A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis

Abstract

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.

WAC Regulates mTOR Activity by Acting as an Adaptor for the TTT and Pontin/Reptin Complexes

“…We generated mosaic eyes that contain homozygous mutant cells using the FLP-FRT system (Xu and Rubin, 1993) and recorded electroretinograms (ERGs) from mutant clones (Jaiswal et al, 2015; Sandoval et al, 2014; Tian et al, 2015; Wang et al, 2014; Xiong et al, 2012; Zhang et al, 2013). The ERG response consists of a depolarization, which is a measure of the strength of phototransduction, and the on and off transients, which gauge synaptic transmission.…”

TMEM175 Is an Organelle K+ Channel Regulating Lysosomal Function

“…Native lysosomal Ca 2+ currents are small or undetectable (data not shown); the ion's contribution to DJ is perhaps mainly through its regulation of other conductances. For the Ca 2+ permeability, Ca V proteins normally found on the plasma membrane are also functional in lysosomes (Tian et al, 2015). In addition, overexpressing several ion channels such as TRPMLs and P2X4 generates lysosomal Ca 2+ -permeable channels and leads to Ca 2+ release (Dong et al, 2010;LaPlante et al, 2004).…”

“…Hyperpolarizing DJ can increase Ca 2+ release from voltage-independent Ca 2+ -permeable channels such as TRPs and P2X4 Xu and Ren, 2015). Hypothetically, depolarizing DJ can also activate lysosomal Ca 2+ release from Ca V s (Tian et al, 2015). Finally, DJ may influence the insertion of membrane proteins with charged residues and regulate the fusion between organelles, either directly by acting on charged lipids and protein residues or indirectly by stimulating Ca 2+ release to promote organelle fusion.…”

Regulation of lysosomal ion homeostasis by channels and transporters

“…A recent study, however, unexpectedly found the presence of the pore-forming α1A subunits of voltage-gated Ca 2+ channels in lysosomes of both fruit flies and mice (Tian et al, 2015). Mutations of the α1A subunits, as well as that of the auxiliary α2δ2 subunits of the voltage-gated Ca 2+ channels, disrupted autophagy and the fusion between lysosomes and endosomes and/or autophagic vacuoles in fly photoreceptor cells and mouse cerebellar neurons (Tian et al, 2015).…”