RESUMO
Endoplasmic reticulum (ER) remodeling is vital for cellular organization. ER-phagy, a selective autophagy targeting ER, plays an important role in maintaining ER morphology and function. The FAM134 protein family, including FAM134A, FAM134B, and FAM134C, mediates ER-phagy. While FAM134B mutations are linked to hereditary sensory and autonomic neuropathy in humans, the physiological role of the other FAM134 proteins remains unknown. To address this, we investigate the roles of FAM134 proteins using single and combined knockouts (KOs) in mice. Single KOs in young mice show no major phenotypes; however, combined Fam134b and Fam134c deletion (Fam134b/cdKO), but not the combination including Fam134a deletion, leads to rapid neuromuscular and somatosensory degeneration, resulting in premature death. Fam134b/cdKO mice show rapid loss of motor and sensory axons in the peripheral nervous system. Long axons from Fam134b/cdKO mice exhibit expanded tubular ER with a transverse ladder-like appearance, whereas no obvious abnormalities are present in cortical ER. Our study unveils the critical roles of FAM134C and FAM134B in the formation of tubular ER network in axons of both motor and sensory neurons.
Assuntos
Axônios , Retículo Endoplasmático , Proteínas de Membrana , Animais , Humanos , Camundongos , Axônios/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos KnockoutRESUMO
The discovery of the multiple roles of mitochondria-endoplasmic reticulum (ER) juxtaposition in cell biology often relied upon the exploitation of Mitofusin (Mfn) 2 as an ER-mitochondria tether. However, this established Mfn2 function was recently questioned, calling for a critical re-evaluation of Mfn2's role in ER-mitochondria cross-talk. Electron microscopy and fluorescence-based probes of organelle proximity confirmed that ER-mitochondria juxtaposition was reduced by constitutive or acute Mfn2 deletion. Functionally, mitochondrial uptake of Ca2+ released from the ER was reduced following acute Mfn2 ablation, as well as in Mfn2-/- cells overexpressing the mitochondrial calcium uniporter. Mitochondrial Ca2+ uptake rate and extent were normal in isolated Mfn2-/- liver mitochondria, consistent with the finding that acute or chronic Mfn2 ablation or overexpression did not alter mitochondrial calcium uniporter complex component levels. Hence, Mfn2 stands as a bona fide ER-mitochondria tether whose ablation decreases interorganellar juxtaposition and communication.
Assuntos
Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Embrião de Mamíferos/citologia , Retículo Endoplasmático/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Deleção de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fígado/metabolismo , Camundongos Knockout , Mitocôndrias/ultraestrutura , Sondas Moleculares/metabolismoRESUMO
Retinal explants and mixed primary cultures are currently used to investigate retinal ganglion cells (RGCs) pathophysiology and pharmacology, but information on yield, quality and quantity of contaminant cells for the available RGCs enrichment techniques is lacking. Here we compare two methods of mouse primary RGCs purification and show that mitochondrial and autophagy parameters can be measured in rapidly purified RGCs. RGCs were purified from P0 mouse eyes using two methods based on the surface antigen Thy1. In a two-step immunopanning purification, a subtraction plate bound macrophage antiserum removed contaminant macrophages and endothelial cells; unbound RGCs were then affinity selected using a plate-bound antiThy1 antibody. In an immunopanning-magnetic separation, macrophage-antiserum bound cells were first subtracted and then RGCs were positively selected using an antiThy1 antibody bound to a magnetic column. The two-steps immunopanning yielded low amounts of 90% pure RGCs, whereas RGCs represented 30% of the 6-fold more cells collected by immunopanning-magnetic separation. RGCs purified with both methods could be microelectroporated to image expressed mitochondria and autophagosomes fluorescent probes and to show that expression of pathogenic Optic atrophy 1 mutants causes mitochondrial fragmentation. Thus, these two methods purify primary mouse RGCs amenable to studies of cell morphology, mitochondrial biology and autophagy.
Assuntos
Autofagia , Mitocôndrias/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Camundongos Endogâmicos C57BLRESUMO
Transporting and translating mRNAs in axons is crucial for neuronal viability. Local synthesis of nuclear-encoded mitochondrial proteins protects long-lived axonal mitochondria from damage; however, the regulatory factors involved are largely unknown. We show that CLUH, which binds mRNAs encoding mitochondrial proteins, prevents peripheral neuropathy and motor deficits in the mouse. CLUH is enriched in the growth cone of developing spinal motoneurons and is required for their growth. The lack of CLUH affects the abundance of target mRNAs and the corresponding mitochondrial proteins more prominently in axons, leading to ATP deficits in the growth cone. CLUH interacts with ribosomal subunits, translation initiation, and ribosome recycling components and preserves axonal translation. Overexpression of the ribosome recycling factor ABCE1 rescues the mRNA and translation defects, as well as the growth cone size, in CLUH-deficient motoneurons. Thus, we demonstrate a role for CLUH in mitochondrial quality control and translational regulation in axons, which is essential for their development and long-term integrity and function.
