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1.
Life Sci Alliance ; 5(4)2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35022247

RESUMO

In motor neurons of sporadic amyotrophic lateral sclerosis (ALS) patients, the RNA editing at the glutamine/arginine site of the GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors is defective or incomplete. As a result, AMPA receptors containing the abnormally expressed, unedited isoform of GluA2 are highly Ca2+-permeable, and are responsible for mediating abnormal Ca2+ influx, thereby triggering motor neuron degeneration and cell death. Thus, blocking the AMPA receptor-mediated, abnormal Ca2+ influx is a potential therapeutic strategy for treatment of sporadic ALS. Here, we report a study of the efficacy and safety of two RNA aptamers targeting AMPA receptors on the ALS phenotype of AR2 mice. A 12-wk continuous, intracerebroventricular infusion of aptamers to AR2 mice reduced the progression of motor dysfunction, normalized TDP-43 mislocalization, and prevented death of motor neurons. Our results demonstrate that the use of AMPA receptor aptamers as a novel class of AMPA receptor antagonists is a promising strategy for developing an ALS treatment approach.


Assuntos
Esclerose Lateral Amiotrófica , Aptâmeros de Nucleotídeos/genética , Receptores de AMPA , Animais , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Masculino , Camundongos , Neurônios Motores/metabolismo , Edição de RNA/genética , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/genética , Receptores de AMPA/metabolismo
2.
Int J Mol Sci ; 20(13)2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31252669

RESUMO

Recent progress in the research for underlying mechanisms in neurodegenerative diseases, including Alzheimer disease (AD), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS) has led to the development of potentially effective treatment, and hence increased the need for useful biomarkers that may enable early diagnosis and therapeutic monitoring. The deposition of abnormal proteins is a pathological hallmark of neurodegenerative diseases, including ß-amyloid in AD, α-synuclein in PD, and the transactive response DNA/RNA binding protein of 43kDa (TDP-43) in ALS. Furthermore, progression of the disease process accompanies the spreading of abnormal proteins. Extracellular proteins and RNAs, including mRNA, micro RNA, and circular RNA, which are present as a composite of exosomes or other forms, play a role in cell-cell communication, and the role of extracellular molecules in the cell-to-cell spreading of pathological processes in neurodegenerative diseases is now in the spotlight. Therefore, extracellular proteins and RNAs are considered potential biomarkers of neurodegenerative diseases, in particular ALS, in which RNA dysregulation has been shown to be involved in the pathogenesis. Here, we review extracellular proteins and RNAs that have been scrutinized as potential biomarkers of neurodegenerative diseases, and discuss the possibility of extracellular RNAs as diagnostic and therapeutic monitoring biomarkers of sporadic ALS.


Assuntos
Esclerose Lateral Amiotrófica/sangue , Ácidos Nucleicos Livres/sangue , Esclerose Lateral Amiotrófica/genética , Animais , Biomarcadores/sangue , Ácidos Nucleicos Livres/genética , Humanos , Edição de RNA
3.
J Clin Neurol ; 15(1): 62-67, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30618218

RESUMO

BACKGROUND AND PURPOSE: Disruption of nucleoporins has been reported in the motor neurons of patients with sporadic amyotrophic lateral sclerosis (sALS). However, the precise changes in the morphology of nucleoporins associated with the pathology of the 43-kDa TAR DNA-binding protein (TDP-43) in the disease process remain unknown. We investigated the expression of nucleoporins that constitute the nuclear pore complex (NPC) in spinal motor neurons that exhibit sALS in relation to TDP-43 pathology, which is a reliable neuropathological hallmark of sALS. METHODS: Paraffin-embedded sections of the lumbar spinal cord were obtained for immunofluorescence analysis from seven control subjects and six sALS patients. Anti-TDP-43 antibody, anti-nucleoporin p62 (NUP62) antibody, and anti-karyopherin beta 1 (KPNB1) antibody were applied as primary antibodies, and then visualized using appropriate secondary antibodies. The sections were then examined under a fluorescence microscope. RESULTS: NUP62 and KPNB1 immunoreactivity appeared as a smooth round rim bordering the nuclear margin in normal spinal motor neurons that exhibited nuclear TDP-43 immunoreactivity. sALS spinal motor neurons with apparent TDP-43 mislocalization demonstrated irregular, disrupted nuclear staining for NUP62 or KPNB1. Some atrophic sALS spinal motor neurons with TDP-43 mislocalization presented no NUP62 immunoreactivity. CONCLUSIONS: Our findings suggest a close relationship between NPC alterations and TDP-43 pathology in the degenerative process of the motor neurons of sALS patients.

