura/genética , Genes Ligados ao Cromossomo X/genética , Humanos , Deficiência Intelectual/patologia , Deficiência Intelectual Ligada ao Cromossomo X/patologia , Peptídeos/química , Peptídeos/genética , Ligação Proteica/genética , Conformação Proteica , Splicing de RNA/genética , Relação Estrutura-Atividade
11.
Mol Psychiatry ; 23(10): 2090-2110, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30283027

RESUMO

Early-phase pathologies of Alzheimer's disease (AD) are attracting much attention after clinical trials of drugs designed to remove beta-amyloid (Aß) aggregates failed to recover memory and cognitive function in symptomatic AD patients. Here, we show that phosphorylation of serine/arginine repetitive matrix 2 (SRRM2) at Ser1068, which is observed in the brains of early phase AD mouse models and postmortem end-stage AD patients, prevents its nuclear translocation by inhibiting interaction with T-complex protein subunit α. SRRM2 deficiency in neurons destabilized polyglutamine binding protein 1 (PQBP1), a causative gene for intellectual disability (ID), greatly affecting the splicing patterns of synapse-related genes, as demonstrated in a newly generated PQBP1-conditional knockout model. PQBP1 and SRRM2 were downregulated in cortical neurons of human AD patients and mouse AD models, and the AAV-PQBP1 vector recovered RNA splicing, the synapse phenotype, and the cognitive decline in the two mouse models. Finally, the kinases responsible for the phosphorylation of SRRM2 at Ser1068 were identified as ERK1/2 (MAPK3/1). These results collectively reveal a new aspect of AD pathology in which a phosphorylation signal affecting RNA splicing and synapse integrity precedes the formation of extracellular Aß aggregates and may progress in parallel with tau phosphorylation.


Assuntos
Doença de Alzheimer/genética , Proteínas de Transporte/genética , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Transporte Ativo do Núcleo Celular , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Cognição , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas , Deficiência Intelectual/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Cultura Primária de Células , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas tau/metabolismo
12.
Hum Mol Genet ; 25(20): 4432-4447, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28173122

RESUMO

DNA damage and repair is a critical domain of many neurodegenerative diseases. In this study, we focused on RpA1, a candidate key molecule in polyQ disease pathologies, and tested the therapeutic effect of adeno-associated virus (AAV) vector expressing RpA1 on mutant Ataxin-1 knock-in (Atxn1-KI) mice. We found significant effects on motor functions, normalized DNA damage markers (γH2AX and 53BP1), and improved Purkinje cell morphology; effects that lasted for 50 weeks following AAV-RpA1 infection. In addition, we confirmed that AAV-RpA1 indirectly recovered multiple cellular functions such as RNA splicing, transcription and cell cycle as well as abnormal morphology of dendrite and dendritic spine of Purkinje cells in Atxn1-KI mice. All these results suggested a possibility of gene therapy with RpA1 for SCA1.


Assuntos
Ataxina-1/genética , Reparo do DNA , Mutação , Proteína de Replicação A/metabolismo , Ataxias Espinocerebelares/metabolismo , Animais , Ciclo Celular , DNA/metabolismo , Dano ao DNA , Dependovirus , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Terapia Genética , Camundongos , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Células de Purkinje/fisiologia , RNA/metabolismo , Splicing de RNA , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia , Ataxias Espinocerebelares/fisiopatologia , Transcrição Gênica
13.
Hum Mol Genet ; 25(21): 4749-4770, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28171658

