Upregulation Of Macroautophagy Research Articles

Proteome-wide modulation of degradation dynamics in response to growth arrest

In dividing cells, cytoplasmic dilution is the dominant route of clearance for long-lived proteins whose inherent degradation is slower than the cellular growth rate. Thus, as cells transition from a dividing to a nondividing state, there is a propensity for long-lived proteins to become stabilized relative to short-lived proteins, leading to alterations in the abundance distribution of the proteome. However, it is not known if cells mount a compensatory response to counter this potentially deleterious proteostatic disruption. We used a proteomic approach to demonstrate that fibroblasts selectively increase degradation rates of long-lived proteins as they transition from a proliferating to a quiescent state. The selective degradation of long-lived proteins occurs by the concurrent activation of lysosomal biogenesis and up-regulation of macroautophagy. Through this mechanism, quiescent cells avoid the accumulation of aged long-lived proteins that would otherwise result from the absence of cytoplasmic dilution by cell division.

Functional links between microtubules, autophagy and leaf starch degradation in plants

ABSTRACTMounting evidence suggests that microtubules play important roles in several aspects of autophagy in mammalian cells, such as autophagosome biogenesis, autophagosome trafficking and autolysosome formation. However, little research attention has been paid to the engagement of microtubules in plant autophagy. Recently, we reported novel findings in Nicotiana benthamiana that disruption of microtubules reduces autophagosome formation during upregulation of macroautophagy and triggers a specific type of chloroplast autophagy (SEX chlorophagy), which is closely associated with the starch-excess phenotype of leaves. These findings reveal important functional links between microtubules, autophagy and leaf starch degradation in plants.

Protective role of PARK2/Parkin in sepsis-induced cardiac contractile and mitochondrial dysfunction

Mitochondrial quality control plays a vital role in the maintenance of optimal mitochondrial function. However, its roles and regulation remain ill-defined in cardiac pathophysiology. Here, we tested the hypothesis that PARK2/Parkin, an E3-ligase recently described as being involved in the regulation of cardiac mitophagy, is important for (1) the maintenance of normal cardiac mitochondrial function; and (2) adequate recovery from sepsis, a condition known to induce reversible mitochondrial injury through poorly understood mechanisms. Investigations of mitochondrial and cardiac function were thus performed in wild-type and Park2-deficient mice at baseline and at 2 different times following administration of a sublethal dose of E. coli lipopolysaccharide (LPS). LPS injection induced cardiac and mitochondrial dysfunctions that were followed by complete recovery in wild-type mice. Recovery was associated with morphological and biochemical evidence of mitophagy, suggesting that this process is implicated in cardiac recovery from sepsis. Under baseline conditions, multiple cardiac mitochondrial dysfunctions were observed in Park2-deficient mice. These mild dysfunctions did not result in a visibly distinct cardiac phenotype. Importantly, Park2-deficient mice exhibited impaired recovery of cardiac contractility and constant degradation of mitochondrial metabolic functions. Interestingly, autophagic clearance of damaged mitochondria was still possible in the absence of PARK2 likely through compensatory mechanisms implicating PARK2-independent mitophagy and upregulation of macroautophagy. Together, these results thus provide evidence that in vivo, mitochondrial autophagy is activated during sepsis, and that compensation for a lack of PARK2 is only partial and/or that PARK2 exerts additional protective roles in mitochondria.

TNF-α upregulates macroautophagic processing of APP/β-amyloid in a human rhabdomyosarcoma cell line

Sporadic inclusion body myositis is a chronic progressive, inflammatory disorder of the skeletal muscle. No effective treatment is available for this debilitating condition and the complex disease pathology is far from being understood. The major hallmark of the pathomechanisms is the co-occurrence of inflammatory as well as degenerative cascades including aggregates consisting of β-amyloid within skeletal muscle fibers. Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to be upregulated via the proinflammatory cytokine TNF-α in human skeletal muscle cells. In a human cell line from rhabdomyosarcoma as a model to study muscle cells, we here show that TNF-α-mediated upregulation of macroautophagy modulates APP and β-amyloid load and can be blocked by inhibition of macroautophagy. Thus, macroautophagy may be a crucial mediator between inflammation and β-amyloid-associated degeneration in skeletal muscle.

Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.

Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days), and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.

Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

BackgroundHuntington's disease is caused by aggregation of mutant huntingtin (mHtt) protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease activities may contribute to toxicity whereas others are consistent with protection. These discrepancies may be due to a number of mechanisms including distinct effects of the specific intermediate digestion products of mutant huntingtin generated by different proteases. These observations suggested a critical need to investigate the consequence of upregulation of individual lysosomal enzyme in mutant huntingtin accumulation and toxicity.ResultsIn this study, we used molecular approaches to enhance lysosomal protease activities and examined their effects on mutant huntingtin level and toxicity. We found that enhanced expression of lysosomal cathepsins D and B resulted in their increased enzymatic activities and reduced both full-length and fragmented huntingtin in transfected HEK cells. Furthermore, enhanced expression of cathepsin D or B protected against mutant huntingtin toxicity in primary neurons, and their neuroprotection is dependent on macroautophagy.ConclusionsThese observations demonstrate a neuroprotective effect of enhancing lysosomal cathepsins in reducing mutant huntingtin level and toxicity in transfected cells. They highlight the potential importance of neuroprotection mediated by cathepsin D or B through macroautophagy.