4.
Neurosci Res ; 147: 48-57, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30448461

RESUMO

Currently, no reliable biomarkers of amyotrophic lateral sclerosis (ALS) exist. In sporadic ALS, RNA editing at the glutamine/arginine site of GluA2 mRNA is specifically reduced in the motor neurons due to the downregulation of adenosine deaminase acting on RNA 2 (ADAR2). Furthermore, TDP-43 pathology, the pathological hallmark of ALS, is observed in the ADAR2-lacking motor neurons in ALS patients and conditional ADAR2 knockout mice, suggesting a pivotal role of ADAR2 downregulation in the ALS pathogenesis. Extracellular RNAs were shown to represent potential disease biomarkers and the editing efficiencies at their ADAR2-dependent sites may reflect cellular ADAR2 activity, suggesting that these RNAs isolated from the body fluids may represent the biomarkers of ALS. We searched for ADAR2-dependent sites in the mouse motor neurons and human-derived cultured cells and found 10 sites in five host RNAs expressed in SH-SY5Y cells and their culture medium. Of these, the arginine/glycine site of SON mRNA was newly identified as an ADAR2-dependent site. Furthermore, we detected a circular RNA with an ADAR2-dependent site in the SH-SY5Y cells and their culture medium. Therefore, the changes in the editing efficiencies at the identified host RNA sites isolated from the body fluids may represent potential biomarkers of ALS.


Assuntos
Adenosina Desaminase/genética , Esclerose Lateral Amiotrófica/genética , Edição de RNA , RNA Circular/metabolismo , Proteínas de Ligação a RNA/genética , RNA/metabolismo , Adenosina Desaminase/deficiência , Adenosina Desaminase/metabolismo , Animais , Sítios de Ligação , Biomarcadores , Linhagem Celular , Linhagem Celular Tumoral , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/metabolismo , Proteínas de Ligação a RNA/metabolismo
5.
Neurosci Res ; 144: 4-13, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29944911

RESUMO

TAR DNA-binding protein (TDP-43) pathology in the motor neurons is the most reliable pathological hallmark of amyotrophic lateral sclerosis (ALS), and motor neurons bearing TDP-43 pathology invariably exhibit failure in RNA editing at the GluA2 glutamine/arginine (Q/R) site due to down-regulation of adenosine deaminase acting on RNA 2 (ADAR2). Conditional ADAR2 knockout (AR2) mice display ALS-like phenotype, including progressive motor dysfunction due to loss of motor neurons. Motor neurons devoid of ADAR2 express Q/R site-unedited GluA2, and AMPA receptors with unedited GluA2 in their subunit assembly are abnormally permeable to Ca2+, which results in progressive neuronal death. Moreover, analysis of AR2 mice has demonstrated that exaggerated Ca2+ influx through the abnormal AMPA receptors overactivates calpain, a Ca2+-dependent protease, that cleaves TDP-43 into aggregation-prone fragments, which serve as seeds for TDP-43 pathology. Activated calpain also disrupts nucleo-cytoplasmic transport and gene expression by cleaving molecules involved in nucleocytoplasmic transport, including nucleoporins. These lines of evidence prompted us to develop molecular targeting therapy for ALS by normalization of disrupted intracellular environment due to ADAR2 down-regulation. In this review, we have summarized the work from our group on the cell death cascade in sporadic ALS and discussed a potential therapeutic strategy for ALS.


