The absence of Lamp2 triggers autophagy and mitochondrial biogenesis in skeletal muscle

Abstract

Skeletal muscle requires functional mitochondria to provide it with its energy needs. The quality of the organelle is dependent on the synthesis of new mitochondria and the degradation of those that are no longer operative, via the mitophagy pathway. Current research is examining mitophagy impairments at the autophagosome level, yet little is known about the degradation of the organelle at the level of the lysosome. Dysfunctional mitochondria produce high amounts of ROS and have a lower membrane potential, targeting them for degradation. Tagged mitochondria are engulfed in autophagosomes, which then fuse with lysosomes containing hydrolytic enzymes. The fusion of the lysosome and autophagosome is mediated by the lysosomal protein Lamp2, and is essential for the final stage of mitophagy. The purpose of this project is to evaluate the consequences of Lamp2 deficiency on mitochondrial and lysosomal proteins, as well as potential compensatory signaling responses within muscle. Gastrocnemius and quadriceps muscles of Lamp2 KO mice, compared to WT mice, were used for western blotting and enzymatic analyses. Lamp2 KO mice exhibited a significant 1.4‐fold increase in the adapter protein p62, responsible for tethering the autophagosome to the dysfunctional cargo. Similarly, the LC3‐II to LC3‐I ratio was increased by 2.4‐fold in mice lacking Lamp2. COX activity, a measurement of mitochondrial content, was also elevated by 1.3‐fold in KO mice compared to WT counterparts. These results suggest that the absence of Lamp2 leads to an accumulation of autophagosomes containing mitochondria that are not properly degraded. Protein levels of Beclin1 and the E3 ubiquitin ligase Parkin, were augmented in KO mice, possibly in an attempt increase signaling towards autophagosome formation, and the targeting of dysfunctional mitochondria. TFEB, the master regulator of lysosomal and autophagy genes, was increased 2.2‐fold in KO mice compared to WT animals. Interestingly, mTOR phosphorylation was also increased 2‐fold in KO mice. This activation of mTOR suggests that the elevated TFEB levels are largely confined to the cytosol, and may be less transcriptionally active. However, a downstream target of TFEB, Cathepsin D was increased in KO mice, but no changes were observed in the lysosomal marker V‐ATPase between genotypes. PGC‐1a, the master regulator of mitochondrial biogenesis was elevated by 1.8‐fold in the KO mice, suggesting increased signaling towards mitochondrial biogenesis. However, levels of Transcription factor A (Tfam) were similar between genotypes, suggesting that the absence of Lamp2 may dysregulate the coordinated expression of nuclear and mtDNA encoded gene products. This was evident from an increase in UQCRC2, a nuclear‐derived protein, with no changes observed in mtDNA encoded COXI. Thus, these data suggest that Lamp2 is required for the clearance of mitochondria. In its absence, mitochondrial degradation is defective, initiating compensatory signaling responses to mitochondrial biogenesis and autophagy induction to promo...

Methylmalonic acidemia triggers lysosomal-autophagy dysfunctions

“…Accordingly, as seen in our previous surveys, dysfunctional mitochondria in MMA produce high amounts of ROS, with cells showing increased susceptibility to stress [ 18 ]. Such oxidative alterations can be strictly connected with LAMP2 down-regulation in MMA, whereas studies in LAMP2-deficient (KO) mice provided evidence for mitophagy impairment proving that LAMP2 is required for mitochondria clearance and turnover [ 50 ]. On the same basis, LAMP2 alterations co-occurred with increased p62 and LC3 content in LAMP2-KO mice [ 50 ].…”

“…Such oxidative alterations can be strictly connected with LAMP2 down-regulation in MMA, whereas studies in LAMP2-deficient (KO) mice provided evidence for mitophagy impairment proving that LAMP2 is required for mitochondria clearance and turnover [ 50 ]. On the same basis, LAMP2 alterations co-occurred with increased p62 and LC3 content in LAMP2-KO mice [ 50 ]. In accordance with these findings, our results displayed higher levels of LC3 and p62 and LAMP1 in mut0 fibroblasts.…”