Fig 1.
A. In response to a 1 s light pulse, cac alleles lack on transients at day 3 and day 33. B. Quantification of depolarization amplitudes (top) and on amplitudes (bottom). Data are presented as means ± standard error of the mean (SEM). C. The cac alleles identified failed to complement deficiencies Df(1)ED7417 and Df(1)BSC543. The lethality was rescued by 80 kb BAC clone CH321–60D21, narrowing the candidate region down to the area in the red box. We then crossed the cac alleles to existing alleles of cac and show that they fail to complement each other. D. Schematic representation of the molecular lesions of cac alleles. E. Lethal staging of cac alleles.
Fig 2.
Aged cac mutant photoreceptor terminals show morphological defects and accumulate late stage AVs.
A. TEM sections of 3- and 27-days-old photoreceptor terminals of flies. Terminals of 27-days-old flies show terminal expansion, loss of capitate projections (CPs) and active zones (AZs) and increased number of mitochondria per terminal. Scale bars, 500 nm. B. Inset from A representing the photoreceptor terminal boxed in white. Yellow arrow indicates double-membraned autophagosome, red indicates amphisome, and blue indicates two fusion-primed AVs. Scale bars, 500 nm. C-E. Quantification of CPs, AZs, and mitochondria per cartridge, respectively. Data are presented as means ± SEM. F. Quantification of the different AVs per cartridge. The mutant terminals accumulate late stage AVs and fusion-primed AVs (ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). Data are presented as means ± SEM. G. 30-days old fly eye brain complexes stained with neuronal marker Elav (blue) and poly-Ub antibody (red). The right panel is the enlarged images of the boxed regions at the left side. H. The level of p62 is increased in cacJ brain. I. Quantification of H. Data are presented as means ± SEM. (*: p < 0.05).
Fig 3.
Autophagy defects observed in cac mutants are not due to defects in neurotransmitter release, but due to defects in lysosomal fusion.
TEM sections of 1-day-old and 30-days-old photoreceptor terminals of flies raised in 12 h light/dark conditions. A, B, D, and E. stj and Vha100–1 (V0) mutant terminals accumulate AVs upon aging. G, H. n-Syb mutant photoreceptor terminals show increased SVs, but not a significant accumulation of AVs and fusion-primed AVs. J, K, M and N. Vamp7 and fab1 mutant photoreceptor terminals show the highest increase in AV accumulation. C, F, I, L, O. Quantification of the different AVs for each genotype (**: p < 0.01). Eyes from three animals for each genotype at each age were analyzed and the counted cartridge number were listed as “n = ” in the figure. Data are presented as means ± standard deviation (SD). Scale bars, 1 μm.
Fig 4.
Cacna1atg-la and Cacna2d2du-2J mice have autophagy defects.
A. Calbindin and hematoxylin staining of mice cerebella indicates that Cacna1atg-la mice have progressive PC and granule cell loss upon aging. Scale bar, 200 μm. B, D. Calbindin (green) and 4',6-diamidino-2-phenylindole (DAPI)(blue) staining of mice cerebellum indicates that there are swollen PC axons (arrows) in the granular layers of the Cacna1atg-la mice cerebellum at day 25 (B) and the Cacna2d2du-2J mice at day 50 (D). Scale bar, 20 μm. C, E. The TEM of the axons at the granular layers of the cerebellum showing that many PC axons in Cacna1atg-la and Cacna2d2du-2J mice are swollen and accumulated with numerous mitochondria, autophagosome/MVBs-like structures (red arrows), and various cytoplasmic vesicles or aberrant membrane structures, including stacks of cisternal membranes (blue arrows), and folded ER (yellow arrows). Scale bars for right two panels are 2 μm. Other scale bars are 500 nm. F, G. LC3II and p62 are slightly increased in 15-days-old Cacna1atg-la mice and 50-days-old Cacna2d2du-2J mice. b, d are quantification of a and c. Data are presented as means ± SEM. (*: p < 0.05).
Fig 5.
CACNA1A localizes to the lysosomes.
A. CACNA1A co-localizes with LAMP1 in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm. B. CACNA1A is present as punctae on the Vacuolin-1 enlarged LAMP1 positive lysosomes in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm. C. Quantification of LAMP1 positive organelles that are also CACNA1A positive. Data are presented as means ± SD. (ns: not significant). D. CACNA1A is concentrated in the lysosomal fractions isolated from mice cerebella with iodixanol gradient.
Fig 6.
Cacna1a mutant neurons display lysosomal fusion defects.
A. LysoTracker Red staining of the primary cerebella neurons from Cacna1atg-la mice and WT controls. The control cells show big bright punctae, while the mutant cells have much dimmer and smaller punctae. B, C and D. Quantification of LysoTracker relative intensity (“n” represents the counted cell number), LysoTracker positive punctae number and size per cell (“n” represents the counted image number). E. Lysosomal marker LAMP1 co-localizes with autophagosome marker LC3 in CTL but not Cacna1atg-la mutant cells. Scale bar, 20 μm. F. Quantification of LC3 and LAMP1 co-localization. Data are presented as means ± SD. (ns: not significant; ***: p < 0.001).
Fig 7.
The calcium channel activity of CACNA1A on the lysosome, not the plasma-membrane is required for lysosomal fusion.
A. DQ-BSA labeled late endosomes fuse with LAMP1 labeled lysosomes in CTL but not the mutant cells. Scale bar, 20 μm. B. Quantification of DQ-BSA and LAMP1 co-localization in CTL and Cacna1atg-la cells. Data are presented as means ± SD. (ns: not significant; ***: p < 0.001) C. Bepridil but not ω-agatoxin TK (ω-Aga TK) treatment reduces lysosomal fusion in cultured cerebellar cells. DQ-BSA (Green) labeled late endosomes co-localize with LAMP1 (Red) labeled lysosomes with/without ω-Aga TK (1 µM) or Bepridil (10 µM) treatment. Scale bar, 20 μm. D. Quantification of DQ-BSA and LAMP1 co-localization with ω-Aga TK or Bepridil treatment. Data are presented as means ± SD. (ns: not significant; **: p < 0.001).
Fig 8.
Cac is required on the lysosomes in neurons to promote lysosomal fusion.
Cac and its accessory subunits are present on the lysosomes. Under normal conditions of activity requiring basal levels of autophagy, Cac allows for calcium efflux from endo/lysosomes to provide for SNARE-mediated fusion of autophagic vacuoles to lysosomes and subsequently efficient degradation of unwanted cellular material and to maintain neuronal homeostasis. In the absence of Cac, there is a block in the autophagic flux and lysosomal fusion, leading to an accumulation of fusion-primed autophagic vacuoles. This subsequently results in an accumulation of improperly formed proteins or damaged organelles, such as mitochondria in the neurons, and causes neurodegeneration.