Assuntos
Adenosina Desaminase/deficiência , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/terapia , Neurônios Motores/patologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Morte Celular/fisiologia , DNA Complementar/administração & dosagem , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Knockout , Neurônios Motores/metabolismo , Proteínas de Ligação a RNA
6.
Genes (Basel) ; 8(2)2017 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-28208729

RESUMO

Transactive response DNA-binding protein (TDP-43) pathology, and failure of A-to-I conversion (RNA editing) at the glutamine/arginine (Q/R) site of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit GluA2, are etiology-linked molecular abnormalities that concomitantly occur in the motor neurons of most patients with amyotrophic lateral sclerosis (ALS). Adenosine deaminase acting on RNA 2 (ADAR2) specifically catalyzes GluA2 Q/R site-RNA editing. Furthermore, conditional ADAR2 knockout mice (AR2) exhibit a progressive ALS phenotype with TDP-43 pathology in the motor neurons, which is the most reliable pathological marker of ALS. Therefore, the evidence indicates that ADAR2 downregulation is a causative factor in ALS, and AR2 mice exhibit causative molecular changes that occur in ALS. We discuss the contributors to ADAR2 downregulation and TDP-43 pathology in AR2 mouse motor neurons. We describe mechanisms of exaggerated Ca2+ influx amelioration via AMPA receptors, which is neuroprotective in ADAR2-deficient motor neurons with normalization of TDP-43 pathology in AR2 mice. Development of drugs to treat diseases requires appropriate animal models and a sensitive method of evaluating efficacy. Therefore, normalization of disrupted intracellular environments resulting from ADAR2 downregulation may be a therapeutic target for ALS. We discuss the development of targeted therapy for ALS using the AR2 mouse model.

7.
Sci Rep ; 7: 39994, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-28045133

RESUMO

Nuclear dysfunction in motor neurons has been hypothesized to be a principal cause of amyotrophic lateral sclerosis (ALS) pathogenesis. Here, we investigated the mechanism by which the nuclear pore complex (NPC) is disrupted in dying motor neurons in a mechanistic ALS mouse model (adenosine deaminase acting on RNA 2 (ADAR2) conditional knockout (AR2) mice) and in ALS patients. We showed that nucleoporins (Nups) that constituted the NPC were cleaved by activated calpain via a Ca2+-permeable AMPA receptor-mediated mechanism in dying motor neurons lacking ADAR2 expression in AR2 mice. In these neurons, nucleo-cytoplasmic transport was disrupted, and the level of the transcript elongation enzyme RNA polymerase II phosphorylated at Ser2 was significantly decreased. Analogous changes were observed in motor neurons lacking ADAR2 immunoreactivity in sporadic ALS patients. Therefore, calpain-dependent NPC disruption may participate in ALS pathogenesis, and inhibiting Ca2+-mediated cell death signals may be a therapeutic strategy for ALS.


Assuntos
Esclerose Lateral Amiotrófica/patologia , Calpaína/metabolismo , Neurônios Motores/metabolismo , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosforilação , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Receptores de AMPA/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , alfa Carioferinas/metabolismo
8.
Sci Rep ; 6: 28649, 2016 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-27350567

RESUMO

Both TDP-43 pathology and failure of RNA editing of AMPA receptor subunit GluA2, are etiology-linked molecular abnormalities that concomitantly occur in the motor neurons of the majority of patients with amyotrophic lateral sclerosis (ALS). AR2 mice, in which an RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is conditionally knocked out in the motor neurons, exhibit a progressive ALS phenotype with TDP-43 pathology in the motor neurons through a Ca(2+)-permeable AMPA receptor-mediated mechanism. Therefore, amelioration of the increased Ca(2+) influx by AMPA receptor antagonists may be a potential ALS therapy. Here, we showed that orally administered perampanel, a selective, non-competitive AMPA receptor antagonist significantly prevented the progression of the ALS phenotype and normalized the TDP-43 pathology-associated death of motor neurons in the AR2 mice. Given that perampanel is an approved anti-epileptic drug, perampanel is a potential candidate ALS drug worthy of a clinical trial.


Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Piridonas/farmacologia , Edição de RNA , Receptores de AMPA/antagonistas & inibidores , Administração Oral , Esclerose Lateral Amiotrófica/genética , Animais , Anticonvulsivantes/farmacologia , Cálcio/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Feminino , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/metabolismo , Nitrilas , Fenótipo , Receptores de AMPA/genética , Medula Espinal/metabolismo
9.
J Clin Neurosci ; 32: 128-9, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27343041

RESUMO

Mutations in the fused in sarcoma (FUS) gene can cause amyotrophic lateral sclerosis (ALS), and FUS gene mutations have been reported in sporadic ALS patients with basophilic cytoplasmic inclusions. Deficiency of adenosine deaminase acting on RNA 2 (ADAR2), an enzyme that specifically catalyzes GluA2 Q/R site-editing, has been reported in considerable proportions of spinal motor neurons of the majority of sporadic ALS patients. We describe the relationship between GluA2 Q/R site-editing efficiency and FUS-positive inclusions in a patient with FUS(P525L). A 24-year-old woman with ALS presented with basophilic cytoplasmic inclusions, significantly reduced GluA2 Q/R site-editing efficiency in the spinal motor neurons, and markedly decreased ADAR2 mRNA levels. Neuropathologic examination showed that not all spinal motor neurons expressed ADAR2 and revealed FUS-positive cytoplasmic inclusions in motor neurons irrespective of ADAR2 immunoreactivity. There were no phosphorylated transactive response (TAR) DNA-binding protein 43 kDa (TDP-43)-positive inclusions, indicating that there was no tight correlation between ADAR2 deficiency and TDP-43 deposition. ADAR2 deficiency can occur in ALS patients with a FUS(P525L) mutation and is unrelated to the presence of FUS-positive inclusions. FUS-associated ALS may share neurodegenerative characteristics with classical sporadic ALS.


Assuntos
Adenosina Desaminase/genética , Esclerose Lateral Amiotrófica/genética , Mutação , Proteína FUS de Ligação a RNA/genética , Proteínas de Ligação a RNA/genética , Esclerose Lateral Amiotrófica/patologia , Feminino , Humanos , Neurônios Motores/patologia , Adulto Jovem
10.
Neurology ; 87(3): 299-308, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27343066

RESUMO

OBJECTIVE: We aimed to analyze the clinical and histopathologic features of cancer-associated myositis (CAM) in relation to anti-transcriptional intermediary factor 1 γ antibody (anti-TIF1-γ-Ab), a marker of cancer association. METHODS: We retrospectively studied 349 patients with idiopathic inflammatory myopathies (IIMs), including 284 patients with pretreatment biopsy samples available. For the classification of IIMs, the European Neuromuscular Center criteria were applied. Patients with CAM with (anti-TIF1-γ-Ab[+] CAM) and without anti-TIF1-γ-Ab (anti-TIF1-γ-Ab[-] CAM) were compared with patients with IIM without cancers within and beyond 3 years of myositis diagnosis. RESULTS: Cancer was detected in 75 patients, of whom 36 (48%) were positive for anti-TIF1-γ-Ab. In anti-TIF1-γ-Ab(+) patients with CAM, cancers were detected within 1 year of myositis diagnosis in 35 (97%) and before 1 year of myositis diagnosis in 1. All the anti-TIF1-γ-Ab(+) patients with CAM satisfied the dermatomyositis (DM) criteria, including 2 possible DM sine dermatitis cases, and were characterized histologically by the presence of perifascicular atrophy, vacuolated fibers (VFs), and dense C5b-9 deposits on capillaries (dC5b-9). In contrast, 39 anti-TIF1-γ-Ab(-) patients with CAM were classified into various subgroups, and characterized by a higher frequency of necrotizing autoimmune myopathy (NAM). Notably, all 7 patients with CAM classified into the NAM subgroup were anti-TIF1-γ-Ab(-) and exhibited no dC5b-9 or VFs. CONCLUSIONS: CAM includes clinicohistopathologically heterogeneous disease entities. Among CAM entities, anti-TIF1-γ-Ab(+) CAM has characteristically shown a close temporal association with cancer detection and the histopathologic findings of dC5b-9 and VFs, and CAM with NAM is a subset of anti-TIF1-γ-Ab(-) CAM.


Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Autoanticorpos/imunologia , Miosite/complicações , Miosite/imunologia , Neoplasias/complicações , Neoplasias/imunologia , Proteínas Nucleares/imunologia , Autoanticorpos/sangue , Biomarcadores/sangue , Feminino , Humanos , Masculino , Miosite/sangue , Miosite/diagnóstico , Neoplasias/sangue , Neoplasias/diagnóstico , Estudos Retrospectivos
11.
Neurosci Res ; 107: 63-9, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26723245

RESUMO

TAR DNA-binding protein-43 (TDP-43) pathology, which includes the presence of abnormal TDP-43-containing inclusions with a loss of nuclear TDP-43 in affected neurons, is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobar degeneration (FTLD). TDP-43 in the pathological brains and spinal cords of ALS/FTLD patients is abnormally fragmented and phosphorylated. It is believed that the generation of aggregation-prone TDP-43 fragments initiates TDP-43 pathology, and we previously reported that calpain has an important role in the generation of such aggregation-prone TDP-43 fragments. However, the role of phosphorylation in TDP-43 pathology has not been largely elucidated, despite previous observations that several kinases and their kinases are involved in TDP-43 phosphorylation. Here, we investigated the role of TDP-43 phosphorylation in the calpain-dependent cleavage of TDP-43 and found that phosphorylated, full-length TDP-43 and calpain-dependent TDP-43 fragments were more resistant to cleavage by calpain than endogenous full-length TDP-43 was. These results suggest that both phosphorylated and calpain-cleaved TDP-43 fragments persist intracellularly for a length of time that is sufficient for self-aggregation, thereby serving as seeds for inclusions.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Calpaína/metabolismo , Proteínas de Ligação a DNA/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Células HeLa , Humanos , Fosforilação , Agregados Proteicos
13.
Neurosci Lett ; 598: 79-84, 2015 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-25980994

RESUMO

In the motor neurons of amyotrophic lateral sclerosis (ALS) patients, an RNA editing enzyme called adenosine deaminase acting on RNA 2 (ADAR2) is down-regulated and consequently GluA2 mRNAs unedited at the Q/R site is expressed in contrast to normal motor neurons that express only GluA2 edited at this site. Motor neurons of the mice lacking ADAR2 undergo Ca(2+)-permeable AMPA receptor-mediated slow death. We investigated the spinal cords of conditional ADAR2-knockout mice modeling ALS for the involvement of autophagy. In the motor neurons of the early- and late-symptomatic-stage mice, LC3-immunopositivity or immunoreactivity for both LC3- and p62 was observed, whereas the presymptomatic-stage mice showed no LC3- or p62-immunoreactivity. Western blot analyses showed increased expression of autophagy associated proteins in the anterior horn of the early symptomatic-stage mice. Electron-microscopically, autophagy was observed in the motor neurons most frequently in the early-symptomatic-stage mice which showed the severest motor neuron degeneration. Increased autophagy flux was not recognized in the wild-type mice or AR2res (ADAR2(flox/flox)/VAChT-Cre. Fast/GluR-B(R)(/)(R)) mice having motor neurons genetically engineered to express normally edited GluA2 in the absence of ADAR2, which show normal Ca(2+)-permeability of the AMPA receptors in motor neurons. Significantly increased autophagy flux in the degenerating motor neurons of ADAR2-knockout mice likely resulted from Ca(2+) overload.


