Deletion of PIK3C3/Vps34 in sensory neurons causes rapid neurodegeneration by disrupting the endosomal but not the autophagic pathway

Abstract

The lipid kinase PIK3C3 (also called Vps34) regulates both the endosomal and autophagic pathways. However, the effect of inactivating PIK3C3 on neuronal endosomal versus autophagic processes in vivo has not been studied. We generated mice in which Pik3c3 was conditionally deleted in differentiated sensory neurons. Within a few days after Pik3c3 deletion, mutant largediameter myelinated neurons accumulated numerous enlarged vacuoles and ubiquitin-positive aggregates and underwent rapid degeneration. By contrast, Pik3c3-deficient small-diameter unmyelinated neurons accumulated excessive numbers of lysosome-like organelles and degenerated more slowly. These differential degenerative phenotypes are unlikely caused by a disruption in the autophagy pathway, because inhibiting autophagy alone by conditional deletion of Atg7 results in a completely distinct phenotype in all sensory neurons (i.e., formation of very large intracellular inclusion bodies and slow degeneration over a period of several months). More surprisingly, a noncanonical PIK3C3-independent LC3-positive autophagosome formation pathway was activated in Pik3c3-deficient small-diameter neurons. Analyses of Pik3c3/Atg7 double mutant neurons revealed that this unconventional initiation pathway still depends on ATG7. Our studies represent in vivo characterization of PIK3C3 functions in mammals and provide insights into the complexity of neuronal endo-lysosomal and autophagic pathways. P IK3C3 (also known as Vps34) is a class III phosphatidylinositol-3-kinase that specifically catalyzes the formation of phosphatidylinositol-3-phosphate (PI3P) (1). Studies in invertebrate organisms as well as in nonneuronal cells showed that PIK3C3/Vps34 regulates multiple aspects of both the endocytic/ endosomal and autophagic pathways (2-5). In yeast, there are two distinct Vps34 complexes: complex I (Vps34, Vps15, Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Atg6, and Vps38) functions in the vacuolar proteinsorting process (6). In mammals, homologs of Vps15 and Atg6 are p150 and Beclin1, respectively (7), and evidence exists for Beclin1-independent functions of PIK3C3 in the endocytic pathways (8). A mammalian homolog of complex I (PIK3C3, p150, Beclin1, and Atg14L) activates autophagy, and a homolog of complex II (PIK3C3, p150, Beclin1, and UVRAG/Vps38) regulates trafficking at late endosomes (7). Interestingly, Bif-1 and Rubicon can interact with complex II to promote autophagy (9, 10). The chemical inhibitor of PIK3C3 (3-MA or wortmanin) has been frequently used as an inhibitor for autophagy in numerous studies, including those studying autophagy in neurons (11)(12)(13)(14). However, because of the lack of genetic studies on Pik3c3 in mammals, it is not clear how inactivating PIK3C3 in neurons in vivo differentially affects endosomal versus autophagic processes.Neurons are highly susceptible to disruptions in both endocytic and autophagic pathways. Genetic mutations in ubiquitously expressed proteins regulating the endocytic (15,16) Lowe (23,...

Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function

“…Acute deletion of Vps34 in MEFs resulted in blocked autophagic degradation as assessed by various biochemical assays. Furthermore, large GFP-LC3 aggregates were observed in Vps34-null cells and tissues, similar to what was reported (10). Characterization of autophagy flux clearly indicated the accumulation of large-sized LC3 structures results from abrogated flux rather than induction of the onset of autophagy.…”

“…Its versatile molecular function has been attributed to its various binding partners and distinct subcellular localizations (31)(32)(33). Its importance is exemplified by the fact that germ-line loss of Vps34 leads to embryonic lethality (10). Although Vps34 has been shown to play an essential role in autophagy in yeast and Drosophila (34,35), its precise molecular function in mammalian autophagy remained elusive.…”

“…Although Vps34 has been shown to play an essential role in autophagy in yeast and Drosophila (34,35), its precise molecular function in mammalian autophagy remained elusive. A recent loss-of-function study in mouse sensory neurons suggested that functional autophagy flux still exists upon Vps34 deletion (10). Using a conditional loss-offunction mouse model of Vps34, we have examined the role of Vps34 in MEFs, liver, and heart.…”

“…However, bevause of the lack of specificities of these PI3K inhibitors, the precise role of Vps34 in autophagy remains unclear. It was only recently that the physiological role of mammalian Vps34 has been investigated in mouse gene knockout studies (10,11). Surprisingly, it was reported that although genetic deletion of Vps34 in sensory neurons leads to disruption of the endocytic pathway, the autophagic pathway is still intact in these cells, raising the question as to whether Vps34 is necessary for autophagy in mammals (10).…”

Vps15 is required for stress induced and developmentally triggered autophagy and salivary gland protein secretion in Drosophila

“…This contrasts with the reported defects of autophagy initiation in Vps34-deficient liver and heart 31 but agrees with work showing that Vps34 deletion in sensory neurons leaves the autophagy pathway intact. 32 Indeed, our work and others have shown that regulators of autophagy, such as Atg7, may have different regulatory mechanisms in distinct cell types within an animal and that Figure 4 Vps15 is required for developmentally programmed salivary gland degradation. (a and aʹ) Control animals lacking the GAL4 driver (Vps15 IR /+), n = 13, and those with salivary gland-specific knockdown of Vps15 (fkh-GAL4; Vps15 IR ), n = 16, were analyzed by histology for the presence of salivary gland fragments 24 h APF.…”