Development of live-cell probes to investigate tubulin post-translational modifications in neuronal regeneration

开发活细胞探针来研究神经元再生中微管蛋白翻译后修饰

• 批准号: 10648255
• 负责人: Roman Jeno Giger
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10648255
• 关键词: Abbreviations; Acetylation; Amino Acid Sequence; Antibodies; Antigens; Appearance; Area; Axon; Axonal Transport; Axotomy; Binding Proteins; Biogenesis; Biological Assay; Biosensor; C-terminal; Cells; Central Nervous System; Collaborations; Colorado; Complementary DNA; Cytoplasm; Cytoskeleton; Data; Dendrites; Detection; Development; Distal; Dynein ATPase; Elements; Epothilones; Excision; Growth Cones; Hydrolase; Image; Immunoglobulin Fragments; Immunoglobulin G; Injury; Kinesin; Length; Maps; Mass Spectrum Analysis; Metabolic; Microtubule Polymerization; Microtubules; Molecular Motors; Monoclonal Antibodies; Motor; Motor Neurons; Mus; Natural regeneration; Nerve; Nerve Degeneration; Nerve Fibers; Nervous System; Neurites; Neuronal Injury; Neurons; Organelles; Oryctolagus cuniculus; Paclitaxel; Pattern; Peptide Sequence Determination; Peripheral Nervous System; Permeability; Pharmaceutical Preparations; Play; Polymers; Positioning Attribute; Post-Translational Protein Processing; Presynaptic Terminals; Process; Proteins; Regenerative capacity; Regulation; Research; Retinal Ganglion Cells; Reverse engineering; Role; Sensory; Site; Spinal Ganglia; Stretching; Synapses; Tail; Therapeutic Agents; Time; Tissues; Trauma; Tubulin; Universities; Wallerian Degeneration; Work; alpha Tubulin; axon growth; axon guidance; axon injury; axon regeneration; axonal degeneration; beta Tubulin; central nervous system injury; chemotherapy; differential expression; disability; injured; injury and repair; nerve supply; neuron development; neuron regeneration; neuronal cell body; neuronal growth; novel therapeutics; painful neuropathy; pharmacologic; regeneration potential; regenerative growth; repaired; response to injury; restoration; retrograde transport; sample fixation; spatiotemporal; tyrosyltubulin ligase

PROJECT SUMMARY/ABSTRACT Damage to the peripheral nervous system (PNS) can be incurred through a wide spectrum of conditions including trauma, metabolic imbalances, and chemotherapy. Neuronal injuries occur when their long and fragile processes (i.e. axons) are damaged, for example by stretching or severing. Compared to PNS neurons, the regenerative capacity of injured central nervous system (CNS) neurons is extremely limited, typically resulting in life-long disability. Regenerative growth of axons can occur in the injured peripheral nervous system, however, restoration of function is often incomplete, resulting in loss of motor and sensory function and frequently the development of neuropathic pain. Regeneration of an injured axon is thought to involve changes in microtubule dynamics and post- translational modifications (PTMs). Microtubules are dynamic polymers of α,β-tubulin and are the core structural element of axons and dendrites. Microtubules also serve as tracks for molecular motor proteins (kinesin, cytoplasmic dynein) that transport various cargos between synaptic terminals and the neuronal cell soma. Following injury, microtubules undergo rapid fragmentation, a process known as Wallerian degeneration. In developing neurons and during axon regeneration, microtubules in the neuronal growth cone and axon shaft play key roles in axon guidance and axon elongation. Regulation of MT function is largely achieved by the differential expression of α- and β-tubulin isotypes, and by PTMs of tubulin subunits within the microtubule polymer. Studies of microtubule isotypes and PTMs inherently rely on antibodies that require fixation and permeabilization of the cell or tissue, which severely limits our understanding of the spatiotemporal component of tubulin PTMs. We have developed a pipeline that allows us to generate live-cell probes for tubulin PTMs. Here, we will generate probes for tubulin isotypes and PTMs relevant to neuronal degeneration and regeneration and use them to map the spatiotemporal patterns of changes to the microtubule cytoskeleton following injury to the PNS and CNS.

项目概要/摘要 周围神经系统 (PNS) 的损伤可能由多种情况引起 包括创伤、代谢失衡和化疗时发生的神经元损伤。 与 PNS 神经元相比，脆弱的突起（即轴突）受到拉伸或切断等损害。 受损的中枢神经系统 (CNS) 神经元的再生能力极其有限，通常 受伤的周围神经可能会发生轴突的再生生长，从而导致终身残疾。 然而，系统功能的恢复往往不完全，导致运动和感觉功能丧失 并经常出现神经性疼痛。 受伤轴突的再生被认为涉及微管动力学和术后恢复的变化。 翻译修饰 (PTM) 是 α,β-微管蛋白的动态聚合物，是核心。 轴突和树突的结构元件也充当分子运动蛋白的轨道。 （驱动蛋白、细胞质动力蛋白）在突触末梢和神经元细胞之间运输各种物质 损伤后，微管会发生快速断裂，这一过程被称为华勒氏体。 在发育中的神经元和轴突再生过程中，神经元生长锥中的微管发生变性。 轴突轴在轴突引导和轴突伸长中起着关键作用。 MT功能的调节很大程度上是通过α-和β-微管蛋白同种型的差异表达来实现的， 以及微管聚合物内微管蛋白亚基的 PTM 微管同种型和 PTM 的研究。 本质上依赖于需要固定和透化细胞或组织的抗体，这严重限制了 我们对微管蛋白 PTM 时空成分的理解我们开发了一个管道，可以实现这一点。 我们生成微管蛋白 PTM 的活细胞探针 在这里，我们将生成微管蛋白同种型和 PTM 的探针。 与神经退化和再生相关，并用它们来绘制神经退化和再生的时空模式 PNS 和 CNS 损伤后微管细胞骨架发生变化。