The role of core circadian regulator Bmal1 in axonal regeneration and nerve repair

项目摘要 周围神经（PN）损伤后的轴突再生通常是不完全的。目前尚无有效 除了手术重建之外的治疗，这只对一小部分病例有益。 因此，了解修复机制至关重要。在这里，我们研究了一个以前未知的函数， 在PN损伤后门控神经再生反应的核心昼夜节律调节因子Bmal 1。这项研究是基于 我们的新数据表明，神经元特异性缺失Bmal 1加速了PN损伤后的轴突再生。这 我们实验室在破译密码方面取得了一系列进展， 控制神经元内在轴突生长潜能的转录网络，即，转录因子如何 (TFs)与表观遗传因子合作重塑染色质景观以诱导再生- 相关基因（RAGs）。我们最近的工作利用了我们的全基因组DNA图谱 羟甲基化（5 hmC）在再生背根神经节（DRG）神经元中的动力学。有趣的是，我们 发现在PN损伤后显示5 hmC变化的基因组位点中Bmal 1结合基序的富集， 提示Bmal 1与5 mC/5 hmC转化酶Tet 3的相互作用。事实上，小鼠中的Bmal 1 cKO 显示Bmal 1-Tet 3 - 5 hmC轴调节与轴突生长、代谢和免疫相关的基因， 交互.此外，试点数据首次显示了DRG中Tet 3和5 hmC的昼夜表观遗传节律 神经元是反相的Bmal 1转录振荡，并对应于一天中的时间差异， 再生反应在这里，我们将测试Bmal 1作为轴突抑制剂发挥作用的中心假设 再生和通过调节5 hmC重编程的损伤免疫激活的看门人。在 目的1，我们将描述Bmal 1门控的再生基因程序，并检查Bmal 1门控的再生基因程序的作用。 SR9009对Bmal 1转录的药理学抑制作用，SR9009是一种具有CNS渗透性的强效Rev-Erb激动剂 能力。从机制上讲，我们将测试一个“两次打击模型”，其中Bmal 1缺失启动DRG神经元，但Bmal 1缺失启动DRG神经元。 RAG诱导需要损伤信号。在目标2中，我们将描述Tet 3/5 hmC表观遗传节律性， 与“神经突生长时钟”的相关性，以及通过测试工作模型的潜在机制， Bmal 1控制Tet 3表达以及Tet 3募集到5 hmC重编程的靶位点。我们将 然后绘制PN损伤对Bmal 1-Tet 3 - 5 hmC节律性的短期和长期影响， 轴突重新连接后节律恢复正常。在目标3中，我们将重点放在促进作用的体内研究 Bmal 1抑制神经修复，包括运动感觉功能。我们还将解决 影响，性别差异，并定时Bmal 1抑制后不久PN损伤。总而言之，我们的建议 连接Bmal 1昼夜节律通路，Tet 3/5 hmC表观遗传重编程，损伤触发的潜力 免疫反应和轴突再生，从而推进神经再生和开放的基础科学 平移路径

Project Summary Axon regeneration after peripheral nerve (PN) injury is often incomplete. There is currently no effective treatment beyond surgical reconstruction, which is only beneficial for a small percentage of cases. Understanding the repair mechanisms is thus crucial. Here, we investigate a previously unknown function of the core circadian regulator Bmal1 in gating neuroregenerative responses after PN injury. The study is based on our new data that neuron-specific deletion of Bmal1 accelerates axon regeneration after PN injury. This exciting finding was made in the context of a series of advances from our laboratory in deciphering transcriptional networks that control neuronal intrinsic axon growth potential, i.e., how transcription factors (TFs) cooperate with epigenetic factors to reshape the chromatin landscape for induction of regeneration- associated genes (RAGs). Our most recent work has leveraged our genome-wide mapping of DNA hydroxymethylation (5hmC) dynamics in regenerating dorsal root ganglia (DRG) neurons. Intriguingly, we discovered enrichment of the Bmal1 binding motifs in genomic loci displaying 5hmC changes after PN injury, suggesting an interaction of Bmal1 with the 5mC/5hmC converting enzyme Tet3. Indeed, Bmal1 cKO in mice showed that the Bmal1-Tet3-5hmC axis regulates genes linked to axon growth, metabolism, and immune interactions. Moreover, pilot data show for the first-time a diurnal epigenetic rhythm of Tet3 and 5hmC in DRG neurons that is anti-phasic to Bmal1 transcriptional oscillation and corresponds to time-of-day differences in regenerative responses. Here, we will test the central hypothesis that Bmal1 functions as an inhibitor of axon regeneration and a gatekeeper of injury-trigged immune activation via regulation of 5hmC reprogramming. In Aim 1, we will characterize Bmal1-gated regenerative gene programs and examine the effect of pharmacological inhibition of Bmal1 transcription by SR9009, a potent Rev-Erb agonist with CNS penetration capability. Mechanistically, we will test a “two-hit model” wherein Bmal1 deletion primes DRG neurons, but an injury signal is required for RAGs induction. In Aim 2, we will characterize Tet3/5hmC epigenetic rhythmicity, correlation with “neurite outgrowth clock”, and the underlying mechanisms by testing the working model that Bmal1 controls Tet3 expression as well as Tet3 recruitment to target loci for 5hmC reprogramming. We will then map short- and long-term impact of PN injury on Bmal1-Tet3-5hmC rhythmicity and whether these rhythms return to normal upon axonal reconnection. In Aim 3, we pivot to in vivo study of the promoting effect of Bmal1 inhibition on nerve repair, including motosensory functions. We will also address sustainability of the effect, sex differences, and timed Bmal1 inhibition shortly after PN injury. In sum, our proposal has the potential of connecting Bmal1 circadian pathway, Tet3/5hmC epigenetic reprogramming, injury-triggered immune responses, and axon regeneration, thus advancing basic science of nerve regeneration and opening translational paths.