RESUMO
Progressive degeneration of motor neurons (MNs) is the hallmark of amyotrophic lateral sclerosis (ALS). Limb-innervating lateral motor column MNs (LMC-MNs) seem to be particularly vulnerable and are among the first MNs affected in ALS. Here, we report association of this differential susceptibility with reduced expression of the mir-17â¼92 cluster in LMC-MNs prior to disease onset. Reduced mir-17â¼92 is accompanied by elevated nuclear PTEN in spinal MNs of presymptomatic SOD1G93A mice. Selective dysregulation of the mir-17â¼92/nuclear PTEN axis in degenerating SOD1G93A LMC-MNs was confirmed in a double-transgenic embryonic stem cell system and recapitulated in human SOD1+/L144F-induced pluripotent stem cell (iPSC)-derived MNs. We further show that overexpression of mir-17â¼92 significantly rescues human SOD1+/L144F MNs, and intrathecal delivery of adeno-associated virus (AAV)9-mir-17â¼92 improves motor deficits and survival in SOD1G93A mice. Thus, mir-17â¼92 may have value as a prognostic marker of MN degeneration and is a candidate therapeutic target in SOD1-linked ALS. VIDEO ABSTRACT.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Neurônios Motores/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Adenoviridae , Animais , Linhagem Celular Tumoral , Extremidades/inervação , Humanos , Células-Tronco Pluripotentes Induzidas , Injeções Espinhais , Proteínas de Membrana/genética , Camundongos , Mutação/genética , Neuroproteção , PTEN Fosfo-Hidrolase/genética , RNA Longo não Codificante , Superóxido Dismutase-1/genéticaRESUMO
Proteolytic processing of amyloid precursor protein (APP) C-terminal fragments (CTFs) by γ-secretase underlies the pathogenesis of Alzheimer's disease (AD). An RNA interference screen using APP-CTF [99-residue CTF (C99)]- and Notch-specific γ-secretase interaction assays identified a unique ErbB2-centered signaling network that was predicted to preferentially govern the proteostasis of APP-C99. Consistently, significantly elevated levels of ErbB2 were confirmed in the hippocampus of human AD brains. We then found that ErbB2 effectively suppressed autophagic flux by physically dissociating Beclin-1 from the Vps34-Vps15 complex independent of its kinase activity. Down-regulation of ErbB2 by CL-387,785 decreased the levels of C99 and secreted amyloid-ß in cellular, zebrafish, and mouse models of AD, through the activation of autophagy. Oral administration of an ErbB2-targeted CL-387,785 for 3 wk significantly improves the cognitive functions of APP/presenilin-1 (PS1) transgenic mice. This work unveils a noncanonical function of ErbB2 in modulating autophagy and establishes ErbB2 as a therapeutic target for AD.
Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Autofagia , Encéfalo/patologia , Presenilina-1/metabolismo , Receptor ErbB-2/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Encéfalo/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Presenilina-1/genética , Proteostase , Receptor ErbB-2/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
Motor neurons (MNs) are unique because they project their axons outside of the CNS to innervate the peripheral muscles. Limb-innervating lateral motor column MNs (LMC-MNs) travel substantially to innervate distal limb mesenchyme. How LMC-MNs fine-tune the balance between survival and apoptosis while wiring the sensorimotor circuit en route remains unclear. Here, we show that the mir-17â¼92 cluster is enriched in embryonic stem cell (ESC)-derived LMC-MNs and that conditional mir-17â¼92 deletion in MNs results in the death of LMC-MNs in vitro and in vivo. mir-17â¼92 overexpression rescues MNs from apoptosis, which occurs spontaneously during embryonic development. PTEN is a primary target of mir-17â¼92 responsible for LMC-MN degeneration. Additionally, mir-17â¼92 directly targets components of E3 ubiquitin ligases, affecting PTEN subcellular localization through monoubiquitination. This miRNA-mediated regulation modulates both target expression and target subcellular localization, providing LMC-MNs with an intricate defensive mechanism that controls their survival.
Assuntos
MicroRNAs/metabolismo , Neurônios Motores/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Animais , Apoptose/fisiologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , Neurônios Motores/citologia , Neurônios Motores/enzimologia , PTEN Fosfo-Hidrolase/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Mutations in presenilin-1 (PS1) are tightly associated with early-onset familial Alzheimer's disease (FAD), which is characterized by extracellular amyloid plaques and the accumulation of intracellular Tau. In addition to being the catalytic subunit of γ-secretase, PS1 has been shown to regulate diverse cellular functions independent of its proteolytic activity. We found that cells deficient in PS1 exhibit reduced levels of p62 protein, a cargo-receptor shuttling Tau for degradation. The downregulation of PS1 led to a significant decrease in both the protein and mRNA transcript of p62, concomitant with attenuated p62 promoter activity. This PS1-dependent regulation of p62 expression was mediated through an Akt/AP-1 pathway independent of the proteolytic activity of PS1/γ-secretase. This p62-mediated Tau degradation was significantly impaired in PS1-deficient cells, which can be rescued by ectopic expression of either p62 or wild-type PS1 but not mutant PS1 containing FAD-linked mutations. Our study suggests a novel function for PS1 in modulating p62 expression to control the proteostasis of Tau.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Regulação da Expressão Gênica , Presenilina-1/fisiologia , Proteólise , Proteínas tau/antagonistas & inibidores , Proteínas tau/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular Tumoral , Regulação para Baixo/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Mutação , Fenótipo , Presenilina-1/deficiência , Presenilina-1/genética , Proteína Sequestossoma-1 , Peixe-Zebra/genética , Proteínas tau/genéticaRESUMO
Gamma-secretase is a multiprotein complex composed of presenilin (PS), nicastrin (NCT), Aph-1, and Pen-2, and it catalyzes the final proteolytic step in the processing of amyloid precursor protein to generate amyloid-beta. Our previous results showed that tumor necrosis factor-alpha (TNF-alpha) can potently stimulate gamma-secretase activity through a c-Jun N-terminal kinase (JNK)-dependent pathway. Here, we demonstrate that TNF-alpha triggers JNK-dependent serine/threonine phosphorylation of PS1 and NCT to stimulate gamma-secretase activity. Blocking of JNK activity with a potent JNK inhibitor (SP600125) reduces TNF-alpha-triggered phosphorylation of PS1 and NCT. Consistent with this, we show that activated JNKs can be copurified with gamma-secretase complexes and that active recombinant JNK2 can promote the phosphorylation of PS1 and NCT in vitro. Using site-directed mutagenesis and a synthetic peptide, we clearly show that the Ser(319)Thr(320) motif in PS1 is an important JNK phosphorylation site that is critical for the TNF-alpha-elicited regulation of gamma-secretase. This JNK phosphorylation of PS1 at Ser(319)Thr(320) enhances the stability of the PS1 C-terminal fragment that is necessary for gamma-secretase activity. Together, our findings strongly suggest that JNK is a critical intracellular mediator of TNF-alpha-elicited regulation of gamma-secretase and governs the pivotal step in the assembly of functional gamma-secretase.