Heat-shock chaperone HSPB1 mitigates poly-glycine-induced neurodegeneration via restoration of autophagic flux

The CGG repeat expansions in the 5’-UTR regions of certain genes have been implicated in various neurodegenerative and muscular disorders. However, the underlying pathogenic mechanisms are not well understood. In this study, we explore the role of the small molecular chaperone HSPB1 in counteracting neurodegeneration induced by poly-glycine (poly-G) aggregates. Employing a reporter system, we demonstrate that CGG repeat expansions within the 5’-UTR of the <i>GIPC1</i> gene produce poly-G proteins, by repeat-associated non-AUG (RAN) translation. Through proximity labeling and subsequent mass spectrometry analysis, we characterize the composition of poly-G insoluble aggregates and reveal that these aggregates sequester key macroautophagy/autophagy receptors, SQSTM1/p62 and TOLLIP. This sequestration disrupts MAP1LC3/LC3 recruitment and impairs autophagosome formation, thereby compromising the autophagic pathway. Importantly, we show that HSPB1 facilitates the dissociation of these receptors from poly-G aggregates and consequently restores autophagic function. Overexpressing HSPB1 alleviates poly-G-induced neurodegeneration in mouse models. Taken together, these findings highlight a mechanistic basis for the neuroprotective effects of HSPB1 and suggest its potential as a therapeutic target in treating poly-G-associated neurodegenerative diseases. <b>Abbreviations</b>: AD: Alzheimer disease; AIF1/Iba1: allograft inflammatory factor 1; Baf A₁: bafilomycin A₁; BFP: blue fluorescent protein; CQ: chloroquine; EIF2A/eIF-2α: eukaryotic translation initiation factor 2A; FRAP: fluorescence recovery after photobleaching; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; HSPB1: heat shock protein family B (small) member 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; NOTCH2NLC: notch 2 N-terminal like C; PD: Parkinson disease; PFA: paraformaldehyde; poly-A: poly-alanine; poly-G: poly-glycine; poly-R: poly-arginine; RAN translation: repeat-associated non-AUG translation; RBFOX3/NeuN: RNA binding fox-1 homolog 3; STED: stimulated emission depletion; TARDBP/TDP-43: TAR DNA binding protein; TG: thapsigargin; TOLLIP: toll interacting protein