Abstract
We have reported that conventional protein kinase Cγ (cPKCγ)-modulated neuron-specific autophagy improved the neurological outcome of mice following ischemic stroke through the Akt-mechanistic target of rapamycin (mTOR) pathway. However, its detailed molecular mechanism remains unclear. In this study, primary cortical neurons from postnatal one-day-old C57BL/6J cPKCγ wild-type (cPKCγ+/+) and knockout (cPKCγ−/−) mice suffering oxygen glucose deprivation/reperfusion (OGD/R) were used to simulate ischemia/reperfusion injury in vitro. A block of autophagic flux was observed in cPKCγ+/+ neurons under OGD/R exposure, characterized by accumulation of p62. Immunofluorescent results showed a decrease in colocalization between LC3 and Atg14 or Stx17 in cPKCγ+/+ neurons when compared with cPKCγ−/− neurons after OGD/R. However, the colocalization between LC3 and Lamp2 was barely decreased, indicating the presence of autolysosomes. The larger lysotracker-positive structures were also significantly increased. These results suggest that cPKCγ-induced inhibition of autophagy occurred at the stages of autophagosome formation, Stx17 anchoring, and the degradation of autolysosomes in particular. In addition, cPKCγ-modulated phosphorylation of mTOR at Ser 2481 was dependent on the site of Ser 2448, which may have blocked autophagic flux. cPKCγ-modulated sequential reactivation of mTOR inhibited autophagic flux in neurons exposed to OGD/R, which may provide endogenous interventional strategies for stroke, especially ischemia/reperfusion injury.
Highlights
Given that recombinant tissue plasminogen activator thrombolysis is a recommended form of therapy for acute ischemic stroke, the mechanism of ischemia and ischemia/reperfusion injury has become a popular topic of research
We further found that the light chain 3 (LC3) conversion in conventional protein kinase Cγ (cPKCγ)−/− neurons was higher than that in cPKCγ+/+ neurons, and the phosphorylation level of mechanistic target of rapamycin (mTOR) at Ser 2481, as the auto-phosphorylation site described above, was lower than that of wild-type under the conditions of normoxia and 1 h oxygen glucose deprivation/reperfusion (OGD/R) 0–1 h treatments
The p62 accumulation was associated with autophagy inhibition in cPKCγ+/+ neurons after 1 h OGD/R 1 h treatments
Summary
Given that recombinant tissue plasminogen activator (rtPA) thrombolysis is a recommended form of therapy for acute ischemic stroke, the mechanism of ischemia and ischemia/reperfusion injury has become a popular topic of research. We found that autophagy is induced in ischemic injury through the Akt-mTOR pathway, while the level of autophagy in cPKCγ wild-type mice is significantly lower than in knockout mice after reperfusion [2]. The detailed molecular mechanism underlying cPKCγ-modulated autophagy and their mediated neuroprotection against ischemic injuries remain unclear. Numerous researchers have focused on the late stage of autophagy (autolysosome formation and degradation) on the basis of studies which focused on the early stage (autophagy initiation and autophagosome formation) [9,10,11,12]. We used oxygen glucose deprivation/reperfusion (OGD/R) treatment of primary cortical neurons to simulate ischemia/reperfusion injury in vitro to understand whether cPKCγ-modulated mTOR activation can block autophagy flux and its possible mechanism
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