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. 2014 Nov 24;207(4):453-62.
doi: 10.1083/jcb.201406026.

Reduced Synaptic Vesicle Protein Degradation at Lysosomes Curbs TBC1D24/sky-induced Neurodegeneration

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Free PMC article

Reduced Synaptic Vesicle Protein Degradation at Lysosomes Curbs TBC1D24/sky-induced Neurodegeneration

Ana Clara Fernandes et al. J Cell Biol. .
Free PMC article

Abstract

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans.

Figures

Figure 1.
Figure 1.
Heterozygous loss of dor/VPS18 suppresses sky/TBC1D24-induced neurodegeneration. (A) Schematic representation of ERG recordings to isolate sky suppressors. Genotypes in the eye and the rest of the body. (B) Quantification of the sum of the on and off transients in ERG recordings (arrowheads in C) of the screened flies. Amplitudes of sky and dor/+; sky2 are indicated. (n = 3–18.) Error bars: SEM. One-way analysis of variance (ANOVA; post hoc Dunnett’s test). (C) Mean ERGs recorded from 5–12 flies with the following genotypes in the eyes: FRT40A (control), sky2, dor36/+; sky2, dor30/+; sky2, and dor35/+; sky2. Note the partial rescue of on and off transient amplitude defect in sky2 mutants when dor is heterozygous. (D) Western blot to assess Dor levels in homozygous dor mutant larvae using anti-Dor and anti–α-Tubulin (Tub) antibodies. (n = 3.) (E) Western blot of dor mutants, using anti–insect Cathepsin L (CatL) and anti-Acon. (n = 3.) (F and G) Retina sections of flies with FRT40A (control), sky2, and dor+/−; sky2 mutant eyes not exposed to light (0 d) or exposed for 7 (only shown in G) or 10 d to constant light (10 d) and quantification of the area of vacuoles normalized to retina area (G; n = 5–11 sections). Arrowheads indicate vacuoles, and asterisks indicate red pigment clones in the eyes (result of mitotic recombination—see Materials and methods). Bars, 50 µm. Error bars: SEM. One-way ANOVA (post hoc Tukey’s test): ***, P < 0.001.
Figure 2.
Figure 2.
Reduced endolysosomal trafficking suppresses the increased synaptic transmission defects in sky mutants. (A and B) Traces (A) and quantification (B) of EJC amplitudes recorded from larval fillets in HL3 with 0.5 mM CaCl2 in FRT40A controls, FRT19A/+; sky1/2 mutants, dor35/+; sky1/2, dor36/+; sky1/2, and dor35/+; sky1/2; daGAL4 UAS-dor (dor+), expressing Dor ubiquitously (n = 5–12). (C and D) Images of FM 1–43 labeling in NMJs on muscle 6 and 7 (C) and quantification (D) of patches of FM 1–43 (arrowheads) after 1-min 90 mM KCl stimulation (n = 12–20). (E and F) Traces (E) and quantification (F) of EJCs recorded from larval fillets in HL3 with 0.5 mM CaCl2 in FRT40A controls (n = 7), sky1/2 mutants (n = 8), sky1/2; UAS-Rab7RNAi nSybGAL4 (Rab7RNAi; n = 20), and sky1/2; UAS-dVPS39RNAi nSybGAL4 (dVPS39RNAi; n = 12), expressing the RNAi constructs in all neurons. (G and H) Images of FM 1–43 labeling in NMJs on muscle 6 and 7 (G) and quantification (H) of patches of FM 1–43 (arrowheads) after 1-min 90 mM KCl stimulation (n = 12–16). Bars, 5 µm. Error bars: SEM. One-way ANOVA (post hoc Tukey’s and Dunnett’s test): *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 3.
Figure 3.
Loss of dor function causes defects in vesicle fusion efficiency. (A and B) EJCs (A) recorded in HL-3 with 0.5 mM CaCl2 from FRT19A control (white), dor35 and dor36 (orange), dor35 and dor36 expressing wild-type Dor ubiquitously (daGAL4; green), or presynaptically at the NMJ (vGlutGAL4, purple). (B) Quantification of the mean EJC amplitude (n = 8–17). (C–E) mEJCs, cumulative probability mEJC histograms (C and E), and quantification of mean mEJC amplitudes (D; n = 7–15). (F) Quantal content; EJC amplitude/mEJC amplitude (see also A; n = 8–17). For B, D, and F, error bars show SEM, with one-way ANOVA (post hoc Dunnett’s test) compared with control: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G and H) Images of FM 1–43 labeling after 1 min of 90 mM KCl stimulation at larval NMJs (G) and quantification of labeling intensity (H; n = 12–30). Bar, 5 µm. Error bars: SEM. t test: **, P < 0.01; ***, P < 0.001. (I and J) Raw data traces of EJC recordings made in HL-3 with 5 mM calcium and stimulated at 60 Hz in FRT19A controls, dor35, and dor36 mutants (I) and quantification of the cumulative released quantal content in such recordings versus time (J). The y intercept of the slope of the trend line (dotted lines) at steady state (points 400–600 ms) provides a measure of the mean RRP size (indicated on the y axis: control: 1,041 ± 109 quanta; dor36: 745 ± 56 quanta; dor36: 705 ± 54 quanta; n = 6–8). Error bars: SEM.
Figure 4.
Figure 4.
Synaptic vesicle protein timers reveal slower synaptic vesicle protein turnover in dor mutants and faster turnover in sky mutants. (A) Schematic of the FT nSyb construct (FT::nSyb). (B) FT::nSyb expression using vGlutGAL4 showing the red and blue forms of the timer at the larval NMJ and the ventral nerve cord (VNC). The blue fluorescence is higher than the red in the VNC. (C) NMJ boutons expressing the FT::nSyb using vGlutGAL4 labeled with antibodies to dsRed (for the FT) and to CSP, a synaptic vesicle protein. (D) FT::nSyb localization (red form) at control boutons and shits1 mutant boutons stimulated at ∼34°C for 10 min using 90 mM KCl. (E and F) Images of FM 1–43 (E) and quantification of labeling intensity (F) at boutons in controls and larva expressing the FT::nSyb using vGlutGAL4. (G) Schematic representation of the distribution of the red (old) and blue (young) FT::nSyb in control (left) and dor (middle) or sky (right) mutants. (H and I) Images (H) and quantification (I) of the ratio of red over blue fluorescence intensities at synaptic boutons shown using the indicated lookup table. FT::nSyb was expressed using vGlutGAL4 in controls, in animals expressing Rab35CA, in dor36, dor35 and sky1/2 mutants as well as in dor35/+; sky1/2 mutants (n = 20–50). Bars: (B [NMJ], D, and E) 5 µm; (B, VNC) 50 µm; (H) 2 µm. Error bars: SEM. In I, all statistical comparisons are to dor35 or dor36, except for the double mutant to sky1/2. One-way ANOVA (post hoc Tukey’s test): ***, P < 0.0001.

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