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.

HSPB1【热休克蛋白家族B（小）成员1（heat shock protein family B (small) member 1）】与HSPB8是维持神经元蛋白质稳态的核心分子伴侣，可通过抑制蛋白质聚集发挥功能。突变型HSPB1和HSPB8主要损伤外周神经元，引发2型轴索性夏科-马里-唐神经病（CMT2）。巨自噬/自噬（Macroautophagy/autophagy）是突变型HSPB1和HSPB8导致神经元功能障碍的共同机制。在2F型夏科-马里-唐神经病患者来源的诱导多能干细胞分化运动神经元中，突变型HSPB1可降低自噬体形成效率。同样，模拟2L型CMT的HSPB8^K141N敲入小鼠模型会出现轴突变性与肌肉萎缩，且伴有SQSTM1/p62阳性沉积。本研究发现，从HSPB8^K141N/绿色荧光蛋白（green fluorescent protein, GFP）-LC3小鼠模型中分离的小鼠胚胎成纤维细胞，在雷帕霉素靶蛋白（MTOR）抑制条件下，自噬体生成能力显著下降。为修复HSPB1和HSPB8模型中的自噬缺陷，本研究通过高通量自噬体定量分析，对Spectrum Collection再利用化合物库进行筛选，以寻找能在经典MTOR抑制基础上进一步增强自噬活性的分子。随后，我们在由HSPB1^P182L和HSPB8^K141N患者来源诱导多能干细胞分化得到的运动神经元中验证了候选化合物的效果，重点检测了自噬诱导、神经突网络密度、轴突变性及线粒体形态四项指标。本研究鉴定出可特异性刺激HSPB8^K141N细胞中自噬体形成、且不影响自噬流的活性分子。其中两款最优先导化合物可诱导自噬并减轻轴突变性，进而促进2型CMT患者来源运动神经元的神经元网络成熟。基于上述发现，本次表型筛选结果显示，piplartine可同时修复HSPB1和HSPB8模型中的自噬缺陷，证实诱导自噬可作为治疗CMT神经病及其他伴侣蛋白病的有效策略。