RESUMO

Neuronal cell death in neurodegenerative diseases is not fully understood. Here we report that mutant huntingtin (Htt), a causative gene product of Huntington's diseases (HD) selectively induces a new form of necrotic cell death, in which endoplasmic reticulum (ER) enlarges and cell body asymmetrically balloons and finally ruptures. Pharmacological and genetic analyses revealed that the necrotic cell death is distinct from the RIP1/3 pathway-dependent necroptosis, but mediated by a functional deficiency of TEAD/YAP-dependent transcription. In addition, we revealed that a cell cycle regulator, Plk1, switches the balance between TEAD/YAP-dependent necrosis and p73/YAP-dependent apoptosis by shifting the interaction partner of YAP from TEAD to p73 through YAP phosphorylation at Thr77. In vivo ER imaging with two-photon microscopy detects similar ER enlargement, and viral vector-mediated delivery of YAP as well as chemical inhibitors of the Hippo pathway such as S1P recover the ER instability and necrosis in HD model mice. Intriguingly S1P completely stops the decline of motor function of HD model mice even after the onset of symptom. Collectively, we suggest approaches targeting the signalling pathway of TEAD/YAP-transcription-dependent necrosis (TRIAD) could lead to a therapeutic development against HD.


Assuntos
Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Necrose/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular , Morte Celular , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/metabolismo , Humanos , Doença de Huntington/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Necrose/genética , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Cultura Primária de Células , Ligação Proteica , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Hum Mol Genet ; 24(2): 540-58, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25231903

RESUMO

Using a high-end mass spectrometry, we screened phosphoproteins and phosphopeptides in four types of Alzheimer's disease (AD) mouse models and human AD postmortem brains. We identified commonly changed phosphoproteins in multiple models and also determined phosphoproteins related to initiation of amyloid beta (Aß) deposition in the mouse brain. After confirming these proteins were also changed in and human AD brains, we put the proteins on experimentally verified protein-protein interaction databases. Surprisingly, most of the core phosphoproteins were directly connected, and they formed a functional network linked to synaptic spine formation. The change of the core network started at a preclinical stage even before histological Aß deposition. Systems biology analyses suggested that phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) by overactivated kinases including protein kinases C and calmodulin-dependent kinases initiates synapse pathology. Two-photon microscopic observation revealed recovery of abnormal spine formation in the AD model mice by targeting a core protein MARCKS or by inhibiting candidate kinases, supporting our hypothesis formulated based on phosphoproteome analysis.


Assuntos
Doença de Alzheimer/metabolismo , Fosfoproteínas/metabolismo , Sinapses/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Substrato Quinase C Rico em Alanina Miristoilada , Fosfoproteínas/genética , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Transdução de Sinais
15.
Proc Jpn Acad Ser B Phys Biol Sci ; 93(6): 361-377, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28603208

RESUMO

The concept of neurodegenerative diseases and the therapeutics targeting these intractable diseases are changing rapidly. Protein aggregation as the top of pathological cascade is now challenged, and many alternative ideas are proposed. Early molecular pathologies before microscopic detection of diseases protein aggregates, which I propose to call "Ultra-Early Phase pathologies or phase 0 pathologies", are the focus of research that might explain the failures of clinical trials with anti-Aß antibodies against Alzheimer's disease. In this review article, I summarize the critical issues that should be successfully and consistently answered by a new concept of neurodegeneration. For reevaluating old concepts and reconstructing a new concept of neurodegeneration that will replace the old ones, non-biased comprehensive approaches including proteome combined with systems biology analyses will be a powerful tool. I introduce our recent efforts in this orientation that have reached to the stage of non-clinical proof of concept applicable to clinical trials.


Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos/química , Anticorpos/imunologia , Encéfalo/metabolismo , Linhagem Celular , Proteína HMGB1/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/química , Doenças Neurodegenerativas/patologia , Neurônios/citologia , Neurônios/metabolismo , Agregação Patológica de Proteínas
16.
Hum Mol Genet ; 23(5): 1345-64, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24179173

