4E,lower panels), suggesting that these two pathways are not involved in CRMP5-induced mitochondrial autophagy. Next, we investigated whether the CRMP5 protein is located in mitochondria simultaneously with LC3 by triple labeling of D-glutamine cells using MT (stainedred) and anti-FLAG (stainedblue) and anti-LC3 (stainedgreen) antibodies (Fig. the number of lysosomes and double membrane vesicles termed autophagosomes, and enhanced the occurrence of microtubule-associated protein 1 light chain 3 (LC3) at the mitochondrial level. Moreover, the lipidated form of LC3, LC3-II, which triggers autophagy by insertion into autophagosomes, enhanced mitophagy initiation. Lysosomal marker translocates at the mitochondrial level, suggesting autophagosome-lysosome fusion, and induced the reduction of mitochondrial content via lysosomal degradation. We show that during early developmental stages the strong expression of endogenous CRMP5, which inhibits dendrite growth, correlated with a decrease of mitochondrial content. In contrast, the knockdown or a decrease of CRMP5 expression at later stages enhanced mitochondrion numbers in cultured neurons, suggesting that CRMP5 modulated these numbers. Our study elucidates a novel regulatory mechanism that utilizes D-glutamine CRMP5-induced mitophagy to orchestrate proper dendrite outgrowth and neuronal function. == Introduction == Mitophagy is a regulated catabolic mechanism whereby cells degrade their damaged mitochondria via autophagy (13). This process seems to be the primary mechanism to ensure mitochondrial quality control that protects cells from damaged mitochondria and from the release of potentially proapoptotic molecules (46). However, Ornipressin Acetate mitophagy is also an actor in the removal of undamaged mitochondria during developmental stages to regulate the changes in steady-state mitochondrial number (6). The process of autophagic degradation is initiated by the sequestration of cytosolic components, such as mitochondria, into double membrane vesicles termed autophagosomes. Many autophagy-related genes (Atg),2identified in yeast, are thought to play similar roles in mammalian cells. Among them,Atg12andAtg8(LC3 counterparts in mammals) are crucial for autophagy (7). As for Atg8, the conversion of cytosolic LC3-I to phosphatidylethanolamine-conjugated LC3-II in mammalian cells contributes to the formation of autophagosomes and the activation of autophagy (7,8). Autophagosomes in turn fuse D-glutamine with endosomes and/or lysosomes to form autolysosomes for the hydrolytic degradation of sequestered material (7). Resulting macromolecules are then transported back into the cytosol for reuse. Whether the autophagic pathway exerts anti- or prodeath roles in neurons under pathological conditions remains unclear (9). Nevertheless, it is increasingly accepted that correct neuronal function is dependent on the trafficking and dynamics of mitochondria, and disruptions in mitochondrial function lead to various neurodegenerative disorders (10,11), such as Parkinson disease (2). Studies on the molecular mechanisms underlying mitophagy have led to the identification of new proteins involved in mitochondrial dynamics. Dynamin-related protein-1 (DRP-1) promotes mitochondrial fission upon recruitment to the outer mitochondrial membrane (12). The protein Parkin, which is commonly mutated in Parkinson disease, translocates to mitochondria after dissipation of the mitochondrial membrane potential (m) and ensures the removal of damaged mitochondria via mitophagy (2,13,14). Other proteins interacting with or functioning in the same pathway as Parkin, such as the PTEN-induced putative kinase 1 (PINK1) and Nix (15,16), have been identified. Collapsin response mediator proteins (CRMPs) are a family of five cytosolic proteins (CRMP15) that are highly expressed in the developing brain (17,18). CRMPs act as signaling molecules involved in the regulation of microtubule polymerization, actin bundling, and endocytosis, leading to neuronal differentiation. CRMP2 was originally identified as the intracellular mediator of Semaphorin 3A signaling that induces growth cone collapse (19). It is now accepted that CRMP2, the best studied member of the CRMP family, is involved in different functions, such as the regulation of neuronal polarity, axon elongation, vesicle trafficking, and synaptic physiology (2022). Fewer studies relate to CRMP5, which is highly expressed in developing brain but decreases in adult brain because at postnatal stages its expression is restrained to the brain areas that retain neurogenesis (23). CRMP5 exhibits spatiotemporal expression in the cortex, hippocampus, and cerebellum D-glutamine and in the postmitotic neuronal precursors, suggesting that it plays a role in process extension (24). Another study has reported that it exerts a role in the regulation of filopodial dynamics and growth cone development (25). The results obtained recently with CRMP5-deficient mice stress the role of CRMP5 in the development and synaptic plasticity of cerebellar Purkinje cells (26). We reported CRMP5 inhibition of neurite outgrowth, especially at the dendritic level,.