Dysregulated autophagy is linked to BAX oligomerization and subsequent cytochrome c release in 6-hydroxydopmaine-treated neuronal cells.

Autophagy and apoptosis are essential physiological pathways that are required to maintain cellular homeostasis. Therefore, it is suggested that dysregulation in both pathways is linked to several disease states. Moreover, the crosstalk between autophagy and apoptosis plays an important role in pathophysiological processes associated with several neurodegenerative disorders. We have previously reported that 6-hydroxydopamine (6-OHDA)-triggered reactive oxygen species (ROS) induces dysregulated autophagy, and that a dysregulated autophagic flux contributes to caspase-dependent neuronal apoptosis. Based on our previous findings, we specifically aimed to elucidate the molecular mechanisms underlying the potential role of dysregulated autophagy in apoptotic neurodegeneration. The disuccinimidyl suberate (DSS) cross-linking assay and immunological analyses indicated that exposure of several types of cells to 6-OHDA resulted in BAX activation and subsequent oligomerization. Pharmacological inhibition and genetic perturbation of autophagy prevented 6-OHDA-induced BAX oligomerization and subsequent release of mitochondrial cytochrome c into the cytosol and caspase activation. These events were independent of expression levels of XIAP. Taken together, our results suggest that BAX oligomerization comprises a critical step by which 6-OHDA-induced dysregulated autophagy mediates neuronal apoptosis.

Buffering of cytosolic calcium plays a neuroprotective role by preserving the autophagy-lysosome pathway during MPP+-induced neuronal death.

Parkinson's disease (PD) is a chronic neurodegenerative disease with no cure. Calbindin, a Ca2+-buffering protein, has been suggested to have a neuroprotective effect in the brain tissues of PD patients and in experimental models of PD. However, the underlying mechanisms remain elusive. Here, we report that in 1-methyl-4-phenylpyridinium (MPP+)-induced culture models of PD, the buffering of cytosolic Ca2+ by calbindin-D28 overexpression or treatment with a chemical Ca2+ chelator reversed impaired autophagic flux, protecting cells against MPP+-mediated neurotoxicity. When cytosolic Ca2+ overload caused by MPP+ was ameliorated, the MPP+-induced accumulation of autophagosomes decreased and the autophagic flux significantly increased. In addition, the accumulation of damaged mitochondria and p62-positive ubiquitinated protein aggregates, following MPP+ intoxication, was alleviated by cytosolic Ca2+ buffering. We showed that MPP+ treatment suppressed autophagic degradation via raising the lysosomal pH and therefore reducing cytosolic Ca2+ elevation restored the lysosomal pH acidity and normal autophagic flux. These results support the notion that functional lysosomes are required for Ca2+-mediated cell protection against MPP+-mediated neurotoxicity. Thus, our data suggest a novel process in which the modulation of Ca2+ confers neuroprotection via the autophagy-lysosome pathway. This may have implications for the pathogenesis and future therapeutic targets of PD.

Dysregulated autophagy contributes to caspase-dependent neuronal apoptosis.

Autophagy is a regulated, intracellular degradation process that delivers unnecessary or dysfunctional cargo to the lysosome. Autophagy has been viewed as an adaptive survival response to various stresses, whereas in other cases, it promotes cell death. Therefore, both deficient and excessive autophagy may lead to cell death. In this study, we specifically attempted to explore whether and how dysregulated autophagy contributes to caspase-dependent neuronal cell death induced by the neurotoxin 6-hydroxydopamine (6-OHDA). Ultrastructural and biochemical analyses indicated that MN9D neuronal cells and primary cultures of cortical neurons challenged with 6-OHDA displayed typical features of autophagy. Cotreatment with chloroquine and monitoring autophagic flux by a tandem mRFP-EGFP-tagged LC3 probe indicated that the autophagic phenomena were primarily caused by dysregulated autophagic flux. Consequently, cotreatment with an antioxidant but not with a pan-caspase inhibitor significantly blocked 6-OHDA-stimulated dysregulated autophagy. These results indicated that 6-OHDA-induced generation of reactive oxygen species (ROS) played a critical role in triggering neuronal death by causing dysregulated autophagy and subsequent caspase-dependent apoptosis. The results of the MTT reduction, caspase-3 activation, and TUNEL assays indicated that pharmacological inhibition of autophagy using 3-methyladenine or deletion of the autophagy-related gene Atg5 significantly inhibited 6-OHDA-induced cell death. Taken together, our results suggest that abnormal induction of autophagic flux promotes apoptotic neuronal cell death, and that the treatments limiting dysregulated autophagy may have a strong neuroprotective potential.

The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons.

Cyclin-dependent kinase 5 (CDK5) plays a pivotal role in neural development and neurodegeneration. CDK5 activity can be regulated by posttranslational modifications, including phosphorylation and S-nitrosylation. In this study, we demonstrate a novel mechanism by which the acetylation of CDK5 at K33 (Ac-CDK5) results in the loss of ATP binding and impaired kinase activity. We identify GCN5 and SIRT1 as critical factor controlling Ac-CDK5 levels. Ac-CDK5 achieved its lowest levels in rat fetal brains but was dramatically increased during postnatal periods. Intriguingly, nuclear Ac-CDK5 levels negatively correlated with neurite length in embryonic hippocampal neurons. Either treatment with the SIRT1 activator SRT1720 or overexpression of SIRT1 leads to increases in neurite length, whereas SIRT1 inhibitor EX527 or ectopic expression of acetyl-mimetic (K33Q) CDK5 induced the opposite effect. Furthermore, the expression of nuclear-targeted CDK5 K33Q abolished the SRT1720-induced neurite outgrowth, showing that SIRT1 positively regulates neurite outgrowth via deacetylation of nuclear CDK5. The CDK5 activity-dependent increase of neurite length was mediated by enhanced transcriptional regulation of BDNF via unknown mechanism(s). Our findings identify a novel mechanism by which acetylation-mediated regulation of nuclear CDK5 activity plays a critical role in determining neurite length in embryonic neurons.

Breaking down autophagy and the Ubiquitin Proteasome System.

Autophagy is an evolutionarily conserved catabolic process that is involved in cellular homeostasis and stress responses. Although basal levels of autophagy are essential for cellular homeostasis, dysregulated autophagy is linked to neurodegeneration. Recent studies using genetic or neurotoxin-based models of Parkinson's disease (PD) detect autophagy. We demonstrate that neurotoxins induce autophagy in dopaminergic neuronal cell line and primary cultured neurons. Based on previous reports, including ones from our laboratory, which show that elevated reactive oxygen species (ROS) and cytosolic calcium are implicated in dopaminergic neurodegeneration, we reasoned that these triggers may play critical roles in determining dysregulated autophagy. Similarly, we have demonstrated that ROS-mediated signals play an essential role in 6-hydroxydopamine (6-OHDA)-induced apoptosis, whereas MPP+ causes elevations in cytosolic calcium and calpain activation. By using these experimental models, we specifically address the question as to whether an increase in ROS or cytosolic calcium governs abnormal flux of autophagy as well as the ubiquitin proteasome system (UPS). So far, our data support a notion that ROS and cytosolic calcium act on a distinct flux of autophagy and the UPS. Our data also raise the possibility of interplay between autophagy and other cell death modes (e.g., caspase- or calpain-dependent cell death) during dopaminergic neurodegeneration.