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1.
Genes Dev ; 32(13-14): 929-943, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29950492

RESUMO

While a mutation in C9ORF72 is the most common genetic contributor to amyotrophic lateral sclerosis (ALS), much remains to be learned concerning the function of the protein normally encoded at this locus. To elaborate further on functions for C9ORF72, we used quantitative mass spectrometry-based proteomics to identify interacting proteins in motor neurons and found that its long isoform complexes with and stabilizes SMCR8, which further enables interaction with WDR41. To study the organismal and cellular functions for this tripartite complex, we generated Smcr8 loss-of-function mutant mice and found that they developed phenotypes also observed in C9orf72 loss-of-function animals, including autoimmunity. Along with a loss of tolerance for many nervous system autoantigens, we found increased lysosomal exocytosis in Smcr8 mutant macrophages. In addition to elevated surface Lamp1 (lysosome-associated membrane protein 1) expression, we also observed enhanced secretion of lysosomal components-phenotypes that we subsequently observed in C9orf72 loss-of-function macrophages. Overall, our findings demonstrate that C9ORF72 and SMCR8 have interdependent functions in suppressing autoimmunity as well as negatively regulating lysosomal exocytosis-processes of potential importance to ALS.


Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Autoimunidade/genética , Proteínas de Transporte/metabolismo , Exocitose/genética , Lisossomos/metabolismo , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Proteínas de Transporte/genética , Regulação da Expressão Gênica/genética , Humanos , Linfonodos/patologia , Proteína 1 de Membrana Associada ao Lisossomo/genética , Macrófagos/patologia , Camundongos , Camundongos Knockout , Mutação , Isoformas de Proteínas , Estabilidade Proteica , Esplenomegalia/genética
2.
PLoS Pathog ; 18(12): e1010956, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36454879

RESUMO

In multiple system atrophy (MSA), the α-synuclein protein misfolds into a self-templating prion conformation that spreads throughout the brain, leading to progressive neurodegeneration. While the E46K mutation in α-synuclein causes familial Parkinson's disease (PD), we previously discovered that this mutation blocks in vitro propagation of MSA prions. Recent studies by others indicate that α-synuclein adopts a misfolded conformation in MSA in which a Greek key motif is stabilized by an intramolecular salt bridge between residues E46 and K80. Hypothesizing that the E46K mutation impedes salt bridge formation and, therefore, exerts a selective pressure that can modulate α-synuclein strain propagation, we asked whether three distinct α-synuclein prion strains could propagate in TgM47+/- mice, which express human α-synuclein with the E46K mutation. Following intracranial injection of these strains, TgM47+/- mice were resistant to MSA prion transmission, whereas recombinant E46K preformed fibrils (PFFs) transmitted neurological disease to mice and induced the formation of phosphorylated α-synuclein neuropathology. In contrast, heterotypic seeding following wild-type (WT) PFF-inoculation resulted in preclinical α-synuclein prion propagation. Moreover, when we inoculated TgM20+/- mice, which express WT human α-synuclein, with E46K PFFs, we observed delayed transmission kinetics with an incomplete attack rate. These findings suggest that the E46K mutation constrains the number of α-synuclein prion conformations that can propagate in TgM47+/- mice, expanding our understanding of the selective pressures that impact α-synuclein prion replication.


Assuntos
Atrofia de Múltiplos Sistemas , Príons , Humanos , Camundongos , Animais , alfa-Sinucleína/genética , Príons/genética , Camundongos Transgênicos , Mutação
3.
PLoS Pathog ; 16(2): e1008222, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32017806