RESUMO

DNA damage repair is implicated in neurodegenerative diseases; however, the relative contributions of various DNA repair systems to the pathology of these diseases have not been investigated systematically. In this study, we performed a systematic in vivo screen of all available Drosophila melanogaster homolog DNA repair genes, and we tested the effect of their overexpression on lifespan and developmental viability in Spinocerebellar Ataxia Type 1 (SCA1) Drosophila models expressing human mutant Ataxin-1 (Atxn1). We identified genes previously unknown to be involved in CAG-/polyQ-related pathogenesis that function in multiple DNA damage repair systems. Beyond the significance of each repair system, systems biology analyses unraveled the core networks connecting positive genes in the gene screen that could contribute to SCA1 pathology. In particular, RpA1, which had the largest effect on lifespan in the SCA1 fly model, was located at the hub position linked to such core repair systems, including homologous recombination (HR). We revealed that Atxn1 actually interacted with RpA1 and its essential partners BRCA1/2. Furthermore, mutant but not normal Atxn1 impaired the dynamics of RpA1 in the nucleus after DNA damage. Uptake of BrdU by Purkinje cells was observed in mutant Atxn1 knockin mice, suggesting their abnormal entry to the S-phase. In addition, chemical and genetic inhibitions of Chk1 elongated lifespan and recovered eye degeneration. Collectively, we elucidated core networks for DNA damage repair in SCA1 that might include the aberrant usage of HR.


Assuntos
Dano ao DNA , Reparo do DNA , Drosophila/genética , Redes Reguladoras de Genes , Ataxias Espinocerebelares/genética , Animais , Animais Geneticamente Modificados , Ataxina-1 , Ataxinas , Ciclo Celular/genética , Quinase 1 do Ponto de Checagem , Modelos Animais de Doenças , Feminino , Vetores Genéticos/genética , Humanos , Longevidade/genética , Masculino , Mutagênese Insercional , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Proteínas Quinases/metabolismo , Células de Purkinje/metabolismo , Transdução de Sinais , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/mortalidade , Biologia de Sistemas
17.
Hum Mol Genet ; 21(5): 1099-110, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22100762

RESUMO

The spinocerebellar ataxia type 7 (SCA7) gene product, Ataxin-7 (ATXN7), localizes to the nucleus and has been shown to function as a component of the TATA-binding protein-free TAF-containing-SPT3-TAF9-GCN5-acetyltransferase transcription complex, although cytoplasmic localization of ATXN7 in affected neurons of human SCA7 patients has also been detected. Here, we define a physiological function for cytoplasmic ATXN7. Live imaging reveals that the intracellular distribution of ATXN7 dynamically changes and that ATXN7 distribution frequently shifts from the nucleus to the cytoplasm. Immunocytochemistry and immunoprecipitation demonstrate that cytoplasmic ATXN7 associates with microtubules (MTs), and expression of ATXN7 stabilizes MTs against nocodazole treatment, while ATXN7 knockdown enhances MT degradation. Interestingly, normal and mutant ATXN7 similarly associate with and equally stabilize MTs. Taken together, these findings provide a novel physiological function of ATXN7 in the regulation of cytoskeletal dynamics, and suggest that abnormal cytoskeletal regulation may contribute to SCA7 disease pathology.


Assuntos
Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Tubulina (Proteína)/metabolismo , Ataxina-7 , Ciclo Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Microtúbulos/ultraestrutura , Mitose , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Transfecção
18.
EMBO J ; 29(14): 2446-60, 2010 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-20531390

RESUMO

Non-cell-autonomous effect of mutant proteins expressed in glia has been implicated in several neurodegenerative disorders, whereas molecules mediating the toxicity are currently not known. We identified a novel molecule named multiple alpha-helix protein located at ER (Maxer) downregulated by mutant ataxin-1 (Atx1) in Bergmann glia. Maxer is an endoplasmic reticulum (ER) membrane protein interacting with CDK5RAP3. Maxer anchors CDK5RAP3 to the ER and inhibits its function of Cyclin D1 transcription repression in the nucleus. The loss of Maxer eventually induces cell accumulation at G1 phase. It was also shown that mutant Atx1 represses Maxer and inhibits proliferation of Bergmann glia in vitro. Consistently, Bergmann glia are reduced in the cerebellum of mutant Atx1 knockin mice before onset. Glutamate-aspartate transporter reduction in Bergmann glia by mutant Atx1 and vulnerability of Purkinje cell to glutamate are both strengthened by Maxer knockdown in Bergmann glia, whereas Maxer overexpression rescues them. Collectively, these results suggest that the reduction of Maxer mediates functional deficiency of Bergmann glia, and might contribute to the non-cell-autonomous pathology of SCA1.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso , Neuroglia/metabolismo , Proteínas Nucleares , Sequência de Aminoácidos , Animais , Ataxina-1 , Ataxinas , Proteínas de Ciclo Celular , Proliferação de Células , Transportador 1 de Aminoácido Excitatório/genética , Transportador 1 de Aminoácido Excitatório/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neuroglia/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Distribuição Tecidual , Proteínas Supressoras de Tumor
19.
Nat Cell Biol ; 9(4): 402-14, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17384639

