RESUMO
The pathogenesis of Alzheimer's disease (AD) is associated with proteolytic processing of the amyloid precursor protein (APP) to an amyloidogenic peptide termed Aß. Although mutations in APP and the secretase enzymes that mediate its processing are known to result in familial forms of AD, the mechanisms underlying the more common sporadic forms of the disease are still unclear. Evidence suggests that the susceptibility of APP to amyloidogenic processing is related to its intracellular localization, and that secretase-independent degradation may prevent the formation of cytotoxic peptide fragments. Recently, single nucleotide polymorphisms in the UBQLN1 gene have been linked to late-onset AD, and its protein product, ubiquilin-1, may regulate the maturation of full-length APP. Here we show that ubiquilin-1 inhibits the maturation of APP by sequestering it in the early secretory pathway, primarily within the Golgi apparatus. This sequestration significantly delayed the proteolytic processing of APP by secretases and the proteasome. These effects were mediated by ubiquilin-1-stimulated K63-linked polyubiquitination of lysine 688 in the APP intracellular domain. Our results reveal the mechanistic basis by which ubiquilin-1 regulates APP maturation, with important consequences for the pathogenesis of late-onset AD.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Lisina/metabolismo , Chaperonas Moleculares/metabolismo , Poliubiquitina/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Ubiquitinação , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Lisossomos/metabolismo , Células PC12 , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Via SecretóriaRESUMO
Long-term outcomes of persistent pulmonary hypertension of newborn (PPHN) depend on disease severity, duration of ventilation, and associated anomalies. Congenital diaphragmatic hernia survivors may have respiratory morbidities and developmental delay. The presence of PPHN is associated with increased mortality in hypoxic-ischemic encephalopathy, though the effects on neurodevelopment are less clear. Preterm infants can develop pulmonary hypertension (PH) early in the postnatal course or later in the setting of bronchopulmonary dysplasia (BPD). BPD-PH is associated with higher mortality, particularly within the first year. Evidence suggests that both early and late PH in preterm infants are associated with neurodevelopmental impairment.
Assuntos
Displasia Broncopulmonar , Hérnias Diafragmáticas Congênitas , Hipertensão Pulmonar , Lactente , Recém-Nascido , Humanos , Óxido Nítrico , Recém-Nascido Prematuro , Hipertensão Pulmonar/epidemiologia , Hipertensão Pulmonar/terapia , Displasia Broncopulmonar/epidemiologia , Displasia Broncopulmonar/terapia , Hérnias Diafragmáticas Congênitas/complicações , Hérnias Diafragmáticas Congênitas/epidemiologia , Hérnias Diafragmáticas Congênitas/terapiaRESUMO
Neonatal seizures are common among patients with acute brain injury or critical illness and can be difficult to diagnose and treat. The most common etiology of neonatal seizures is hypoxic-ischemic encephalopathy, with other common causes including ischemic stroke and intracranial hemorrhage. Neonatal clinicians can use a standardized approach to patients with suspected or confirmed neonatal seizures that entails laboratory testing, neuromonitoring, and brain imaging. The primary goals of management of neonatal seizures are to identify the underlying cause, correct it if possible, and prevent further brain injury. This article reviews recent evidence-based guidelines for the treatment of neonatal seizures and discusses the long-term outcomes of patients with neonatal seizures.
Assuntos
Convulsões , Humanos , Recém-Nascido , Convulsões/diagnóstico , Convulsões/etiologia , Convulsões/terapia , Hipóxia-Isquemia Encefálica/diagnóstico , Hipóxia-Isquemia Encefálica/terapiaRESUMO
Alzheimer disease (AD) is associated with extracellular deposition of proteolytic fragments of amyloid precursor protein (APP). Although mutations in APP and proteases that mediate its processing are known to result in familial, early onset forms of AD, the mechanisms underlying the more common sporadic, yet genetically complex forms of the disease are still unclear. Four single-nucleotide polymorphisms within the ubiquilin-1 gene have been shown to be genetically associated with AD, implicating its gene product in the pathogenesis of late onset AD. However, genetic linkage between ubiquilin-1 and AD has not been confirmed in studies examining different populations. Here we show that regardless of genotype, ubiquilin-1 protein levels are significantly decreased in late onset AD patient brains, suggesting that diminished ubiquilin function may be a common denominator in AD progression. Our interrogation of putative ubiquilin-1 activities based on sequence similarities to proteins involved in cellular quality control showed that ubiquilin-1 can be biochemically defined as a bona fide molecular chaperone and that this activity is capable of preventing the aggregation of amyloid precursor protein both in vitro and in live neurons. Furthermore, we show that reduced activity of ubiquilin-1 results in augmented production of pathogenic amyloid precursor protein fragments as well as increased neuronal death. Our results support the notion that ubiquilin-1 chaperone activity is necessary to regulate the production of APP and its fragments and that diminished ubiquilin-1 levels may contribute to AD pathogenesis.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Células HeLa , Humanos , Chaperonas Moleculares/genética , Células PC12 , RatosRESUMO
Fas receptor is a member of the tumor necrosis factor-alpha family of death receptors that mediate physiologic apoptotic signaling. To investigate the molecular mechanisms regulating calcium mobilization during Fas-mediated apoptosis, we have analyzed the sequential steps leading to altered calcium homeostasis and cell death in response to activation of the Fas receptor. We show that Fas-mediated apoptosis requires endoplasmic reticulum-mediated calcium release in a mechanism dependent on phospholipase C-gamma1 (PLC-gamma1) activation and Ca2+ release from inositol 1,4,5-trisphosphate receptor (IP3R) channels. The kinetics of Ca2+ release were biphasic, demonstrating a rapid elevation caused by PLC-gamma1 activation and a delayed and sustained increase caused by cytochrome c binding to IP3R. Blocking either phase of Ca2+ mobilization was cytoprotective, highlighting PLC-gamma1 and IP3R as possible therapeutic targets for disorders associated with Fas signaling.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fosfolipase C gama/metabolismo , Receptor fas/fisiologia , Apoptose , Linhagem Celular , Citocromos c/metabolismo , Citocromos c/fisiologia , Proteína Ligante Fas/metabolismo , Proteína Ligante Fas/fisiologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Modelos Biológicos , Transdução de Sinais , Receptor fas/metabolismoRESUMO
Single nucleotide polymorphisms in the ubiquilin-1 gene may confer risk for late-onset Alzheimer disease (AD). We have shown previously that ubiquilin-1 functions as a molecular chaperone for the amyloid precursor protein (APP) and that protein levels of ubiquilin-1 are decreased in the brains of AD patients. We have recently found that ubiquilin-1 regulates APP trafficking and subsequent secretase processing by stimulating non-degradative ubiquitination of a single lysine residue in the cytosolic domain of APP. Thus, ubiquilin-1 plays a central role in regulating APP biosynthesis, trafficking and ultimately toxicity. As ubiquilin-1 and other ubiquilin family members have now been implicated in the pathogenesis of numerous neurodegenerative diseases, these findings provide mechanistic insights into the central role of ubiquilin proteins in maintaining neuronal proteostasis.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Relacionadas à Autofagia , Humanos , Modelos Moleculares , Transporte Proteico , ProteóliseRESUMO
Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed α, ß, and γ-secretases. Sequential proteolytic cleavage of APP with ß and γ-secretases leads to the production of a small proteolytic peptide, termed Aß, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-1. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro, however it obtains stable secondary structure when bound to Fe65. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis, to modulate signaling pathways, and to regulate calcium signaling. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.