RESUMO

Multiple system atrophy (MSA), a progressive neurodegenerative disease characterized by autonomic dysfunction and motor impairment, is caused by the self-templated misfolding of the protein α-synuclein. With no treatment currently available, we sought to characterize the spread of α-synuclein in a transgenic mouse model of MSA prion propagation to support drug discovery programs for synucleinopathies. Brain homogenates from MSA patient samples or mouse-passaged MSA were inoculated either by standard freehand injection or stereotactically into TgM83+/- mice, which express human α-synuclein with the A53T mutation. Following disease onset, brains from the mice were tested for biologically active α-synuclein prions using a cell-based assay and examined for α-synuclein neuropathology. Inoculation studies using homogenates prepared from brain regions lacking detectable α-synuclein neuropathology transmitted neurological disease to mice. Terminal animals contained similar concentrations of α-synuclein prions; however, a time-course study where mice were terminated every five days through disease progression revealed that the kinetics of α-synuclein prion replication in the mice were variable. Stereotactic inoculation into the thalamus reduced variability in disease onset in the mice, although incubation times were consistent with standard inoculations. Using human samples with and without neuropathological lesions, we observed that α-synuclein prion formation precedes neuropathology in the brain, suggesting that disease in patients is not limited to brain regions containing neuropathological lesions.


Assuntos
Encéfalo/metabolismo , Atrofia de Múltiplos Sistemas/metabolismo , Mutação Puntual , alfa-Sinucleína/metabolismo , Animais , Encéfalo/patologia , Feminino , Humanos , Cinética , Masculino , Camundongos , Camundongos Transgênicos , Atrofia de Múltiplos Sistemas/genética , Atrofia de Múltiplos Sistemas/patologia , Príons/genética , Príons/metabolismo , alfa-Sinucleína/genética
4.
Acta Neuropathol ; 144(4): 677-690, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36018376

RESUMO

In multiple system atrophy (MSA), the protein α-synuclein misfolds into a prion conformation that self-templates and causes progressive neurodegeneration. While many point mutations in the α-synuclein gene, SNCA, have been identified as the cause of heritable Parkinson's disease (PD), none have been identified as causing MSA. To examine whether MSA prions can transmit disease to mice expressing wild-type (WT) human α-synuclein, we inoculated transgenic (Tg) mice denoted TgM20+/- with brain homogenates prepared from six different deceased MSA patients. All six samples transmitted CNS disease to the mice, with an average incubation period of ~ 280 days. Interestingly, TgM20+/- female mice developed disease > 60 days earlier than their male counterparts. Brains from terminal mice contained phosphorylated α-synuclein throughout the hindbrain, consistent with the distribution of α-synuclein inclusions in MSA patients. In addition, using our α-syn-YFP cell lines, we detected α-synuclein prions in brain homogenates prepared from terminal mice that retained MSA strain properties. To our knowledge, the studies described here are the first to show that MSA prions transmit neurological disease to mice expressing WT SNCA and that the rate of transmission is sex dependent. By comparison, TgM20+/- mice inoculated with WT preformed fibrils (PFFs) developed severe neurological disease in ~ 210 days and exhibited robust α-synuclein neuropathology in both limbic regions and the hindbrain. Brain homogenates from these animals exhibited biological activities that are distinct from those found in MSA-inoculated mice when tested in the α-syn-YFP cell lines. Differences between brains from MSA-inoculated and WT PFF-inoculated mice potentially argue that α-synuclein prions from MSA patients are distinct from the PFF inocula and that PFFs do not replicate MSA strain biology.


Assuntos
Atrofia de Múltiplos Sistemas , Príons , Animais , Feminino , Humanos , Corpos de Inclusão/patologia , Masculino , Camundongos , Camundongos Transgênicos , Atrofia de Múltiplos Sistemas/patologia , Príons/genética , Príons/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
5.
Proc Natl Acad Sci U S A ; 115(2): 409-414, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29279394

RESUMO

In the neurodegenerative disease multiple system atrophy (MSA), α-synuclein misfolds into a self-templating conformation to become a prion. To compare the biological activity of α-synuclein prions in MSA and Parkinson's disease (PD), we developed nine α-synuclein-YFP cell lines expressing point mutations responsible for inherited PD. MSA prions robustly infected wild-type, A30P, and A53T α-synuclein-YFP cells, but they were unable to replicate in cells expressing the E46K mutation. Coexpression of the A53T and E46K mutations was unable to rescue MSA prion infection in vitro, establishing that MSA α-synuclein prions are conformationally distinct from the misfolded α-synuclein in PD patients. This observation may have profound implications for developing treatments for neurodegenerative diseases.


