Scale bar: 200?m. mitochondria fragmentation, which alleviated mitochondrial dysfunction under oxidative stress. Furthermore, we showed that conditional expression of LAMP2A, the key CMA regulator, in dopaminergic (DA) neurons helped maintain mitochondria morphology and protected DA neuronal viability in a rodent C527 PD model. Our work uncovers a critical role of CMA in maintaining proper mitochondria dynamics, and loss of this regulatory control may occur in PD and underlie its pathogenic process. Abbreviations: CMA: chaperone-mediated autophagy; DA: dopaminergic; DNM1L: dynamin 1 like; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; HSPA8: heat shock protein family A (Hsp70) member 8; LAMP2A: lysosomal associated membrane protein 2A; MARCHF5: membrane-associated ring-CH-type finger 5; MMP: mitochondria membrane potential; OCR: oxygen consumption rate; 6-OHDA: 6-hydroxydopamine; PD: Parkinson disease; SNc: substantia nigra pars compacta; TEM: transmission electron microscopy; TH: tyrosine hydroxylase; TMRE: tetramethylrhodamine ethyl ester perchlorate; WT: wild type. and siRNAs to knockdown endogenous LAMP2A. Analysis of whole cell lysates revealed that knockdown of LAMP2A was accompanied with an increase in the level of MARCHF5 while other mitochondrial proteins such as MFN2 and HSPD1 (heat shock protein family D (Hsp60) member 1) remained unchanged, indicating that the increase of MARCHF5 was a specific effect of LAMP2A knockdown (Figure 1E). Consistently, the level of mitochondria-associated MARCHF5 was also higher after LAMP2A knockdown (Figure 1F). On the contrary, LAMP2A overexpression significantly reduced the level of MARCHF5 and this reduction was completely reversed by lysosome hydrolase inhibitors, suggesting that MARCHF5 is degraded directly by CMA (Figure 1G). In line with above findings, knocking down LAMP2A also abolished serum withdrawal-induced down-regulation C527 of MARCHF5 (Figure 1H). Collectively, these data support MARCHF5 as a CMA target. Open in a separate window Figure 1. MARCHF5 level is regulated by LAMP2A. (A) Scheme of screening for proteins modulated by LAMP2A. SN4741 cells were transfected with small interfering RNA (siRNA) for or non-specific control siRNA (NC) for 48?h and collected for MS/MS analysis. (B) Identification of MARCHF5 as a candidate CMA Rabbit Polyclonal to EMR1 substrate. Partial list shows proteins whose levels were upregulated following LAMP2A knockdown. denotes known CMA substrates identified previously. (C) Change of MARCHF5 protein level after prolonged serum starvation. Enriched mitochondria (Mito) and total cell lysate (TCL) derived from SN4741 cells following serum starvation for the indicated periods of time were blotted. Right panel shows the relative MARCHF5 level normalized to HSPD1. (D) Lysosome-dependent degradation of MARCHF5 after serum starvation. SN4741 cells were serum-starved for 24?h with concurrent treatment as indicated (N/L: a combination of 20?mM NH4Cl and 100?M leupeptin; 3-MA: 10?mM 3-methyladenine). Right panel shows the relative MARCHF5 level normalized to HSPD1. (E) The effect of LAMP2A knockdown on MARCHF5 level. SN4741 cells were transfected with three different siRNAs for 48?h. TCL were blotted for the indicated proteins. Right panel shows the quantification of MARCHF5 levels normalized to ACTB. (F) Change of MARCHF5 in different subcellular compartments. MARCHF5 level was blotted in TCL, cytoplasm (Cyto) and Mito fractions derived from SN4741 cells with LAMP2A knocked down (siRNA-215 was used here). Right panel shows the relative mitochondria-associated MARCHF5 level normalized to COX4I1. (G) The effect of LAMP2A overexpression on MARCHF5. SN4741 cells were transfected with the empty vector C527 (EV) or MYC-LAMP2A plasmids for C527 24?h with or without lysosome inhibitors. The Mito and TCL extractions were blotted for the indicated proteins. Right panel shows the quantification of MARCHF5 level normalized to HSPD1. OE refers to MYC-LAMP2A overexpression. (H) The effect of LAMP2A knockdown on serum starvation-induced change of MARCHF5. Mito and TCL extractions from serum-starved SN4741 cells transfected with NC or siRNAs were blotted. Right panel shows the relative MARCHF5 level normalized to HSPD1. Data are expressed as mean S.E.M. (=?3 independent experiments. Two-tailed Students =?3 independent experiments. Two-tailed Students siRNAs were treated with 6-OHDA (80?M) for 24?h and.