臺灣博碩士論文加值系統

Advillin 為一種在感覺神經元專一表現的蛋白質，其功用為調節和控制肌動蛋白微絲的結構。目前已知該蛋白質於發育早期大量表現於幾乎所有形式的體感覺神經元，然而，Advillin在成體準確的功能與角色仍尚未釐清。在本研究中，我們發現Advillin於成鼠感覺神經節其神經元族群的表現上具有特化性，主要表現於異凝集素B4 (IB4)這群非胜肽生成型的體感覺亞型神經元。無論於細胞培養或活體實驗中，皆顯示基因剔除Advillin專一地損減IB4這類軸突神經元纖維的再生。在神經元纖維再生的過程裡，Advillin主要表現於神經生長錐(growth cone)上，並存在於絲狀偽足(filopodia)的尖端結構，影響神經元纖維尋找路徑的能力，並透過與局部接合蛋白質複合體(focal adhesion complex)相關蛋白質諸如Myosin IIa、Vinculin和Focal adhesion kinase的結合和調控，來形塑和調節生長錐的形成。此外，我們更進一步地發現，在神經元纖維回縮的過程中，有部分含有Advillin的局部接合蛋白質複合體會由纖維尖端脫落於介質上，而這現象促使我們在帶有神經性疼痛的小鼠腦脊髓液中偵測到Advillin。為了瞭解Advillin所調控的神經纖維再生在神經性疼痛所扮演的角色，我們將Advillin基因剔除鼠和活體基因減弱的實驗運用在神經性疼痛的小鼠模式上，包含experimental autoimmune encephalomyelitis (EAE)此發炎性的、奧沙利鉑(oxaliplatin)此化療性的和慢性壓迫性損傷(chronic constriction injury, CCI)此類周邊神經局部損傷的神經性疼痛模式。其結果顯示無論基因剔除或基因減弱皆能延續或惡化神經損傷所引起的機械性痛覺過敏(mechanical hyperalgesia)、機械性觸摸痛(mechanical allodynia)和冷覺觸摸痛(cold allodynia)。更重要的是，在EAE 和CCI此兩種神經性疼痛小鼠模式中，Advillin基因剔除能導致脊髓背角(spinal-cord dorsal horn)上的IB4神經重塑(neural plasticity)型態發生異常。本篇論文由Advillin出發，替神經生長錐的形成、神經纖維的再生和神經性疼痛提供了一個嶄新的整合性方向，並點出了IB4此類非胜肽生成型的神經元於其中的角色。

Advillin is a sensory neuron-specific protein which regulates organization of the actin cytoskeleton. Studies indicate advillin expressed at high levels in all types of somatosensory neurons in early development. The precise role of advillin in adulthood, however, is largely unexplored. Here we reveal advillin protein expression mostly restricted to isolectin B4-positive (IB4+) neurons, classified as non-peptidergic nociceptors, in adult dorsal root ganglia (DRG). Advillin knockout (KO) specifically impaired axonal regeneration in adult IB4+ DRG neurons in vitro and in vivo. During axon regeneration, advillin was expressed at the very tips of filopodia, influenced neurite pathfinding, and modulated formation of the growth cone by interacting with and/or regulating focal adhesion-related proteins, such as myosin IIa, vinculin and activated focal adhesion kinase. The advillin-containing focal adhesion protein complex was shed from neurite tips during neurite retraction and was detectable in cerebrospinal fluid of mice with neuropathy. To test the role of advillin-mediated regeneration in neuropathic pain, advillin null mice and mice with in vivo advillin knockdown were applied to inflammation-induced (experimental autoimmune encephalomyelitis), chemotherapy-induced (oxaliplatin), and local peripheral nerve injury-induced (chronic constriction injury) neuropathic pain models. Knockout or knockdown of advillin aggravated neuropathic pain, including mechanical hyperalgesia, mechanical allodynia, and cold allodynia. Most importantly, advillin KO disturbed EAE-induced and CCI-induced neural plasticity of IB4+ afferents in the spinal-cord dorsal horn. Our study highlights a role for advillin in growth cone formation, axon regeneration, and neuropathic pain associated with IB4+ DRG neurons in adulthood.