Assuntos
Atrofia de Múltiplos Sistemas/genética , Doença de Parkinson/genética , Mutação Puntual , Príons/genética , Animais , Linhagem Celular , Células HEK293 , Humanos , Camundongos Transgênicos , Príons/metabolismo , Príons/patogenicidade , Dobramento de Proteína , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
6.
Acta Neuropathol ; 137(3): 437-454, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30690664

RESUMO

Previously, we reported that intracranial inoculation of brain homogenate from multiple system atrophy (MSA) patient samples produces neurological disease in the transgenic (Tg) mouse model TgM83+/-, which uses the prion protein promoter to express human α-synuclein harboring the A53T mutation found in familial Parkinson's disease (PD). In our studies, we inoculated MSA and control patient samples into Tg mice constructed using a P1 artificial chromosome to express wild-type (WT), A30P, and A53T human α-synuclein on a mouse α-synuclein knockout background [Tg(SNCA+/+)Nbm, Tg(SNCA*A30P+/+)Nbm, and Tg(SNCA*A53T+/+)Nbm]. In contrast to studies using TgM83+/- mice, motor deficits were not observed by 330-400 days in any of the Tg(SNCA)Nbm mice after inoculation with MSA brain homogenates. However, using a cell-based bioassay to measure α-synuclein prions, we found brain homogenates from Tg(SNCA*A53T+/+)Nbm mice inoculated with MSA patient samples contained α-synuclein prions, whereas control mice did not. Moreover, these α-synuclein aggregates retained the biological and biochemical characteristics of the α-synuclein prions in MSA patient samples. Intriguingly, Tg(SNCA*A53T+/+)Nbm mice developed α-synuclein pathology in neurons and astrocytes throughout the limbic system. This finding is in contrast to MSA-inoculated TgM83+/- mice, which develop exclusively neuronal α-synuclein aggregates in the hindbrain that cause motor deficits with advanced disease. In a crossover experiment, we inoculated TgM83+/- mice with brain homogenate from two MSA patient samples or one control sample first inoculated, or passaged, in Tg(SNCA*A53T+/+)Nbm animals. Additionally, we performed the reverse experiment by inoculating Tg(SNCA*A53T+/+)Nbm mice with brain homogenate from the same two MSA samples and one control sample first passaged in TgM83+/- animals. The TgM83+/- mice inoculated with mouse-passaged MSA developed motor dysfunction and α-synuclein prions, whereas the mouse-passaged control sample had no effect. Similarly, the mouse-passaged MSA samples induced α-synuclein prion formation in Tg(SNCA*A53T+/+)Nbm mice, but the mouse-passaged control sample did not. The confirmed transmission of α-synuclein prions to a second synucleinopathy model and the ability to propagate prions between two distinct mouse lines while retaining strain-specific properties provides compelling evidence that MSA is a prion disease.


Assuntos
Atrofia de Múltiplos Sistemas/patologia , Doenças Priônicas/patologia , Doenças Priônicas/transmissão , Príons/metabolismo , alfa-Sinucleína/metabolismo , Animais , Humanos , Camundongos , Camundongos Transgênicos
8.
Proc Natl Acad Sci U S A ; 113(48): E7701-E7709, 2016 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-27849576

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.


Assuntos
Esclerose Lateral Amiotrófica/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/fisiologia , Proteína FUS de Ligação a RNA/fisiologia , Linhagem Celular , Humanos , Neurônios Motores/metabolismo , Transporte Proteico , Transcrição Gênica
9.
N Engl J Med ; 373(13): 1251-61, 2015 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-26398074

RESUMO

A 50-year-old man with a history of cardiomyopathy and progressive muscle weakness was admitted with cardiogenic shock. Electroencephalography showed total suppression of cerebral activity; ventilator support was withdrawn, and he died. An autopsy was performed.