Assuntos
Adenosina Desaminase/metabolismo , Esclerose Lateral Amiotrófica/patologia , Autofagia , Cálcio/metabolismo , Neurônios Motores/patologia , Proteínas de Ligação a RNA/metabolismo , Medula Espinal/patologia , Adenosina Desaminase/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Progressão da Doença , Regulação para Baixo , Camundongos Knockout , Neurônios Motores/metabolismo , Proteínas de Ligação a RNA/genética , Receptores de AMPA/metabolismo , Medula Espinal/metabolismo
14.
Brain Res ; 1584: 28-38, 2014 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-24355598

RESUMO

TAR DNA-binding protein (TDP-43) pathology and reduced expression of adenosine deaminase acting on RNA 2 (ADAR2), which is the RNA editing enzyme responsible for adenosine-to-inosine conversion at the GluA2 glutamine/arginine (Q/R) site, concomitantly occur in the same motor neurons of amyotrophic lateral sclerosis (ALS) patients; this finding suggests a link between these two ALS-specific molecular abnormalities. AMPA receptors containing Q/R site-unedited GluA2 in their subunit assembly are Ca(2+)-permeable, and motor neurons lacking ADAR2 undergo slow death in conditional ADAR2 knockout (AR2) mice, which is a mechanistic ALS model in which the ADAR2 gene is targeted in cholinergic neurons. Moreover, deficient ADAR2 induced mislocalization of TDP-43 similar to TDP-43 pathology seen in the sporadic ALS patients in the motor neurons of AR2 mice. The abnormal mislocalization of TDP-43 specifically resulted from activation of the Ca(2+)-dependent serine protease calpain that specifically cleaved TDP-43 at the C-terminal region, and generated aggregation-prone N-terminal fragments. Notably, the N-terminal fragments of TDP-43 lacking the C-terminus were demonstrated in the brains and spinal cords of ALS patients. Because normalization of either the Ca(2+)-permeability of AMPA receptors or the calpain activity in the motor neurons normalized the subcellular localization of TDP-43 in AR2 mice, it is likely that exaggerated calpain-dependent TDP-43 fragments played a role at least in the initiation of TDP-43 pathology. Elucidation of the molecular cascade of neuronal death induced by ADAR2 downregulation could provide a new specific therapy for sporadic ALS. In this review, we summarized the work from our group on the role of inefficient GluA2 Q/R site-RNA editing and TDP-43 pathology in sporadic ALS, and discussed possible effects of inefficient ADAR2-mediated RNA editing in general.


Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/metabolismo , Edição de RNA , Receptores de AMPA/genética , Adenosina Desaminase/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Calpaína/metabolismo , Morte Celular/genética , Humanos , Camundongos , Neurônios Motores/enzimologia , Proteínas de Ligação a RNA/genética , Medula Espinal/patologia
15.
Rinsho Shinkeigaku ; 54(12): 1151-4, 2014.
Artigo em Japonês | MEDLINE | ID: mdl-25672733

RESUMO

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease affecting healthy middle-aged individuals. Mislocalization of TAR DNA binding protein of 43 kDa (TDP-43) or TDP-43 pathology observed in the spinal motor neurons is the pathological hallmark of ALS. The mechanism generating TDP-43 pathology remained uncertain. Several reports suggested that cleavage of TDP-43 into aggregation-prone fragments might be the earliest event. Therefore, elucidation of the protease(s) that is responsible for TDP-43 cleavage in the motor neurons is awaited. ALS-specific molecular abnormalities other than TDP-43 pathology in the motor neurons of sporadic ALS patients include inefficient RNA editing at the GluA2 glutamine/arginine (Q/R) site, which is specifically catalyzed by adenosine deaminase acting on RNA 2 (ADAR2). We have developed the conditional ADAR2 knockout (AR2) mice, in which the ADAR2 gene is targeted in motor neurons. We found that Ca(2+)-dependent cysteine protease calpain cleaved TDP-43 into aggregation-prone fragments, which initiated TDP-43 mislocalization in the motor neurons expressing abnormally abundant Ca(2+)-permeable AMPA receptors. Here we summarized the molecular cascade leading to TDP-43 pathology observed in the motor neurons of AR2 mice and discussed possible roles of dysregulation of calpain-dependent cleavage of TDP-43 in TDP-43 pathology observed in neurological diseases in general.


