【研究背景】

     遗传性痉挛性截瘫（Hereditary Spastic Paraplegia, HSP）是一组具有高度临床和遗传异质性的中枢神经系统退行性疾病，以缓慢进行性下肢痉挛及皮质脊髓束变性为核心特征。目前已鉴定超80个HSP致病基因，其中SPAST（SPG4）是最常见的常染色体显性遗传致病基因，占单纯型HSP病例的40%以上。SPAST编码的spastin蛋白通过调控微管动态平衡维持神经元轴突运输功能，其突变（如无义、移码及剪接异常）可通过单倍剂量不足或显性负效应引发轴突退行性病变。然而，内含子区非经典剪接位点突变的致病机制尚不明确，且SPAST缺陷对神经元早期发育及信号通路的调控作用仍需系统解析。

【研究目的】

     本研究旨在构建中国HSP患者的基因突变谱，揭示基因型与表型的关联性；明确SPAST c.1004+5G>A突变导致RNA剪接异常的分子路径，验证其致病性；通过斑马鱼、hiPSCs诱导脊髓运动神经元及HEK293T SPAST-KO细胞模型，揭示SPAST缺陷对轴突发育、微管及细胞周期的影响。阐明SPAST缺陷通过MAPK通路失活与微管网络紊乱协同调控轴突退行及增殖抑制的级联反应，为HSP的精准诊断、预后评估及靶向治疗提供实验依据。

【实验方法】

1．通过全外显子组测序，系统筛查HSP患者的致病基因突变，结合家系共分离分析及ACMG指南评估突变致病性，一代测序验证关键位点。

2．通过琼脂糖凝胶电泳及Sanger测序鉴定SPAST c.1004+5G>A突变导致异常剪接产物，并进行保守性分析。

3．分别构建野生型和突变型SPAST质粒，转染至HeLa和HEK293T细胞中，通过Western blot和免疫荧光分析蛋白表达及亚细胞定位。

4．环己酰亚胺（CHX）追踪实验检测野生型与突变型spastin蛋白的半衰期，评估无义介导mRNA降解（NMD）逃逸效应。

5．设计靶向斑马鱼spast基因剪接位点的吗啉寡核苷酸（MO），显微注射至Tg(hb9:eGFP)转基因胚胎，通过qPCR验证敲降效率；观察运动神经元轴突发育（荧光成像）、运动行为（轨迹追踪及加速度分析）及脊髓超微结构变化。

6．利用 shRNA 慢病毒转染的方式构建 SPAST基因敲减的iPSC模型,并通过多阶段定向分化生成脊髓运动神经元，qPCR和Western blot技术检测敲降效率；动态观察不同阶段的生长发育情况，免疫荧光染色（Map2、Hb9标记）评估轴突生长、分支复杂度及胞体形态变化。

7．利用CRISPR/Cas9技术构建斑马鱼spast^+/-稳定品系，成功搭建体内研究平台。

8．利用CRISPR/Cas9技术构建HEK293T稳定敲除细胞系（SPAST-KO），EdU流式细胞术检测增殖能力，碘化丙啶（PI）染色分析细胞周期分布。转录组测序筛选差异表达基因，KEGG富集分析揭示MAPK通路异常，Western blot验证ERK/JNK总蛋白表达水平。

【研究结果】

1．临床队列与突变鉴定，SPAST (SPG4)，NIPA1 (SPG6)，SPG7，KIF5A (SPG10)，KIF1A (SPG30)，REEP1 (SPG31)，PNPLA6 (SPG39)，IBA57 (SPG74)，和UBAP1 (SPG80)具有高度的临床和表型异质性。

2．在16例HSP患者中，SPAST基因突变占比最高（38%），包括错义突变（c.212T>A，c.64C>G）、无义突变（c.716T>A）、移码突变（c.1417_1418del）、复合杂合突变（c.1027G>C / c.1267G>C）及内含子剪接突变（c.1004+5G>A）。

3．SPAST c.1004+5G>A突变首次报道，家系共分离分析显示其与疾病表型完全共分离，SPRS评分随年龄增长显著升高，家系共分离及RNA剪接验证显示其导致外显子6跳跃，生成截短spastin蛋白（p.Leu295*），可逃逸无义介导mRNA降解并异常累积。

