Autophagy induction by piplartine ameliorates axonal degeneration caused by mutant HSPB1 and HSPB8 in Charcot-Marie-Tooth type 2 neuropathies.

HSPB1 [heat shock protein family B (small) member 1] and HSPB8 are essential molecular chaperones for neuronal proteostasis, as they prevent protein aggregation. Mutant HSPB1 and HSPB8 primarily harm peripheral neurons, resulting in axonal Charcot-Marie-Tooth neuropathies (CMT2). Macroautophagy/autophagy is a shared mechanism by which HSPB1 and HSPB8 mutations cause neuronal dysfunction. Autophagosome formation is reduced in mutant HSPB1-induced pluripotent stem-cell-derived motor neurons from CMT type 2F patients. Likewise, the HSPB8^K141N knockin mouse model, mimicking CMT type 2 L, exhibits axonal degeneration and muscle atrophy, with SQSTM1/p62-positive deposits. We show here that mouse embryonic fibroblasts isolated from a HSPB8^K141N/green fluorescent protein (GFP)-LC3 model have diminished autophagosome production under conditions of MTOR inhibition. To correct the autophagic deficits in the HSPB1 and HSPB8 models, we screened by high-throughput autophagosome quantification the repurposing Spectrum Collection library for molecules that could boost the autophagic activity above the canonical MTOR inhibition. Hit compounds were validated on motor neurons obtained by differentiation of HSPB1^P182L and HSPB8^K141N patient-derived induced pluripotent stem cells, focusing on autophagy induction as well as neurite network density, axonal degeneration, and mitochondrial morphology. We identified molecules that specifically stimulate autophagosome formation in the HSPB8^K141N cells, without affecting autophagy flux. Two top lead compounds induced autophagy and reduced axonal degeneration, thus promoting neuronal network maturation in the CMT2 patient-derived motor neurons. Based on these findings, the phenotypical screen revealed that piplartine rescued autophagy deficiencies in both the HSPB1 and HSPB8 models, demonstrating autophagy induction as an effective therapeutic strategy for CMT neuropathies and other chaperonopathies.