Extended Data Fig. 4: CMA contributes to lysosomal degradation of proteins important for synaptic neurotransmission.
Comparative proteomics of whole-brain ex vivo slices from 9 months old male and female control (Ctrl) and L2AKO mice incubated with or without lysosomal protease inhibitors (20 mM ammonium chloride and 400 μM leupeptin, NL) for 20 h. a, STRING plots of pathways enriched in proteins no longer degraded in lysosomes in the L2AKO female or male mice brain compared to controls. b, IPA analysis pathways with significant enrichment in proteins undergoing LAMP2A-dependent lysosomal degradation (CMA substrates) in both females and males. c,d, IPA analysis for cellular pathways with enrichment of proteins displaying CMA degradation in females (c) or males (d). e, Heat maps showing fold change of pre-synaptic protein levels in aggregates (pellet) compared to soluble cytosol in CKL2AKO male mice brain relative to Ctrl. n = 3 mice per group. f, Synapsin-I (Syn1), Synapsin-II (Syn2) and Synaptotagmin-1 (Syt1) mRNA levels by qRT-PCR of female and male Ctrl and L2AKO mice hippocampus. g, Representative immunoblots (left) for p62 in whole-brain ex vivo slices from 9 months old female and male Ctrl and L2AKO mice incubated with lysosomal protease inhibitors (20 mM ammonium chloride and 400 μM leupeptin, NL) for 20 h. Lysosomal degradation, calculated as the increase in protein levels upon NL addition, is shown on right. Ponceau staining is shown as loading control for the immunoblots. h, Chymotrypsin-like activity of the 20S and 26S proteasome in female and male Ctrl and L2AKO mice whole-brain expressed as area under the curve. n = [5] (f); [6] (g), [4] (h) mice per group. No statistical significance detected for any of the indicated measurements using wo-tailed unpaired Student’s t-test. All GO terms are statistically enriched with P < 0.05. Data are presented as mean values ±SEM. P values in b–d were calculated using the hypergeometric test.