Assuntos
Esclerose Lateral Amiotrófica/genética , Calpaína/fisiologia , Proteínas de Ligação a DNA/metabolismo , Animais , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Neurônios Motores/metabolismo , Edição de RNA , Receptores de AMPA/fisiologia , Medula Espinal/citologia
16.
EMBO Mol Med ; 5(11): 1710-9, 2013 11.
Artigo em Inglês | MEDLINE | ID: mdl-24115583

RESUMO

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease, and the lack of effective therapy results in inevitable death within a few years of onset. Failure of GluA2 RNA editing resulting from downregulation of the RNA-editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) occurs in the majority of ALS cases and causes the death of motor neurons via a Ca(2+) -permeable AMPA receptor-mediated mechanism. Here, we explored the possibility of gene therapy for ALS by upregulating ADAR2 in mouse motor neurons using an adeno-associated virus serotype 9 (AAV9) vector that provides gene delivery to a wide array of central neurons after peripheral administration. A single intravenous injection of AAV9-ADAR2 in conditional ADAR2 knockout mice (AR2), which comprise a mechanistic mouse model of sporadic ALS, caused expression of exogenous ADAR2 in the central neurons and effectively prevented progressive motor dysfunction. Notably, AAV9-ADAR2 rescued the motor neurons of AR2 mice from death by normalizing TDP-43 expression. This AAV9-mediated ADAR2 gene delivery may therefore enable the development of a gene therapy for ALS.


Assuntos
Adenosina Desaminase/genética , Esclerose Lateral Amiotrófica/enzimologia , Terapia Genética , Neurônios Motores/enzimologia , Proteínas de Ligação a RNA/genética , Adenosina Desaminase/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/terapia , Animais , Encéfalo/citologia , Encéfalo/enzimologia , Encéfalo/virologia , Dependovirus/genética , Dependovirus/fisiologia , Modelos Animais de Doenças , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Ligação a RNA/metabolismo
17.
Neurosci Res ; 77(4): 234-41, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24080146

RESUMO

TDP-43 is a discriminative protein that is found as intracellular aggregations in the neurons of the cerebral cortex and spinal cord of patients with amyotrophic lateral sclerosis (ALS); however, the mechanisms of neuron loss and its relation to the aggregations are still unclear. In this study, we generated a useful model to produce TDP-43 aggregations in the motor cortex using in utero electroporation on mouse embryos. The plasmids used were full-length TDP-43 and C-terminal fragments of TDP-43 (wild-type or M337V mutant) tagged with GFP. For the full-length TDP-43, both wild-type and mutant, electroporated TDP-43 localized mostly in the nucleus, and though aggregations were detected in embryonic brains, they were very rarely observed at P7 and P21. In contrast, TDP-43 aggregations were generated in the brains electroporated with the C-terminal TDP-43 fragments as previously reported in in vitro experiments. TDP-43 protein was distributed diffusely-not only in the nucleus, but also in the cytoplasm-and the inclusion bodies were ubiquitinated and included phosphorylated TDP-43, which reflects the human pathology of ALS. This model using in utero electroporation of pathogenic genes into the brain of the mouse will likely become a useful model for studying ALS and also for evaluation of agents for therapeutic purpose, and may be applicable to other neurodegenerative diseases, as well.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Córtex Motor/metabolismo , Esclerose Lateral Amiotrófica/embriologia , Animais , Eletroporação , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Córtex Motor/embriologia , Gravidez
18.
Am J Pathol ; 183(1): 211-25, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23680655