4．突变型spastin蛋白水平表达增高，并在核周形成聚集沉积，与微管共定位，干扰微管动态平衡。CHX实验证实突变蛋白半衰期延长（>12小时），稳定性显著增强。

5．MO spast敲降斑马鱼呈现轴突寻路错误、分支紊乱及脊髓运动神经元荧光强度降低，运动轨迹受限，加速度异常，与人类 SAPST基因突变引起的痉挛性截瘫临床症状相似。

6．iPSC诱导的SPAST敲降脊髓运动神经元，在分化早期（Day 12）即出现轴突生长迟缓，成熟期（Day 27）胞体肥大、分支复杂度降低，免疫荧光显示轴突缺失与树突微管结构紊乱，提示微管动态失衡干扰神经突触发育。

7．SPAST基因敲除诱导G2/M期阻滞，EdU阳性细胞比例下降，抑制细胞增殖，转录组测序揭示MAPK通路（ERK/JNK）显著抑制，下游基因CCNB1、VIM表达下调，协同微管动态失衡抑制增殖，SPAST-KO组ERK/JNK总蛋白表达水平显著降低。

【研究结论】

本研究首次在中国HSP队列中鉴定出SPAST c.1004+5G>A突变，明确其通过“外显子跳跃-截短蛋白累积-微管动态失衡”三级级联反应致病，为SPG4型HSP的基因诊断提供新依据。多模型联用揭示病理机制：斑马鱼敲减模型复现轴突发育异常及运动功能障碍，精准模拟人类表型；hiPSCs神经元模型揭示SPAST缺陷导致脊髓运动神经元发育障碍的时空机制，提示微管动态失衡干扰神经突触发育；SPAST-KO细胞阐明MAPK通路失活与细胞周期阻滞的协同效应。创新性构建CRISPR/Cas9介导的斑马鱼spast^+/-稳定品系及iPSC诱导神经元模型，为SPG4机制研究与药物筛选提供多层次工具。

    转化医学意义：靶向RNA剪接校正（如反义寡核苷酸）或MAPK通路激活可能成为SPG4的潜在治疗策略，为临床精准干预奠定理论基础

【Background】

Hereditary Spastic Paraplegia (HSP) represents a spectrum of central nervous system neurodegenerative conditions exhibiting both genetic and clinical diversity. These disorders manifest as progressive spastic weakness in the lower extremities accompanied by axonal degeneration within corticospinal pathways. Currently, over 80 HSP pathogenic genes and 100 pathogenic mutations have been identified. Among them, SPAST (SPG4) is the most common autosomal dominant pathogenic gene, accounting for more than 40% of pure HSP cases. The spastin protein encoded by SPAST maintains neuronal axonal transport by regulating microtubule dynamics. Mutations (e.g., nonsense, frameshift, and splicing abnormalities) can cause axonal degenerative lesions through haploinsufficiency or dominant-negative effects. However, the pathogenic mechanisms of non-canonical splice-site mutations in intronic regions remain unclear, and the regulatory roles of SPAST deficiency in early neuronal development and signaling pathways require systematic investigation.

【Objectives】

This study aimed to: Construct the genetic mutation spectrum of Chinese HSP patients and reveal genotype-phenotype correlations. Clarify the molecular mechanism by which the SPAST c.1004+5G>A mutation causes abnormal RNA splicing and validate its pathogenicity. Investigate the effects of SPAST deficiency on axonal development, microtubule dynamics, and cell cycle regulation using zebrafish, iPSC-induced neurons, and HEK293T cell models. Elucidate the synergistic cascade of SPAST deficiency through MAPK pathway inactivation and microtubule network disruption in regulating axonal degeneration and proliferation inhibition, providing experimental evidence for the precise diagnosis, prognostic evaluation, and targeted therapy of HSP.

【Methods】

1．Whole-exome sequencing was performed to systematically screen pathogenic mutations in HSP patients. Mutations were classified according to ACMG guidelines, and key mutation sites were validated via Sanger sequencing.

2．Agarose gel electrophoresis and Sanger sequencing were used to identify abnormal splicing products caused by the SPAST c.1004+5G>A mutation, followed by conservation analysis.

3．Generated both wild-type and mutant SPAST overexpression vectors, which were subsequently delivered into HEK293T and HeLa cell lines via liposome-based transfection. Protein expression and subcellular localization were analyzed via Western blot and immunofluorescence.