Assuntos
Músculo Esquelético/patologia , Distrofia Miotônica/diagnóstico , Choque Cardiogênico/etiologia , Diagnóstico Diferencial , Ecocardiografia , Eletroencefalografia , Evolução Fatal , Parada Cardíaca/etiologia , Humanos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Debilidade Muscular/etiologia , Distrofia Miotônica/complicações , Radiografia
10.
Acta Neuropathol ; 135(1): 49-63, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28849371

RESUMO

In multiple system atrophy (MSA), progressive neurodegeneration results from the protein α-synuclein misfolding into a self-templating prion conformation that spreads throughout the brain. MSA prions are transmissible to transgenic (Tg) mice expressing mutated human α-synuclein (TgM83+/-), inducing neurological disease following intracranial inoculation with brain homogenate from deceased patient samples. Noting the similarities between α-synuclein prions and PrP scrapie (PrPSc) prions responsible for Creutzfeldt-Jakob disease (CJD), we investigated MSA transmission under conditions known to result in PrPSc transmission. When peripherally exposed to MSA via the peritoneal cavity, hind leg muscle, and tongue, TgM83+/- mice developed neurological signs accompanied by α-synuclein prions in the brain. Iatrogenic CJD, resulting from PrPSc prion adherence to surgical steel instruments, has been investigated by incubating steel sutures in contaminated brain homogenate before implantation into mouse brain. Mice studied using this model for MSA developed disease, whereas wire incubated in control homogenate had no effect on the animals. Notably, formalin fixation did not inactivate α-synuclein prions. Formalin-fixed MSA patient samples also transmitted disease to TgM83+/- mice, even after incubating in fixative for 244 months. Finally, at least 10% sarkosyl was found to be the concentration necessary to partially inactivate MSA prions. These results demonstrate the robustness of α-synuclein prions to denaturation. Moreover, they establish the parallel characteristics between PrPSc and α-synuclein prions, arguing that clinicians should exercise caution when working with materials that might contain α-synuclein prions to prevent disease.


Assuntos
Atrofia de Múltiplos Sistemas/metabolismo , Príons/metabolismo , Animais , Transporte Biológico , Encéfalo/metabolismo , Encéfalo/patologia , Detergentes/farmacologia , Modelos Animais de Doenças , Fixadores , Formaldeído , Células HEK293 , Humanos , Camundongos Transgênicos , Atrofia de Múltiplos Sistemas/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação , Príons/administração & dosagem , Agregados Proteicos , Estabilidade Proteica/efeitos dos fármacos , Sarcosina/análogos & derivados , Sarcosina/farmacologia , Aço Inoxidável , alfa-Sinucleína/administração & dosagem , alfa-Sinucleína/efeitos adversos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
11.
Proc Natl Acad Sci U S A ; 112(35): E4949-58, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26286986

RESUMO

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)-YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson's disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.


Assuntos
Doenças Neurodegenerativas/metabolismo , Príons/metabolismo , alfa-Sinucleína/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Doenças Neurodegenerativas/patologia
12.
Proc Natl Acad Sci U S A ; 112(38): E5308-17, 2015 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-26324905

RESUMO

Prions are proteins that adopt alternative conformations that become self-propagating; the PrP(Sc) prion causes the rare human disorder Creutzfeldt-Jakob disease (CJD). We report here that multiple system atrophy (MSA) is caused by a different human prion composed of the α-synuclein protein. MSA is a slowly evolving disorder characterized by progressive loss of autonomic nervous system function and often signs of parkinsonism; the neuropathological hallmark of MSA is glial cytoplasmic inclusions consisting of filaments of α-synuclein. To determine whether human α-synuclein forms prions, we examined 14 human brain homogenates for transmission to cultured human embryonic kidney (HEK) cells expressing full-length, mutant human α-synuclein fused to yellow fluorescent protein (α-syn140*A53T-YFP) and TgM83(+/-) mice expressing α-synuclein (A53T). The TgM83(+/-) mice that were hemizygous for the mutant transgene did not develop spontaneous illness; in contrast, the TgM83(+/+) mice that were homozygous developed neurological dysfunction. Brain extracts from 14 MSA cases all transmitted neurodegeneration to TgM83(+/-) mice after incubation periods of ∼120 d, which was accompanied by deposition of α-synuclein within neuronal cell bodies and axons. All of the MSA extracts also induced aggregation of α-syn*A53T-YFP in cultured cells, whereas none of six Parkinson's disease (PD) extracts or a control sample did so. Our findings argue that MSA is caused by a unique strain of α-synuclein prions, which is different from the putative prions causing PD and from those causing spontaneous neurodegeneration in TgM83(+/+) mice. Remarkably, α-synuclein is the first new human prion to be identified, to our knowledge, since the discovery a half century ago that CJD was transmissible.


