Mitochondrial Protein Import Regulates Cytosol Protein Homeostasis And Neuronal Integrity

Neurodegeneration is characterized by protein aggregate deposits and mitochondrial malfunction.It is unclear if these two phenomena are intrinsically linked.We found that reduction in Tom40(translocase of outer membrane 40)expression,a key subunit of the translocase of the outer mitochondrial membrane complex,led to abnormal accumulation of ubiquitin(Ub)-positive protein aggregates engulfed by Atg8a-positive membranes.Further analysis indicated that other autophagy markers were also abnormally accumulated.Autophagy was induced but the majority of autophagosomes failed to fuse with lysosomes when Tom40 was down regulated.In Tom40 RNAi tissues,ultrastructural analysis revealed that autophagosome-like(AL)structures were ten times larger than starvation induced autophagosomes.The giant AL structures were often not sealed.Furthermore,in these tissues,Atg5 downregulation abolished AL structure formation,but the Ub-positive aggregates remained,whereas knock down of Syx17,a gene required for autophagosome-lysosome fusion,led to the disappearance of giant AL structures and accumulation of small autophagosomes and phagophores near the Ub-positive aggregates.These findings suggest that giant AL structures in Tom40 RNAi tissues are formed through fusion between phagophores and small autophagosomes.The protein aggregates contained many mitochondrial pre-proteins,cytosolic proteins,and proteasome subunits.Proteasome activity was greatly reduced in Tom40 RNAi tissues and led to the accumulation of Ub-positive aggregates.Tom40 knockdown also led to reduced levels of ATP and increased levels of ROS.The simultaneous inhibition of proteasome activity,reduction in ATP production,and increase in ROS,but none of these conditions alone,can mimic the imbalanced proteostasis phenotypes observed in Tom40 RNAi cells.Knockdown of Ref(2)P or ectopic expression of Pink1 and Park greatly reduced aggregate formation in Tom40 RNAi tissues.In nerve tissues,reduction in Tom40 activity leads to aggregate formation and neurodegeneration.Surprisingly,rather than diminishing the neurodegenerative phenotypes,overexpression of Pinkl enhanced them.We proposed that defects in mitochondrial protein import may be the key to linking imbalanced proteostasis and mitochondrial defects—two common features of neurodegeneration.