RESUMO

Nuclear dysfunction is a key feature of the pathology of polyglutamine (polyQ) diseases. It has been suggested that mutant polyQ proteins impair functions of nuclear factors by interacting with them directly in the nucleus. However, a systematic analysis of quantitative changes in soluble nuclear proteins in neurons expressing mutant polyQ proteins has not been performed. Here, we perform a proteome analysis of soluble nuclear proteins prepared from neurons expressing huntingtin (Htt) or ataxin-1 (AT1) protein, and show that mutant AT1 and Htt similarly reduce the concentration of soluble high mobility group B1/2 (HMGB1/2) proteins. Immunoprecipitation and pulldown assays indicate that HMGBs interact with mutant AT1 and Htt. Immunohistochemistry showed that these proteins were reduced in the nuclear region outside of inclusion bodies in affected neurons. Compensatory expression of HMGBs ameliorated polyQ-induced pathology in primary neurons and in Drosophila polyQ models. Furthermore, HMGBs repressed genotoxic stress signals induced by mutant Htt or transcriptional repression. Thus, HMGBs may be critical regulators of polyQ disease pathology and could be targets for therapy development.


Assuntos
Proteína HMGB1/fisiologia , Proteína HMGB2/fisiologia , Doenças Neurodegenerativas/metabolismo , Proteínas Nucleares/fisiologia , Proteômica/métodos , Animais , Western Blotting , Morte Celular , Células Cultivadas , Drosophila , Eletroforese em Gel Bidimensional , Proteína HMGB1/análise , Proteína HMGB1/metabolismo , Proteína HMGB2/análise , Proteína HMGB2/metabolismo , Imuno-Histoquímica , Imunoprecipitação , Modelos Biológicos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Neurônios/citologia , Neurônios/metabolismo , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Células de Purkinje/citologia , Células de Purkinje/metabolismo , RNA Interferente Pequeno , Ratos , Ratos Wistar , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
Commun Biol ; 7(1): 413, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38594382

RESUMO

Better understanding of the earliest molecular pathologies of all neurodegenerative diseases is expected to improve human therapeutics. We investigated the earliest molecular pathology of spinocerebellar ataxia type 1 (SCA1), a rare familial neurodegenerative disease that primarily induces death and dysfunction of cerebellum Purkinje cells. Extensive prior studies have identified involvement of transcription or RNA-splicing factors in the molecular pathology of SCA1. However, the regulatory network of SCA1 pathology, especially central regulators of the earliest developmental stages and inflammatory events, remains incompletely understood. Here, we elucidated the earliest developmental pathology of SCA1 using originally developed dynamic molecular network analyses of sequentially acquired RNA-seq data during differentiation of SCA1 patient-derived induced pluripotent stem cells (iPSCs) to Purkinje cells. Dynamic molecular network analysis implicated histone genes and cytokine-relevant immune response genes at the earliest stages of development, and revealed relevance of ISG15 to the following degradation and accumulation of mutant ataxin-1 in Purkinje cells of SCA1 model mice and human patients.


Assuntos
Células-Tronco Pluripotentes Induzidas , Ataxias Espinocerebelares , Animais , Humanos , Camundongos , Citocinas , Células-Tronco Pluripotentes Induzidas/patologia , Camundongos Transgênicos , Células de Purkinje/fisiologia , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia , Ubiquitinas
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