Assuntos
Atrofia de Múltiplos Sistemas/metabolismo , Transtornos Parkinsonianos/metabolismo , Príons/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Animais , Encéfalo/patologia , Éxons , Feminino , Células HEK293 , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Pessoa de Meia-Idade , Atrofia de Múltiplos Sistemas/genética , Doenças Neurodegenerativas/metabolismo , Fosforilação , Polimorfismo de Nucleotídeo Único , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , alfa-Sinucleína/genética
13.
Nat Aging ; 4(7): 984-997, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38907103

RESUMO

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by a progressive loss of motor function linked to degenerating extratelencephalic neurons/Betz cells (ETNs). The reasons why these neurons are selectively affected remain unclear. Here, to understand the unique molecular properties that may sensitize ETNs to ALS, we performed RNA sequencing of 79,169 single nuclei from cortices of patients and controls. In both patients and unaffected individuals, we found significantly higher expression of ALS risk genes in THY1+ ETNs, regardless of diagnosis. In patients, this was accompanied by the induction of genes involved in protein homeostasis and stress responses that were significantly induced in a wide collection of ETNs. Examination of oligodendroglial and microglial nuclei revealed patient-specific downregulation of myelinating genes in oligodendrocytes and upregulation of an endolysosomal reactive state in microglia. Our findings suggest that selective vulnerability of extratelencephalic neurons is partly connected to their intrinsic molecular properties sensitizing them to genetics and mechanisms of degeneration.


Assuntos
Esclerose Lateral Amiotrófica , Neurônios , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/metabolismo , Humanos , Neurônios/metabolismo , Neurônios/patologia , Fatores de Risco , Microglia/metabolismo , Microglia/patologia , Núcleo Celular/metabolismo , Núcleo Celular/genética , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Masculino , Análise de Célula Única , Análise de Sequência de RNA , Feminino , Pessoa de Meia-Idade , Degeneração Neural/genética , Degeneração Neural/patologia , Degeneração Neural/metabolismo
15.
bioRxiv ; 2023 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-37645969

RESUMO

Microtubule-associated protein tau (MAPT/tau) accumulates in a family of neurodegenerative diseases, including Alzheimer's disease (AD). In disease, tau is aberrantly modified by post-translational modifications (PTMs), including hyper-phosphorylation. However, it is often unclear which of these PTMs contribute to tau's accumulation or what mechanisms might be involved. To explore these questions, we focused on a cleaved proteoform of tau (tauC3), which selectively accumulates in AD and was recently shown to be degraded by its direct binding to the E3 ubiquitin ligase, CHIP. Here, we find that phosphorylation of tauC3 at a single residue, pS416, is sufficient to block its interaction with CHIP. A co-crystal structure of CHIP bound to the C-terminus of tauC3 revealed the mechanism of this clash and allowed design of a mutation (CHIPD134A) that partially restores binding and turnover of pS416 tauC3. We find that pS416 is produced by the known AD-associated kinase, MARK2/Par-1b, providing a potential link to disease. In further support of this idea, an antibody against pS416 co-localizes with tauC3 in degenerative neurons within the hippocampus of AD patients. Together, these studies suggest a discrete molecular mechanism for how phosphorylation at a specific site contributes to accumulation of an important tau proteoform.

