Fig 2: Loss of Gba1b leads to GlcCer accumulation.(A) Immunofluorescent images of fly retina of the indicated genotypes and the conditions under which the flies were raised: antibody against GlcCer in green, whereas phalloidin labels rhabdomeres in magenta. (a and b) Flies kept in the dark show very little GlcCer in the ommatidia of y1 w* (basal level). (c and d) After 12 hours of light exposure, GlcCer is synthesized and accumulates in retina of y w flies (n ≥ 9). (e) A cartoon image illustrating the structure of a fly ommatidium. (B) (a, b, e, and f) GlcCer accumulation in the glia of y1 w* flies is reduced after 12 hours of inactivation of neurons in the darkness. (a to d) After 2 days of D/L cycles, similar to y w flies, GlcCer accumulates in the glia of y1 w*; Gba1bT2A-Gal4 flies. However, GlcCer accumulates more in the neurons of y1 w*; Gba1bT2A-Gal4 flies. (c, d, g, and h) The glial accumulation of GlcCer fails to be degraded upon exposure to 12 hours of darkness in retina of y1 w*; Gba1bT2A-Gal4 flies (n ≥ 9). (C) Loss of Gba1b leads to progressive neurodegeneration. Numbers of intact PRs per ommatidium in (B) and (D) were quantified. Error bars represent SEM (n ≥ 9); ***P < 0.001 and ****P < 0.0001. (D) (a, c, d, and f) GlcCer progressively accumulates in the retina of y1 w*; Gba1bT2A-Gal4 flies, which is highly enriched in the glial region. (b and e) CTSL (yellow) represents lysosomes. The number of lysosomes is increased in y1 w*; Gba1bT2A-Gal4 flies compared with y1 w* flies (n ≥ 9). (E) Quantification of relative GlcCer levels in (B) and (D). Error bars represent SEM (n ≥ 9); **P < 0.01, ***P < 0.001, and ****P < 0.0001. All flies that are tested in this experiment are phenotypically white.

Fig 3: Loss of Vps29 causes age-dependent, progressive lysosomal dysfunction in the brain.(A) In the absence of Vps29, glucosylceramide (anti-GlcCer, green) accumulates in the retina. Tissue is counterstained for actin (phalloidin, red) to highlight the photoreceptor rhabdomeres. Glucosylceramide signal was quantified from a 36.94 × 36.94 µm2 region encompassing ~10 ommatidia in 30-day-old animals (n = 10) raised in dark conditions, including GMR-w-RNAi/+; Vps291; GR/+ (controls) and GMR-w-RNAi/+; Vps291. Eye pigment was removed using RNAi against the w gene to reduce auto-fluorescence. A representative ommatidium (boxed) is shown at higher-power (right). Scale bars, 5 µm. (B) The uncleaved, CTSL proform is increased in aged animals lacking Vps29, consistent with diminished lysosomal proteolytic capacity. Western blots from adult fly head homogenates were probed for CTSL and Tubulin (loading control), and quantified based on n = 3 replicate experiments. See also Figure 6—figure supplement 1A. (C) Autophagic flux is reduced in aged animals lacking Vps29, and this phenotype is suppressed by reduction of Rab7. Western blots of adult head homogenates were probed for autophagic markers, including p62, Atg8, polyubiquitin (FK1), or Tubulin (loading control), and quantified based on n = 8 replicate experiments. See also Figure 6—figure supplement 1B,C. Statistical analysis based on Student’s t-test (A, B) and one-way ANOVA (C). All error bars denote SEM. n.s., not significant; *, p<0.05; **, p<0.01.

Fig 4: Arf1 myristoylation-dependent vesicle trafficking regulates CTSL transport.A. Western blot analysis of CTSL in Arf1 knockdown cells. Two different non-overlapping dsRNAs targeting Arf1 were used.B. Schematic of Arf1 function. Myristoylated Arf1 associates with Golgi membranes to recruit coat proteins for vesicle trafficking. COPI mediates retrograde transport from Golgi to ER, Clathrin mediates post-Golgi transport, COPII mediates anterograde transport from ER to Golgi.C. Western blot analysis of CTSL in cells with knockdown of COPI or clathrin subunits.D. Western blot analysis of CTSL in cells with knockdown of COPII subunits.

Fig 5: The JAK–STAT pathway regulates CG5966 and schlank expression in the fat body.a, c, qRT–PCR of CG5966 (a) and schlank (c) mRNA in the fat body of flies with or without ISC Upd3 or Pvf1 expression (esg>Upd3 or Pvf1) at day 8 (n = 3). b, d, CG5966 (b) and schlank (d) mRNA in the fat body of flies with or without fat-body Stat92E-act (constitutive-activated form of Stat92e) expression at day 8 (n = 3). e, g, CG5966 (e) and schlank (g) mRNA in the fat body of control, Yki flies, and Yki flies with fat-body hop depletion at day 6 (n = 3). f, h, CG5966 (f) and schlank (h) mRNA in the fat body of Yki flies with or without fat-body Stat92e depletion at day 6 (n = 3). i, Data from the ChIP-seq database indicating enrichment of Stat92e binding at the CG5966 and schlank promoter and gene regions. j, ChIP revealed the enrichment of HA-tagged Stat92E-act binding at the CG5966 and schlank promoter and gene regions, shown by fold changes relative to control IgG at day 8 (n = 3). Neg, negative control. k, Relative levels of whole-body ceramide levels of control, Yki flies, and Yki flies with fat-body hop or Stat92E depletion at day 6 (n > 3), data were retrieved from lipidomics analysis. l, Live imaging of lysosome and autophagic vesicles in Yki flies with or without fat-body hop or Stat92e depletion at day 7. m-q, Quantification of autophagic vesicle numbers (m) and sizes (n), lysosome numbers (o) and sizes (p), and autolysosome numbers (q) in l (n > 15). r, Western blot showing pro and matured forms of CtsL in control flies, Yki flies with or without fat-body hop or Stat92e depletion at day 7. Rhodamine was used as loading control. The same membrane was probed sequentially with Atg8 antibody in S4a, loading control is shared. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates not significant. Error bars indicate SDs. n indicates the number of biological replicates in each experiment.