誌謝 I
摘要 II
Abstract III
Table of Contents IV
List of figures and tables VII
Abbreviations IX
Chapter 1. Advillin Modulates Neurite Outgrowth of Somatosensory Neurons 1
1.1 Introduction 1
1.1.1 Advillin belongs to gelsolin family, assumed an actin regulating protein 1
1.1.2 Advillin is specifically expressed in sensory neurons 3
1.1.3 Objective 4
1.2 Materials and Methods 5
1.2.1 Animals 5
1.2.2 Antibody preparation 5
1.2.3 Co-immunoprecipitation 6
1.2.4 Mass spectrometry 6
1.2.5 Western blot analysis 7
1.2.6. Plasmid construction 8
1.2.7. Cell culture and transfection 8
1.2.8 Immunofluorescence 9
1.2.9 Live imaging 11
1.2.10 Image quantification 12
1.2.11 Statistical analysis 13
1.3 Results 14
1.3.1 Validation and characterization of advillin antibody by western blot. 14
1.3.2 Identification of advillin interacting protein by using LC-MS/MS analyses 14
1.3.3 The expression profile of advillin protein in DRG neuronal population of adult mice 15
1.3.4 The central and peripheral projections of advillin protein is subtype-specific 16
1.3.5 Advillin immunoreactivity labeled specific axonal structure in Avil+/Cre::GFP mice 17
1.3.6 Advillin is expressed in filopodia tips at active growth cones of regenerative DRG neurons 17
1.3.7 Advillin promotes neurite processing and filopodium formation in Neuro-2a cells. 18
1.3.9 Advillin leads neurite outgrowth and modulates filopodia of living N2a cells 19
1.3.10 Advillin has effect on the dynamics of axonal neurite outgrowth of DRG neurons on live imaging. 20
1.3.11 Advillin modulates subtype-specific neurite behaviors 20
1.3.12 Advillin coordinates the localization of vinculin and focal adhesion kinase in growth cones. 21
1.3.13 Advillin modulates the distribution of myosin IIa in growth cones. 21
1.3.14 Advillin-associated protein complex is shedding from neurite retraction. 22
Chapter 2. A Role of Advillin in the Development of Neuropathic Pain 24
2.1 Introduction 24
2.1.1 Neuropathic pain and peripheral nerve regeneration 24
2.1.2 Neuropathy and axonal regeneration of subtype specific somatosensory neurons 25
2.1.3 Models of neuropathic pain 25
2.1.4 Objective 29
2.2 Materials and Methods 30
2.2.1 Animals 30
2.2.2 Experimental autoimmune encephalomyelitis model 30
2.2.3 Oxaliplatin model 31
2.2.4 CCI-decompression model 31
2.2.5 Measurements of mechanical nocifensive behavior 31
2.2.6 Assessments of cold allodynia 32
2.2.7 Balance-beam walking task and gait behavior 33
2.2.8 Rotarod test 33
2.2.9 In vivo advillin knockdown 34
2.2.10 Western blot analysis 35
2.2.11 Immunohistochemistry 35
2.2.12 Image quantification 36
2.2.13 CSF collection 36
2.2.14 Quantification of advillin in CSF 37
2.2.15 Statistical Analysis 37
2.3 Results 38
2.3.1 Advillin has effect on EAE-induced symptoms 38
2.3.2 Advillin is involved in EAE-induced neuropathic pain 38
2.3.3 Advillin-KO EAE mice reveal elevated vinculin expression in superficial dorsal horn 40
2.3.4 Advillin impacts on neural plasticity of IB4+ central afferents in the EAE mouse model 40
2.3.5 Advillin is promising as a biomarker of EAE-induced neuropathic pain 41
2.3.6 Advillin plays a role in chemotherapy-induced peripheral neuropathy 41
2.3.7 Advillin is detectable in the CSF of mice with oxaliplatin-induced neuropathy 42
2.3.8 Advillin is involved in peripheral neuropathy induced by chronic constriction injury 43
2.3.9 Chronic constriction injury augments Advillin expression in the superficial dorsal horn 44
2.3.10 Advillin affects neural plasticity of IB4+ central afferents in the CCI mouse model 44
2.3.11 Advillin is detectable in the CSF of CCI mice 45
Chapter 3 Discussion 46
3.1 Summary 46
3.2 Non-peptidergic nociceptors and neuropathic pain 46
3.3 Advillin modulates growth cones to mediate neurite outgrowth 47
3.4 Clinical potential 49
3.5 Advillin, neural plasticity, and neuropathic pain 50
3.6 Advillin in neuropathic pain models 52
3.7 Advillin and stress 53
3.8 Conclusion 54
References 55



