Sisto Angela, van Wermeskerken Tamira, Pancher Michael, Gatto Pamela, Asselbergh Bob, Assunção Carreira Ágata Sofia, De Winter Vicky, Adami Valentina, Provenzani Alessandro, Timmerman Vincent
Peripheral Neuropathy Research Group, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
Laboratory of Neuromuscular Pathology, Institute Born Bunge, Faculty of Medicine, University of Antwerp, Antwerp, Belgium.
Autophagy. 2025 May;21(5):1116-1143. doi: 10.1080/15548627.2024.2439649. Epub 2024 Dec 27.
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 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/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 and HSPB8 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 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.
热休克蛋白家族B（小分子）成员1（HSPB1）和热休克蛋白B8（HSPB8）是神经元蛋白质稳态的重要分子伴侣，因为它们可防止蛋白质聚集。突变的HSPB1和HSPB8主要损害周围神经元，导致轴索性夏科-马里-图斯氏神经病（CMT2）。巨自噬/自噬是HSPB1和HSPB8突变导致神经元功能障碍的共同机制。在2F型CMT患者的突变HSPB1诱导的多能干细胞衍生的运动神经元中，自噬体形成减少。同样，模拟2L型CMT的HSPB8基因敲入小鼠模型表现出轴突变性和肌肉萎缩，并伴有SQSTM1/p62阳性沉积物。我们在此表明，从HSPB8/绿色荧光蛋白（GFP）-LC3模型分离的小鼠胚胎成纤维细胞在mTOR抑制条件下自噬体产生减少。为了纠正HSPB1和HSPB8模型中的自噬缺陷，我们通过高通量自噬体定量筛选了重新利用的光谱收集文库，以寻找能够将自噬活性提高到经典mTOR抑制水平以上的分子。对通过HSPB1和HSPB8患者来源的诱导多能干细胞分化获得的运动神经元进行了命中化合物验证，重点关注自噬诱导以及神经突网络密度、轴突变性和线粒体形态。我们鉴定出了在不影响自噬通量的情况下特异性刺激HSPB8细胞中自噬体形成的分子。两种顶级先导化合物诱导自噬并减少轴突变性，从而促进2型CMT患者来源的运动神经元中神经网络成熟。基于这些发现，表型筛选显示，胡椒碱可挽救HSPB1和HSPB8模型中的自噬缺陷，证明自噬诱导是CMT神经病和其他伴侣蛋白病的有效治疗策略。