RESUMO

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a neurodegenerative disorder caused by mutations in the tau gene. Many mutations identified in FTDP-17 have been shown to affect tau exon 10 splicing in vitro, which presumably causes pathologic imbalances in exon 10(-) [3-repeat (3R)] and exon 10(+) [4-repeat (4R)] tau expression and leads to intracellular inclusions of hyperphosphorylated tau in patient brains. However, no reports have investigated this theory using model mice with a tau intronic mutation. Herein, we generated new transgenic mice harboring the tau intron 10 +16C → T mutation. We prepared a transgene construct containing intronic sequences required for exon 10 splicing in the longest tau isoform cDNA. Although mice bearing the construct without the intronic mutation showed normal developmental changes of the tau isoform from 3R tau to equal amounts of 3R and 4R tau, mice with the mutation showed much higher levels of 4R tau at the adult stage. 4R tau was selectively recovered in insoluble brain fractions in their old age. Furthermore, these mice displayed abnormal tau phosphorylation, synapse loss and dysfunction, memory impairment, glial activation, tangle formation, and neuronal loss in an age-dependent manner. These findings provide the first evidence in a mouse model that a tau intronic mutation-induced imbalance of 3R and 4R tau could be a cause of tauopathy.


Assuntos
Éxons , Demência Frontotemporal/genética , Íntrons , Mutação , Splicing de RNA , Tauopatias/genética , Proteínas tau/genética , Animais , Western Blotting , Demência Frontotemporal/patologia , Demência Frontotemporal/fisiopatologia , Marcadores Genéticos , Masculino , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tauopatias/patologia , Tauopatias/fisiopatologia
19.
Nat Commun ; 3: 1307, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23250437

RESUMO

Both mislocalization of TDP-43 and downregulation of RNA-editing enzyme ADAR2 co-localize in the motor neurons of amyotrophic lateral sclerosis patients, but how they are linked is not clear. Here we demonstrate that activation of calpain, a Ca2+-dependent cysteine protease, by upregulation of Ca2+-permeable AMPA receptors generates carboxy-terminal-cleaved TDP-43 fragments and causes mislocalization of TDP-43 in the motor neurons expressing glutamine/arginine site-unedited GluA2 of conditional ADAR2 knockout (AR2) mice that mimic the amyotrophic lateral sclerosis pathology. These abnormalities are inhibited in the AR2res mice that express Ca2+-impermeable AMPA receptors in the absence of ADAR2 and in the calpastatin transgenic mice, but are exaggerated in the calpastatin knockout mice. Additional demonstration of calpain-dependent TDP43 fragments in the spinal cord and brain of amyotrophic lateral sclerosis patients, and high vulnerability of amyotrophic lateral sclerosis-linked mutant TDP43 to cleavage by calpain support the crucial role of the calpain-dependent cleavage of TDP43 in the amyotrophic lateral sclerosis pathology.


Assuntos
Esclerose Lateral Amiotrófica/patologia , Calpaína/fisiologia , Proteínas de Ligação a DNA/fisiologia , Esclerose Lateral Amiotrófica/metabolismo , Animais , Western Blotting , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Camundongos Mutantes , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Neurônios Motores/fisiologia , Regulação para Cima/fisiologia
20.
PLoS One ; 7(8): e43469, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22916266

RESUMO

TDP-43 pathology in spinal motor neurons is a neuropathological hallmark of sporadic amyotrophic lateral sclerosis (ALS) and has recently been shown to be closely associated with the downregulation of an RNA editing enzyme called adenosine deaminase acting on RNA 2 (ADAR2) in the motor neurons of sporadic ALS patients. Because TDP-43 pathology is found more frequently in the brains of elderly patients, we investigated the age-related changes in the TDP-43 localization and ADAR2 activity in mouse motor neurons. We found that ADAR2 was developmentally upregulated, and its mRNA expression level was progressively decreased in the spinal cords of aged mice. Motor neurons normally exhibit nuclear ADAR2 and TDP-43 immunoreactivity, whereas fast fatigable motor neurons in aged mice demonstrated a loss of ADAR2 and abnormal TDP-43 localization. Importantly, these motor neurons expressed significant amounts of the Q/R site-unedited AMPA receptor subunit 2 (GluA2) mRNA. Because expression of unedited GluA2 has been demonstrated as a lethality-causing molecular abnormality observed in the motor neurons, these results suggest that age-related decreases in ADAR2 activity play a mechanistic role in aging and serve as one of risk factors for ALS.


Assuntos
Adenosina Desaminase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/metabolismo , Animais , Eletroquímica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Proteínas de Ligação a RNA , Medula Espinal/citologia
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