List of figures and tables
Table 1. LC MS/MS analysis of co-immunoprecipitation with advillin antibody 67
Table 2. Expression profiles of advillin in neuron subtypes of lumbar dorsal root ganglia (DRG) in adult mice. 69
Figure 1.Validation and characterization of advillin expression by western blotting 70
Figure 2.Exploring the interacting protein by co-IP and LC MS/MS 71
Figure 3.Advillin expression in mouse neonatal spinal cord and DRG 72
Figure 4.Expression of advillin protein in adult DRG neurons 73
Figure 5.Hierarchical clustering and K-means clustering of advillin immunoreactivity. 74
Figure 6.Advillin protein in the central and peripheral projections of adult sensory neurons. 75
Figure 7.Advillin immunoreactivity in Avil+/Cre::GFP mice. 76
Figure 8.Advillin expression in filopodia tips at active growth cones of regenerative DRG neurons 77
Figure 9.Effect of advillin on neurite processing and filopodium formation. 78
Figure 10. Advillin modulating neurite processing in living N2a cells 79
Figure 11. Effect of advillin KO on in vitro DRG neuron regeneration 80
Figure 12. Dynamics of axonal neurite outgrowth of DRG neurons on live imaging. 81
Figure 13. The effect of advillin KO on subtype-specific neurite outgrowth 82
Figure 14. Effect of advillin KO on vinculin and focal adhesion kinase (FAK) in growth cones. 83
Figure 15. Effect of advillin KO on myosin IIa in growth cones. 84
Figure 16. Shedding the advillin-associated protein complex from neurite retraction. 85
Figure 17. Effect of advillin KO on EAE-induced symptoms. 86
Figure 18. Effect of advillin KO on EAE-induced cold allodynia. 87
Figure 19. Validation of advillin knockdown in N2a cells and DRG neurons 88
Figure 20. Experimental design of advillin knockdown and von Frey test 89
Figure 21. Advillin-KO EAE mice in the recovery phase showed elevated vinculin expression in lamina I of superficial dorsal horns. 90
Figure 22. Advillin KO resulted in abnormal neural plasticity in spinal dorsal horn in the
recovery phase of EAE models. 91
Figure 23. The method used for analyzing IB4+ and CGRP+ overlapping areas in the spinal dorsal horn. 92
Figure 24. Assessment of advillin as a biomarker for EAE-induced neuropathic pain 93
Figure 25. Effect of advillin KO and knockdown on oxaliplatin-induced peripheral neuropathy 94
Figure 26. Experimental design of advillin knockdown and cold plate test 95
Figure 27. Assessment of advillin as a biomarker for oxaliplatin-induced neuropathic pain 96
Figure 28. Expression of advillin in the spinal cord dorsal horn after chronic constriction injury and decompression of constriction 97
Figure 29. Effect of advillin KO on neuroplasticity associated with local nerve injury
from compression of the sciatic nerve. 98
Figure 30. Western blot analysis of advillin expression in CSF samples of CCI model. 99
Appendix Figure 1. Effect of advillin KO and knockdown on EAE-induced neuropathic pain. 100
Appendix Figure 2. Effect of advillin KO on neuropathic pain induced by chronic constriction injury 101
Appendix Figure 3. Dissection of the fixed spinal cord from CCI-treated mice 102

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