16.
Front Aging Neurosci ; 14: 1017299, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36408113

RESUMO

Neurodegenerative disorders have been extremely challenging to treat with traditional drug-based approaches and curative therapies are lacking. Given continued progress in stem cell technologies, cell replacement strategies have emerged as concrete and potentially viable therapeutic options. In this review, we cover advances in methods used to differentiate human pluripotent stem cells into several highly specialized types of neurons, including cholinergic, dopaminergic, and motor neurons, and the potential clinical applications of stem cell-derived neurons for common neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, ataxia, and amyotrophic lateral sclerosis. Additionally, we summarize cellular differentiation techniques for generating glial cell populations, including oligodendrocytes and microglia, and their conceivable translational roles in supporting neural function. Clinical trials of specific cell replacement therapies in the nervous system are already underway, and several attractive avenues in regenerative medicine warrant further investigation.

17.
Proc Natl Acad Sci U S A ; 105(48): 18730-4, 2008 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-19028869

RESUMO

The Saccharomyces cerevisiae Mec1-Ddc2 checkpoint kinase complex (the ortholog to human ATR-ATRIP) is an essential regulator of genomic integrity. The S. cerevisiae BRCT repeat protein Dpb11 functions in the initiation of both DNA replication and cell cycle checkpoints. Here, we report a genetic and physical interaction between Dpb11 and Mec1-Ddc2. A C-terminal domain of Dpb11 is sufficient to associate with Mec1-Ddc2 and strongly stimulates the kinase activity of Mec1 in a Ddc2-dependent manner. Furthermore, Mec1 phosphorylates Dpb11 and thereby amplifies the stimulating effect of Dpb11 on Mec1-Ddc2 kinase activity. Thus, Dpb11 is a functional ortholog of human TopBP1, and the Mec1/ATR activation mechanism is conserved from yeast to humans.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular/genética , Dano ao DNA , Reparo do DNA , Ativação Enzimática , Humanos , Hidroxiureia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Inibidores da Síntese de Ácido Nucleico/metabolismo , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
18.
Mol Cell Biol ; 27(9): 3367-77, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17339343

RESUMO

The ATR (ATM and Rad3-related) kinase is essential to maintain genomic integrity. ATR is recruited to DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with the single-stranded DNA binding protein RPA (replication protein A). In this study, a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs was identified by biochemical mapping of the RPA binding site in combination with nuclear magnetic resonance, mutagenesis, and computational modeling. Mutations in the CRD of the Saccharomyces cerevisiae ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA-damaging agents, and partially compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal DNA damage responses. However, the stimulation of ATR kinase activity by binding of topoisomerase binding protein 1 (TopBP1) to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results support the idea of a multistep model for ATR activation that requires separable localization and activation functions of ATRIP.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Exodesoxirribonucleases/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Dano ao DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Humanos , Imageamento por Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia Estrutural de Proteína , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Neuron ; 108(4): 775-783.e4, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33022228

RESUMO

A hexanucleotide repeat expansion at C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Initial studies of bacterial artificial chromosome (BAC) transgenic mice harboring this expansion described an absence of motor and survival phenotypes. However, a recent study by Liu and colleagues described transgenic mice harboring a large repeat expansion (C9-500) and reported decreased survival and progressive motor phenotypes. To determine the utility of the C9-500 animals for understanding degenerative mechanisms, we validated and established two independent colonies of transgene carriers. However, extended studies of these animals for up to 1 year revealed no reproducible abnormalities in survival, motor function, or neurodegeneration. Here, we propose several potential explanations for the disparate nature of our findings from those of Liu and colleagues. Resolving the discrepancies we identify will be essential to settle the translational utility of C9-500 mice. This Matters Arising paper is in response to Liu et al. (2016), published in Neuron. See also the response by Nguyen et al. (2020), published in this issue.


Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Proteína C9orf72/fisiologia , Destreza Motora/fisiologia , Degeneração Neural/fisiopatologia , Sobrevida/fisiologia , Esclerose Lateral Amiotrófica/genética , Animais , Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Modelos Animais de Doenças , Heterozigoto , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo
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