4．Cycloheximide (CHX) tracking experiments were conducted to measure the half-life of wild-type and mutant spastin proteins, assessing escape from nonsense-mediated mRNA decay (NMD).

5．Splice-blocking morpholinos (MO) targeting the zebrafish spast gene were microinjected into Tg(hb9:eGFP) transgenic embryos. Knockdown efficiency was verified by qPCR. Axonal development (fluorescence imaging), motor behavior (trajectory tracking and acceleration analysis), and spinal cord ultrastructural changes were observed.

6．shRNA lentivirus transfection was used to construct SPAST knockdown iPSC models. Spinal motor neurons were generated through multi-stage differentiation. Knockdown efficiency was confirmed by qPCR and Western blot. Axonal growth, branching complexity, and somatic morphology were dynamically assessed, with immunostaining (Map2, Hb9) to evaluate dendritic microtubule structure.

7．CRISPR/Cas9 technology was employed to establish zebrafish spast^+/- stable lines, providing an in vivo research platform.

8．CRISPR/Cas9-generated SPAST-KO HEK293T cells were used for EdU flow cytometry (proliferation) and PI staining (cell cycle distribution). Transcriptome sequencing and KEGG enrichment analysis identified dysregulated pathways (e.g., MAPK). Western blot validated total ERK/JNK protein expression levels.

【Results】

1．Genetic spectrum and phenotypic heterogeneity: Mutations in SPAST (SPG4), NIPA1 (SPG6), SPG7, KIF5A (SPG10), KIF1A (SPG30), REEP1 (SPG31), PNPLA6 (SPG39), IBA57 (SPG74), and UBAP1 (SPG80) were identified, demonstrating significant clinical and phenotypic heterogeneity.

2．High prevalence of SPAST mutations: Among 16 HSP patients, SPAST mutations accounted for 38%, including missense (c.212T>A, c.64C>G), nonsense (c.716T>A), frameshift (c.1417_1418del), compound heterozygous (c.1027G>C / c.1267G>C), and intronic splice-site (c.1004+5G>A) mutations.

3．Novel SPAST splice-site mutation: The c.1004+5G>A mutation, first reported here, exhibited complete co-segregation with the disease phenotype. SPRS scores increased significantly with age (2–37 points). RNA splicing validation confirmed exon 6 skipping, generating a truncated spastin protein (p.Leu295*) that escaped NMD and accumulated abnormally.

4．Cellular pathology: Mutant spastin showed elevated expression levels, formed perinuclear aggregates, colocalized with microtubules, and disrupted microtubule dynamics. CHX experiments demonstrated prolonged mutant protein half-life (>12 hours).

5．Zebrafish phenocopy: spast-MO zebrafish exhibited axonal pathfinding errors, disordered branching, reduced spinal motor neuron fluorescence intensity, restricted movement trajectories, and abnormal acceleration, mimicking human SPAST-associated HSP symptoms.

6．iPSC neuron defects: SPAST knockdown in iPSC-derived spinal motor neurons caused delayed axonal growth at early differentiation stages (Day 12) and somatic hypertrophy with reduced branching complexity at maturity (Day 27). Immunofluorescence revealed axonal loss and dendritic microtubule disorganization, indicating microtubule imbalance disrupting synaptic development.

7．Cell cycle arrest and MAPK suppression: SPAST knockout induced G2/M phase arrest, reduced EdU-positive cell ratios, and inhibited proliferation. Transcriptomics revealed significant MAPK pathway (ERK/JNK) inhibition, with downregulated CCNB1 and VIM expression. Total ERK/JNK protein levels were markedly reduced in SPAST-KO cells.

【Conclusions】

This study identifies SPAST c.1004+5G>A as a novel pathogenic mutation in Chinese HSP patients, driven by a "splicing defect–truncated protein accumulation–microtubule dysregulation" cascade. Multi-model analyses (zebrafish, iPSC neurons, HEK293T) demonstrate that SPAST deficiency disrupts axonal development via microtubule instability and MAPK pathway suppression. The CRISPR/Cas9-engineered spast^+/- zebrafish and iPSC-derived neuron models provide robust platforms for mechanistic studies and drug screening.

Therapeutic strategies targeting RNA splicing correction (e.g., antisense oligonucleotides) or MAPK activation hold promise for SPG4 treatment.