Zocchi Riccardo, Bellacchio Emanuele, Piccione Michela, Scardigli Raffaella, D'Oria Valentina, Petrini Stefania, Baranano Kristin, Bertini Enrico, Sferra Antonella
Unit of Neuromuscular Disorders, Translational Pediatrics and Clinical Genetics, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.
Molecular Genetics and Functional Genomics Research Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.
Front Cell Neurosci. 2023 Jun 23;17:1162363. doi: 10.3389/fncel.2023.1162363. eCollection 2023.
Microtubules are dynamic cytoskeletal structures involved in several cellular functions, such as intracellular trafficking, cell division and motility. More than other cell types, neurons rely on the proper functioning of microtubules to conduct their activities and achieve complex morphologies. Pathogenic variants in genes encoding for α and β-tubulins, the structural subunits of microtubules, give rise to a wide class of neurological disorders collectively known as "tubulinopathies" and mainly involving a wide and overlapping range of brain malformations resulting from defective neuronal proliferation, migration, differentiation and axon guidance. Although tubulin mutations have been classically linked to neurodevelopmental defects, growing evidence demonstrates that perturbations of tubulin functions and activities may also drive neurodegeneration. In this study, we causally link the previously unreported missense mutation p.I384N in TUBA1A, one of the neuron-specific α-tubulin isotype I, to a neurodegenerative disorder characterized by progressive spastic paraplegia and ataxia. We demonstrate that, in contrast to the p.R402H substitution, which is one of the most recurrent TUBA1A pathogenic variants associated to lissencephaly, the present mutation impairs TUBA1A stability, reducing the abundance of TUBA1A available in the cell and preventing its incorporation into microtubules. We also show that the isoleucine at position 384 is an amino acid residue, which is critical for α-tubulin stability, since the introduction of the p.I384N substitution in three different tubulin paralogs reduces their protein level and assembly into microtubules, increasing their propensity to aggregation. Moreover, we demonstrate that the inhibition of the proteasome degradative systems increases the protein levels of TUBA1A mutant, promoting the formation of tubulin aggregates that, as their size increases, coalesce into inclusions that precipitate within the insoluble cellular fraction. Overall, our data describe a novel pathogenic effect of p.I384N mutation that differs from the previously described substitutions in , and expand both phenotypic and mutational spectrum related to this gene.
微管是参与多种细胞功能的动态细胞骨架结构，如细胞内运输、细胞分裂和运动。与其他细胞类型相比，神经元更依赖微管的正常功能来进行其活动并实现复杂的形态。编码微管结构亚基α和β微管蛋白的基因中的致病性变异会引发一大类统称为“微管蛋白病”的神经疾病，主要涉及由神经元增殖、迁移、分化和轴突导向缺陷导致的广泛且重叠的脑畸形。尽管微管蛋白突变传统上与神经发育缺陷有关，但越来越多的证据表明，微管蛋白功能和活性的扰动也可能驱动神经退行性变。在本研究中，我们将神经元特异性α微管蛋白同种型I之一TUBA1A中先前未报道的错义突变p.I384N与一种以进行性痉挛性截瘫和共济失调为特征的神经退行性疾病建立了因果联系。我们证明，与与无脑回畸形相关的最常见的TUBA1A致病性变异之一p.R402H替代不同，当前突变损害了TUBA1A的稳定性，降低了细胞中可用的TUBA1A丰度，并阻止其掺入微管。我们还表明，384位的异亮氨酸是一个对α微管蛋白稳定性至关重要的氨基酸残基，因为在三种不同的微管蛋白旁系同源物中引入p.I384N替代会降低它们的蛋白质水平并减少其组装入微管，增加它们的聚集倾向。此外，我们证明蛋白酶体降解系统的抑制会增加TUBA1A突变体的蛋白质水平，促进微管蛋白聚集体的形成，随着聚集体尺寸的增加，它们会合并成包涵体并沉淀在不溶性细胞部分中。总体而言，我们的数据描述了p.I384N突变与先前描述的替代不同的新致病效应，并扩展了与该基因相关的表型和突变谱。
