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RAB24 facilitates clearance of autophagic compartments during basal conditions.

ABSTRACT: RAB24 belongs to a family of small GTPases and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy shows an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicate that this is due to a hindrance in their clearance. Formation of autophagosomes is shown to be unaffected by RAB24-silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 is also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions.

SUBMITTER: Yla-Anttila P

PROVIDER: S-EPMC4824578 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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RAB24 facilitates clearance of autophagic compartments during basal conditions.

Ylä-Anttila Päivi P Mikkonen Elisa E Happonen Kaisa E KE Holland Petter P Ueno Takashi T Simonsen Anne A Eskelinen Eeva-Liisa EL

Autophagy 20150101 10

RAB24 belongs to a family of small GTPases and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not dur ...[more]

PMID: 26325487

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Project description:Metabolic acidosis, a common complication of CKD, causes mitochondrial stress by undefined mechanisms. Selective autophagy of impaired mitochondria, called mitophagy, contributes toward maintaining cellular homeostasis in various settings. We hypothesized that mitophagy is involved in proximal tubular cell adaptations to chronic metabolic acidosis. In transgenic mice expressing green fluorescent protein-tagged microtubule-associated protein 1 light chain 3 (GFP-LC3), NH4Cl loading increased the number of GFP puncta exclusively in the proximal tubule. In vitro, culture in acidic medium produced similar results in proximal tubular cell lines stably expressing GFP-LC3 and facilitated the degradation of SQSTM1/p62 in wild-type cells, indicating enhanced autophagic flux. Upon acid loading, proximal tubule-specific autophagy-deficient (Atg5-deficient) mice displayed significantly reduced ammonium production and severe metabolic acidosis compared with wild-type mice. In vitro and in vivo, acid loading caused Atg5-deficient proximal tubular cells to exhibit reduced mitochondrial respiratory chain activity, reduced mitochondrial membrane potential, and fragmented morphology with marked swelling in mitochondria. GFP-LC3-tagged autophagosomes colocalized with ubiquitinated mitochondria in proximal tubular cells cultured in acidic medium, suggesting that metabolic acidosis induces mitophagy. Furthermore, restoration of Atg5-intact nuclei in Atg5-deficient proximal tubular cells increased mitochondrial membrane potential and ammoniagenesis. In conclusion, metabolic acidosis induces autophagy in proximal tubular cells, which is indispensable for maintaining proper mitochondrial functions including ammoniagenesis, and thus for adapted urinary acid excretion. Our results provide a rationale for the beneficial effect of alkali supplementation in CKD, a condition in which autophagy may be reduced, and suggest a new therapeutic option for acidosis by modulating autophagy.

Project description:RationaleIncreasing evidence suggests that misfolded proteins and intracellular aggregates contribute to cardiac disease and heart failure. Several cardiomyopathies, including the αB-crystallin R120G mutation (CryAB(R120G)) model of desmin-related cardiomyopathy, accumulate cytotoxic misfolded proteins in the form of preamyloid oligomers and aggresomes. Impaired autophagic function is a potential cause of misfolded protein accumulations, cytoplasmic aggregate loads, and cardiac disease. Atg7, a mediator of autophagosomal biogenesis, is a putative regulator of autophagic function.ObjectiveTo determine whether autophagic induction by Atg7 is sufficient to reduce misfolded protein and aggregate content in protein misfolding-stressed cardiomyocytes.Methods and resultsTo define the gain and loss of function effects of Atg7 expression on CryAB(R120G) protein misfolding and aggregates, neonatal rat cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB or CryAB(R120G) and coinfected with Atg7 adenovirus or with Atg7 silencing siRNAs to produce gain-of or loss-of Atg7 function. Atg7 overexpression effectively induced basal autophagy with no detrimental effects on cell survival, suggesting that Atg7 can activate autophagy with no apparent cytotoxic effects. Autophagic flux assays on CryAB(R120G)-expressing cardiomyocytes revealed reduced autophagic function, which probably contributed to the failure of misfolded proteins and aggregates to be cleared. Coexpression of Atg7 and CryAB(R120G) significantly reduced preamyloid oligomer staining, aggregate content, and cardiomyocyte cytotoxicity. Conversely, Atg7 silencing in the CryAB(R120G) background significantly inhibited the already reduced rate of autophagy and increased CryAB(R120G) aggregate content and cytotoxicity.ConclusionsAtg7 induces basal autophagy, rescues the CryAB(R120G) autophagic deficiency, and attenuates the accumulation of misfolded proteins and aggregates in cardiomyocytes.

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RAB24 facilitates clearance of autophagic compartments during basal conditions.

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RAB24 facilitates clearance of autophagic compartments during basal conditions.

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