Trehalose induces autophagy via lysosomal-mediated TFEB activation in models of motoneuron degeneration.

ABSTRACT

Macroautophagy/autophagy, a defense mechanism against aberrant stresses, in neurons counteracts aggregate-prone misfolded protein toxicity. Autophagy induction might be beneficial in neurodegenerative diseases (NDs). The natural compound trehalose promotes autophagy via TFEB (transcription factor EB), ameliorating disease phenotype in multiple ND models, but its mechanism is still obscure. We demonstrated that trehalose regulates autophagy by inducing rapid and transient lysosomal enlargement and membrane permeabilization (LMP). This effect correlated with the calcium-dependent phosphatase PPP3/calcineurin activation, TFEB dephosphorylation and nuclear translocation. Trehalose upregulated genes for the TFEB target and regulator Ppargc1a, lysosomal hydrolases and membrane proteins (Ctsb, Gla, Lamp2a, Mcoln1, Tpp1) and several autophagy-related components (Becn1, Atg10, Atg12, Sqstm1/p62, Map1lc3b, Hspb8 and Bag3) mostly in a PPP3- and TFEB-dependent manner. TFEB silencing counteracted the trehalose pro-degradative activity on misfolded protein causative of motoneuron diseases. Similar effects were exerted by trehalase-resistant trehalose analogs, melibiose and lactulose. Thus, limited lysosomal damage might induce autophagy, perhaps as a compensatory mechanism, a process that is beneficial to counteract neurodegeneration. Abbreviations ALS amyotrophic lateral sclerosis; AR androgen receptor; ATG autophagy related; AV autophagic vacuole; BAG3 BCL2-associated athanogene 3; BECN1 beclin 1, autophagy related; CASA chaperone-assisted selective autophagy; CTSB cathepsin b; DAPI 4',6-diamidino-2-phenylindole; DMEM Dulbecco's modified Eagle's medium; EGFP enhanced green fluorescent protein; fALS, familial amyotrophic lateral sclerosis; FRA filter retardation assay; GAPDH glyceraldehyde-3-phosphate dehydrogenase; GLA galactosidase, alpha; HD Huntington disease; hIPSCs human induced pluripotent stem cells; HSPA8 heat shock protein A8; HSPB8 heat shock protein B8; IF immunofluorescence analysis; LAMP1 lysosomal-associated membrane protein 1; LAMP2A lysosomal-associated membrane protein 2A; LGALS3 lectin, galactose binding, soluble 3; LLOMe L-leucyl-L-leucine methyl ester; LMP lysosomal membrane permeabilization; Lys lysosomes; MAP1LC3B microtubule-associated protein 1 light chain 3 beta; MCOLN1 mucolipin 1; mRNA messenger RNA; MTOR mechanistic target of rapamycin kinase; NDs neurodegenerative diseases; NSC34 neuroblastoma x spinal cord 34; PBS phosphate-buffered saline; PD Parkinson disease; polyQ polyglutamine; PPARGC1A peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PPP3CB protein phosphatase 3, catalytic subunit, beta isoform; RT-qPCR real-time quantitative polymerase chain reaction; SBMA spinal and bulbar muscular atrophy; SCAs spinocerebellar ataxias; siRNA small interfering RNA; SLC2A8 solute carrier family 2, (facilitated glucose transporter), member 8; smNPCs small molecules neural progenitors cells; SOD1 superoxide dismutase 1; SQSTM1/p62 sequestosome 1; STED stimulated emission depletion; STUB1 STIP1 homology and U-box containing protein 1; TARDBP/TDP-43 TAR DNA binding protein; TFEB transcription factor EB; TPP1 tripeptidyl peptidase I; TREH trehalase (brush-border membrane glycoprotein); WB western blotting; ZKSCAN3 zinc finger with KRAB and SCAN domains 3.