Hu Mengming, Veldman Matthew B
Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA.
Department of Ophthalmology and Visual Science, Medical College of Wisconsin, Milwaukee, USA.
Mol Neurodegener. 2025 Mar 5;20(1):28. doi: 10.1186/s13024-025-00807-2.
Axon regeneration within the mammalian central nervous system is extremely limited. In optic neuropathy conditions like glaucoma, the inability of retinal ganglion cell (RGC) axons to regenerate is a major impediment to functional recovery. In contrast, adult teleost fish such as zebrafish can fully regenerate RGC axons enabling visual recovery from optic nerve (ON) injury making it an ideal model to probe the mechanisms of successful axon regeneration.
Laser Capture Microdissection followed by RNA-sequencing (LCM-seq) was used to identify genes and pathways differentially expressed in RGCs during ON regeneration. We validate these findings by in situ hybridization and qRT-PCR. Using loss- and gain-of-function experiments we demonstrate the necessity of srebf2 for efficient ON regeneration and recovery of visual function. Finally, we use LCM-seq coupled with experimental manipulations to identify downstream srebf2 target genes and test the role of hmgcra/b and mevalonate in this process. Statistical analysis was performed using Student's t-test, two-way ANOVA, or repeated measures with appropriate post-hoc tests where applicable.
LCM-seq comparison of uninjured versus 3-day post ON injury RGCs identified significant upregulation of the cholesterol synthesis pathway during axon regeneration. The master regulator of this pathway, the transcription factor srebf2, is upregulated throughout the regeneration phase. Chemical inhibition or morpholino-based gene knockdown of srebf2 decreased axon regeneration into the ON and optic tectum and delayed recovery of visual behavior over the course of normal optic nerve regeneration without causing a significant loss of RGCs. Constitutively active srebf2 can fully rescue axon regeneration and visual behavior losses caused by inhibition of endogenous srebf2 but does not accelerate regeneration compared to the control group. LCM-seq confirms the expected regulation of predicted srebf2 target genes after loss- or gain-of-function in vivo. Downstream of srebf2, hmgcra/b knockdown or simvastatin treatment delayed axon regeneration and this effect was rescued by supplemental mevalonate. Mevalonate treatment alone was sufficient to accelerate ON regeneration.
These results demonstrate that srebf2 and the downstream mevalonate synthesis pathway plays an important role in regulating efficient axon regeneration in the zebrafish visual system. Involvement of this pathway should be closely examined in failed mammalian ON regeneration.
哺乳动物中枢神经系统内的轴突再生极其有限。在青光眼等视神经病变情况下，视网膜神经节细胞（RGC）轴突无法再生是功能恢复的主要障碍。相比之下，成年硬骨鱼如斑马鱼能够使RGC轴突完全再生，从而从视神经（ON）损伤中恢复视觉，这使其成为探究成功轴突再生机制的理想模型。
采用激光捕获显微切割结合RNA测序（LCM-seq）来鉴定ON再生过程中RGCs中差异表达的基因和通路。我们通过原位杂交和qRT-PCR验证了这些发现。利用功能丧失和功能获得实验，我们证明了srebf2对于高效的ON再生和视觉功能恢复的必要性。最后，我们使用LCM-seq结合实验操作来鉴定srebf2的下游靶基因，并测试hmgcra/b和甲羟戊酸在这一过程中的作用。在适用的情况下，使用学生t检验、双向方差分析或重复测量并进行适当的事后检验进行统计分析。
LCM-seq对未受伤与ON损伤后3天的RGCs进行比较，发现在轴突再生过程中胆固醇合成途径显著上调。该途径的主要调节因子，即转录因子srebf2，在整个再生阶段均上调。srebf2的化学抑制或基于吗啉代的基因敲低减少了轴突向ON和视顶盖的再生，并在正常视神经再生过程中延迟了视觉行为的恢复，而不会导致RGCs的显著损失。组成型活性srebf2可以完全挽救由内源性srebf2抑制引起的轴突再生和视觉行为损失，但与对照组相比不会加速再生。LCM-seq证实了体内功能丧失或获得后预测的srebf2靶基因的预期调控。在srebf2的下游，hmgcra/b敲低或辛伐他汀处理延迟了轴突再生，补充甲羟戊酸可挽救这种效应。单独的甲羟戊酸处理足以加速ON再生。
这些结果表明，srebf2和下游甲羟戊酸合成途径在调节斑马鱼视觉系统中高效的轴突再生中起重要作用。在哺乳动物ON再生失败的情况下，应密切研究该途径的参与情况。