Assuntos
Axônios , Mitocôndrias , Neurônios Motores , Doenças do Sistema Nervoso Periférico , Biossíntese de Proteínas , Animais , Neurônios Motores/metabolismo , Mitocôndrias/metabolismo , Axônios/metabolismo , Camundongos , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/patologia , Cones de Crescimento/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Camundongos KnockoutRESUMO
The highly specialized structure and function of neurons depend on a sophisticated organization of the cytoskeleton, which supports a similarly sophisticated system to traffic organelles and cargo vesicles. Mitochondria sustain crucial functions by providing energy and buffering calcium where it is needed. Accordingly, the distribution of mitochondria is not even in neurons and is regulated by a dynamic balance between active transport and stable docking events. This system is finely tuned to respond to changes in environmental conditions and neuronal activity. In this review, we summarize the mechanisms by which mitochondria are selectively transported in different compartments, taking into account the structure of the cytoskeleton, the molecular motors and the metabolism of neurons. Remarkably, the motor proteins driving the mitochondrial transport in axons have been shown to also mediate their transfer between cells. This so-named intercellular transport of mitochondria is opening new exciting perspectives in the treatment of multiple diseases.
Assuntos
Axônios , Neurônios , Neurônios/metabolismo , Axônios/metabolismo , Mitocôndrias/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismoRESUMO
The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra (SN) (A9 neurons) and the ventral tegmental area (VTA) (A10 cells). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson's disease. Here, we report that gene expression analysis of A9 dopaminergic neurons (DA) identifies transcripts for alpha- and beta-chains of hemoglobin (Hb). Globin immunoreactivity decorates the majority of A9 DA, a subpopulation of cortical and hippocampal astrocytes and mature oligodendrocytes. This pattern of expression was confirmed in different mouse strains and in rat and human. We show that Hb is expressed in the SN of human postmortem brain. By microarray analysis of dopaminergic cell lines overexpressing alpha- and beta-globin chains, changes in genes involved in O(2) homeostasis and oxidative phopshorylation were observed, linking Hb expression to mitochondrial function. Our data suggest that the most famed oxygen-carrying globin is not exclusively restricted to the blood, but it may play a role in the normal physiology of the brain and neurodegenerative diseases.
Assuntos
Neuroglia/metabolismo , Neurônios/metabolismo , Substância Negra/citologia , Área Tegmentar Ventral/citologia , alfa-Globinas/metabolismo , Globinas beta/metabolismo , Animais , Citometria de Fluxo , Proteínas de Fluorescência Verde , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RatosRESUMO
Mitochondrial dysfunction and mitophagy are often hallmarks of neurodegenerative diseases such as autosomal dominant optic atrophy (ADOA) caused by mutations in the key mitochondrial dynamics protein optic atrophy 1 (Opa1). However, the second messengers linking mitochondrial dysfunction to initiation of mitophagy remain poorly characterized. Here, we show in mammalian and nematode neurons that Opa1 mutations trigger Ca2+-dependent mitophagy. Deletion or expression of mutated Opa1 in mouse retinal ganglion cells and Caenorhabditis elegans motor neurons lead to mitochondrial dysfunction, increased cytosolic Ca2+ levels, and decreased axonal mitochondrial density. Chelation of Ca2+ restores mitochondrial density in neuronal processes, neuronal function, and viability. Mechanistically, sustained Ca2+ levels activate calcineurin and AMPK, placed in the same genetic pathway regulating axonal mitochondrial density. Our data reveal that mitophagy in ADOA depends on Ca2+-calcineurin-AMPK signaling cascade.
Assuntos
Atrofia Óptica Autossômica Dominante , Animais , Cálcio , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Mamíferos/metabolismo , Camundongos , Mitofagia/genética , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
In autosomal dominant optic atrophy (ADOA), caused by mutations in the mitochondrial cristae biogenesis and fusion protein optic atrophy 1 (Opa1), retinal ganglion cell (RGC) dysfunction and visual loss occur by unknown mechanisms. Here, we show a role for autophagy in ADOA pathogenesis. In RGCs expressing mutated Opa1, active 5' AMP-activated protein kinase (AMPK) and its autophagy effector ULK1 accumulate at axonal hillocks. This AMPK activation triggers localized hillock autophagosome accumulation and mitophagy, ultimately resulting in reduced axonal mitochondrial content that is restored by genetic inhibition of AMPK and autophagy. In C. elegans, deletion of AMPK or of key autophagy and mitophagy genes normalizes the axonal mitochondrial content that is reduced upon mitochondrial dysfunction. In conditional, RGC specific Opa1-deficient mice, depletion of the essential autophagy gene Atg7 normalizes the excess autophagy and corrects the visual defects caused by Opa1 ablation. Thus, our data identify AMPK and autophagy as targetable components of ADOA pathogenesis.