RESUMO
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multifactorial fatal motoneuron disease without a cure. Ten percent of ALS cases can be pointed to a clear genetic cause, while the remaining 90% is classified as sporadic. Our study was aimed to uncover new connections within the ALS network through a bioinformatic approach, by which we identified C13orf18, recently named Pacer, as a new component of the autophagic machinery and potentially involved in ALS pathogenesis. METHODS: Initially, we identified Pacer using a network-based bioinformatic analysis. Expression of Pacer was then investigated in vivo using spinal cord tissue from two ALS mouse models (SOD1G93A and TDP43A315T) and sporadic ALS patients. Mechanistic studies were performed in cell culture using the mouse motoneuron cell line NSC34. Loss of function of Pacer was achieved by knockdown using short-hairpin constructs. The effect of Pacer repression was investigated in the context of autophagy, SOD1 aggregation, and neuronal death. RESULTS: Using an unbiased network-based approach, we integrated all available ALS data to identify new functional interactions involved in ALS pathogenesis. We found that Pacer associates to an ALS-specific subnetwork composed of components of the autophagy pathway, one of the main cellular processes affected in the disease. Interestingly, we found that Pacer levels are significantly reduced in spinal cord tissue from sporadic ALS patients and in tissues from two ALS mouse models. In vitro, Pacer deficiency lead to impaired autophagy and accumulation of ALS-associated protein aggregates, which correlated with the induction of cell death. CONCLUSIONS: This study, therefore, identifies Pacer as a new regulator of proteostasis associated with ALS pathology.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Autofagia/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos Transgênicos , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
The control of apoptosis in mammals has been historically associated with the activity of the BCL-2 family of proteins at the mitochondria. In the past years, a novel group of cell death regulators have emerged, known as the Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) protein family. This group of proteins is composed of at least six highly conserved members expressed in mammals, with homologs in insects, fish, plants, viruses and yeast. Different studies indicate that all TMBIM family members have inhibitory activities in different setting of apoptosis. Here, we overview and integrate possible mechanisms underlying the impact of the TMBIM protein family in the regulation of cell death, which include activities at diverse subcellular compartments, including death receptor regulation, modulation of endoplasmic reticulum (ER) calcium homeostasis, ER stress signaling, autophagy, reactive oxygen species production, among other effects. The possible intersection between the BCL-2 and TMBIM family in the control of cell death is also discussed, in addition to their implication in the progression of cancer.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Proteínas de Membrana/genética , Família Multigênica , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Estresse do Retículo Endoplasmático/genética , Humanos , Proteínas de Membrana/metabolismo , Modelos Genéticos , Neoplasias/genética , Transdução de Sinais/genéticaRESUMO
Accurate methods to measure autophagic activity in vivo in neurons are not available, and most of the studies are based on correlative and static measurements of autophagy markers, leading to conflicting interpretations. Autophagy is an essential homeostatic process involved in the degradation of diverse cellular components including organelles and protein aggregates. Autophagy impairment is emerging as a relevant factor driving neurodegeneration in many diseases. Moreover, strategies to modulate autophagy have been shown to provide protection against neurodegeneration. Here we describe a novel and simple strategy to express an autophagy flux reporter in the nervous system of adult animals by the intraventricular delivery of adeno-associated viruses (AAV) into newborn mice. Using this approach we efficiently expressed a monomeric tandem mCherry-GFP-LC3 construct in neurons of the peripheral and central nervous system, allowing the measurement of autophagy activity in pharmacological and disease settings.
Assuntos
Autofagia/fisiologia , Sistema Nervoso/metabolismo , Animais , Linhagem Celular , Dependovirus/metabolismo , Vetores Genéticos/metabolismo , Humanos , Camundongos , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Sistema Nervoso/ultraestrutura , Nervo Isquiático/metabolismo , Nervo Isquiático/ultraestrutura , Medula Espinal/metabolismo , Medula Espinal/ultraestruturaRESUMO
Spinal cord injury (SCI) is a major cause of paralysis, and involves multiple cellular and tissular responses including demyelination, inflammation, cell death and axonal degeneration. Recent evidence suggests that perturbation on the homeostasis of the endoplasmic reticulum (ER) is observed in different SCI models; however, the functional contribution of this pathway to this pathology is not known. Here we demonstrate that SCI triggers a fast ER stress reaction (1-3 h) involving the upregulation of key components of the unfolded protein response (UPR), a process that propagates through the spinal cord. Ablation of X-box-binding protein 1 (XBP1) or activating transcription factor 4 (ATF4) expression, two major UPR transcription factors, leads to a reduced locomotor recovery after experimental SCI. The effects of UPR inactivation were associated with a significant increase in the number of damaged axons and reduced amount of oligodendrocytes surrounding the injury zone. In addition, altered microglial activation and pro-inflammatory cytokine expression were observed in ATF4 deficient mice after SCI. Local expression of active XBP1 into the spinal cord using adeno-associated viruses enhanced locomotor recovery after SCI, and was associated with an increased number of oligodendrocytes. Altogether, our results demonstrate a functional role of the UPR in SCI, offering novel therapeutic targets to treat this invalidating condition.
