UC San Diego

Beclin 1 is a critical regulator of autophagy which functions as a scaffold protein to initiate autophagosome formation and maturation. Global Beclin 1 knockout mice are embryonically lethal, while heterozygous Beclin 1 mice display a reduction in autophagy and increased susceptibility to myocardial ischemia/reperfusion injury. Cre-mediated recombination is a powerful tool that is commonly used in research to generate tissue-specific and inducible knockout mouse lines. To specifically delete Beclin 1 in the adult heart, our lab has generated cardiac specific, inducible MerCreMer Beclin 1 f/f mice. In establishing this model, Cre toxicity (rapid cardiac dysfunction and increased mortality) was observed in Beclin1 f/f mice (BMCM) but not in MerCreMer cassette-carrying mice (MCM) upon injection with tamoxifen to induce Cre expression. Here, we assess Cre toxicity in Beclin1-deficiency in vitro and in vivo to determine whether the Beclin1 deficiency increases the sensitivity to Cre expression. We observed a dose-dependent toxicity of Cre induction in Beclin1-deficient MEFs and hearts. Overexpression of Cre did not cause cell death in Rab7-deficient MEFs or Beclin 1-deficient HeLa cells. Our findings suggest that lack of Beclin 1 leads to increased susceptibility to Cre-mediated toxicity.

The R120G mutation in alpha-b-crystallin (CryAB-R120G) causes a proteotoxic cardiomyopathy that is characterized by the accumulation of misfolded protein aggregates. Recently, mitochondria have been shown to import misfolded proteins as a novel mechanism of protein quality control in S. cerevisiae. However, this import mechanism has not been investigated in a mammalian system. Here we disrupt mitochondrial protein import to characterize the potential uptake of CryAB-R120G by mitochondria. We observed that mitochondrial import can be perturbed by knocking down a subunit of the translocase of the mitochondrial outer membrane (Tom20) and that this results in cytosolic accumulation of CryAB-R120G. Our findings suggest that mitochondria play a role in cytosolic proteostasis amid CryAB-R120G-induced aggregation.