Assuntos
Fator 4 Ativador da Transcrição/genética , Proteínas de Ligação a DNA/genética , Traumatismos da Medula Espinal/genética , Medula Espinal/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional/genética , Resposta a Proteínas não Dobradas/genética , Fator 4 Ativador da Transcrição/deficiência , Animais , Axônios/patologia , Contagem de Células , Proteínas de Ligação a DNA/deficiência , Dependovirus , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Vetores Genéticos , Injeções Espinhais , Locomoção , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligodendroglia/patologia , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais/genética , Medula Espinal/patologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Fatores de Transcrição/deficiência , Proteína 1 de Ligação a X-BoxRESUMO
Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Resposta a Proteínas não Dobradas/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose , Drosophila melanogaster , Estresse do Retículo Endoplasmático , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Peixe-Zebra , eIF-2 Quinase/metabolismoRESUMO
A variety of neurological diseases including Huntington's disease (HD), Alzheimer's disease and Parkinson's disease share common neuropathology, primarily featuring the presence of abnormal protein inclusions containing specific misfolded proteins. Mutations leading to expansion of a poly-glutamine track in Huntingtin cause HD, and trigger its misfolding and aggregation. Recent evidence indicates that alterations in the secretory pathway, in particular the endoplasmic reticulum (ER), are emerging features of HD. Although it is not clear how cytoplasmic/nuclear located mutant Huntingtin alters the function of the ER, several reports indicate that mutant Huntingtin affects many essential processes related to the secretory pathway, including inhibition of ER-associated degradation, altered ER/Golgi vesicular trafficking and axonal transport, disrupted autophagy and abnormal ER calcium homeostasis. All these alterations are predicted to have a common pathological outcome associated to disturbance of protein folding and maturation pathways at the ER, generating chronic ER stress and neuronal dysfunction. Here, we review recent evidence involving ER stress in HD pathogenesis and discuss possible therapeutic strategies to target organelle function in the context of disease.
Assuntos
Retículo Endoplasmático/fisiologia , Doença de Huntington/fisiopatologia , Animais , Apoptose , Cálcio/metabolismo , Humanos , Proteína Huntingtina , Doença de Huntington/terapia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Multimerização Proteica , Transporte Proteico , Deficiências na Proteostase , Estresse Fisiológico , Resposta a Proteínas não DobradasRESUMO
Microcin E492 is a low-molecular-weight, channel-forming bacteriocin produced and excreted by Klebsiella pneumoniae RYC492. A 13 kb chromosomal DNA fragment from K. pneumoniae RYC492 was sequenced, and it was demonstrated by random Tn5 mutagenesis that most of this segment, which has at least 10 cistrons, is needed for the production of active microcin and its immunity protein. Genes mceG and mceH correspond to an ABC exporter and its accessory protein, respectively, and they are closely related to the colicin V ABC export system. The microcin E492 system also requires the product of gene mceF as an additional factor for export. Despite the fact that this bacteriocin lacks post-translational modifications, genes mceC, mceI and mceJ are needed for the production of active microcin. Genes mceC and mceI are homologous to a glycosyl transferase and acyltransferase, respectively, whereas mceJ has no known homologue. Mutants in these three genes secrete an inactive form of microcin, able to form ion channels in a phospholipidic bilayer, indicating that the mutation of these microcin genes does not alter the process of membrane insertion. On the other hand, microcin isolated from mutants in genes mceC and mceJ has a lethal effect when incubated with spheroplasts of sensitive cells, indicating that the microcin defects in these mutants are likely to alter receptor recognition at the outer membrane. A model for synthesis and export is proposed as well as a novel maturation pathway that would involve conformational changes to explain the production of active microcin E492.
Assuntos
Bacteriocinas/genética , Genes Bacterianos , Klebsiella pneumoniae/genética , Sequência de Aminoácidos , Antibacterianos/metabolismo , Bacteriocinas/química , Bacteriocinas/metabolismo , Transporte Biológico , Eletrofisiologia , Canais Iônicos/metabolismo , Klebsiella pneumoniae/metabolismo , Dados de Sequência Molecular , Família Multigênica , Mutagênese , Fases de Leitura Aberta/genética , Peptídeos , Alinhamento de SequênciaRESUMO
BACKGROUND: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. RESULTS: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-f), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn(2+)-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide, but not fibronectin, also mediated Mn(2+)-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. CONCLUSIONS: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading.
Assuntos
Antígenos CD/metabolismo , Astrócitos/metabolismo , Adesões Focais/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Antígenos Thy-1/metabolismo , Antígenos Thy-1/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Encéfalo/metabolismo , Adesão Celular , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Integrina beta3 , Camundongos , Dados de Sequência Molecular , Neurônios/metabolismo , Oligopeptídeos/farmacologia , Fosfotirosina/metabolismo , Glicoproteínas da Membrana de Plaquetas/imunologia , Ratos , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Antígenos Thy-1/química , Células Tumorais CultivadasRESUMO
Necrosis, as opposed to apoptosis, is recognized as a nonspecific cell death that induces tissue inflammation and is preceded by cell edema. In non-neuronal cells, the latter has been explained by defective outward pumping of Na(+) caused by metabolic depletion or by increased Na(+) influx via membrane transporters. Here we describe a novel mechanism of swelling and necrosis; namely the influx of Na(+) through oxidative stress-activated nonselective cation channels. Exposure of liver epithelial Clone 9 cells to the free-radical donors calphostin C or menadione induced the rapid activation of an approximately 16-pS nonselective cation channel (NSCC). Blockage of this conductance with flufenamic acid protected the cells against swelling, calcium overload, and necrosis. Protection was also achieved by Gd(3+), an inhibitor of stretch-activated cation channels, or by isosmotic replacement of extracellular Na(+) with N-methyl-D-glucamine. It is proposed that NSCCs, which are ubiquitous although largely inactive in healthy cells, become activated under severe oxidative stress. The ensuing influx of Na(+) initiates a positive feedback of metabolic and electrolytic disturbances leading cells to their necrotic demise.