Farmacologische inhibitie van histone deacetylase 6 (HDAC6) als therapeutische strategie in chemotherapie-geïnduceerde periphere neuropathieën

As cancer is becoming more and more a chronic disease, a large proportion of patients is confronted with devastating side effects of certain anti-cancer drugs. The most common neurological complications are painful peripheral neuropathies. Chemotherapeutics that interfere with microtubules, including plant-derived vinca-alkaloids such as vincristine, can cause these chemotherapy-induced peripheral neuropathies (CIPN). Available treatments focus on symptom alleviation and pain reduction rather than prevention of the neuropathy. The aim of this study was to investigate the potential of specific histone deacetylase 6 (HDAC6) inhibitors as a preventive therapy for CIPN using multiple rodent models for vincristine-induced peripheral neuropathies (VIPN). HDAC6 inhibition increased the level of acetylated α-tubulin in tissues of rodents undergoing vincristine-based chemotherapy, which correlates to a reduced severity of the neurological symptoms, both at the electrophysiological and the behavioral level. Mechanistically, disturbances in axonal transport of mitochondria is considered as an important contributing factor in the pathophysiology of VIPN. As vincristine interferes with the polymerization of microtubules, we investigated whether disturbances in axonal transport could contribute to VIPN. We observed that increasing α-tubulin acetylation through HDAC6 inhibition restores vincristine-induced defects of axonal transport in cultured dorsal root ganglion neurons. Finally, we assured that HDAC6-inhibition offers neuroprotection without interfering with the anti-cancer efficacy of vincristine using a mouse model for acute lymphoblastic leukemia. Taken together, our results emphasize the therapeutic potential of HDAC6 inhibitors with beneficial effects both on vincristine-induced neurotoxicity, as well as on tumor proliferation.TABLE OF CONTENTS A WORD OF APPRECIATION 4 TABLE OF CONTENTS 11 ABBREVIATIONS 15 CHAPTER 1: INTRODUCTION 19 1. Peripheral Neuropathies 19 1.1. Etiology 21 1.1.1. Hereditary peripheral neuropathies 21 1.1.2. Acquired peripheral neuropathies 22 1.2. Possible mechanisms 23 1.2.1. Axonal transport and the cytoskeleton 23 1.2.2. Other mechanisms 27 1.2.2.1. Protein degradation pathways 27 1.2.2.2. Metabolic dysregulation and Schwann cell biology 28 1.2.2.3. Organelle damage (Golgi, endoplasmic reticulum and mitochondria) 29 1.2.2.4. Channelopathies 29 1.2.2.5. Intracellular signalling 30 1.3. Therapeutic strategies 31 2. Histone deacetylases 33 2.1. Protein family and structure 33 2.2. Mechanisms associated with HDAC6 function 34 2.2.1. Microtubule dynamics and the actin cytoskeleton 34 2.2.2. Other mechanisms 35 2.2.2.1. Antioxidant reactivity through peroxiredoxins 35 2.2.2.2. Regulation of the chaperoning activity of HSP90 36 2.2.2.3. Regulation of cellular pathways for protein-induced stress responses 36 2.2.3. Conclusions on HDAC6 mechanisms 40 3. HDAC6 in the context of human disease 41 3.1. HDAC6 in neurodegeneration 41 3.2. HDAC6 in cancer 42 3.3. HDAC6 in immunology 43 3.4. Conclusions 44 CHAPTER 2: AIMS OF THE STUDY 45 CHAPTER 3: MATERIALS & METHODS 47 1. Animals and treatment strategies 47 2. Whole-mount immunofluorescence of zebrafish 47 3. Zebrafish locomotion assay 50 4. Electrophysiology 50 5. Mechanical sensitivity assay and Rotarod 51 6. Histological examination of neuromuscular junctions 52 7. Histological examination of skin biopsies 52 8. DRG cultures 53 9. Axonal transport 53 10. Immunofluorescence of DRG cultures 54 11. Neurite outgrowth assay and live cell imaging. 54 12. Western Blot 55 13. Axon counts 56 14. Tumour progression and survival 57 15. Xenograft model and flow cytometry 58 16. Statistics 59 17. Study Approval 59 CHAPTER 4: RESULTS 61 1. Creation and characterisation of animal models to study VIPN 61 1.1. Proof-of-concept using a zebrafish model for neurodegeneration 61 1.2. Strain susceptibility of rodents to VIPN 62 1.3. Optimal treatment strategy to induce VIPN in rodents 63 1.4. Vincristine induced a purely sensory peripheral neuropathy in rodents 65 1.5. Conclusions 65 2. Inhibition of HDAC6 reduced the severity of VIPN 69 2.1. Proof-of-concept using a zebrafish model for neurodegeneration 69 2.2. Pharmacological inhibition of HDAC6 protects against VIPN in rodents 70 2.3. Conclusions 71 3. Unravelling the role of HDAC6 in the pathophysiology of VIPN in vitro 73 3.1. HDAC6-inhibition increases acetylated α-tubulin and restores axonal transport deficits in DRG neurons treated with vincristine 73 3.2. HDAC6-inhibition counteracts vincristine-associated inhibition on neurite outgrowth 76 3.3. Conclusions 78 4. Validating the role of HDAC6 in the pathophysiology of VIPN in vivo 80 4.1. HDAC6-inhibition increased α-tubulin acetylation in vivo 80 4.2. HDAC6-inhibition preserves axon numbers in sciatic nerve 82 4.3. HDAC6-inhibition conserves innervation of the skin and muscle 83 4.4. Conclusions 86 5. Testing the therapeutic relevance of HDAC6-inhibition: validation in a cancer model 87 5.1. HDAC6-inhibition reduced tumour proliferation and increased cell death in vitro 87 5.2. HDAC6-inhibition reduced tumour proliferation and protects against vincristine neurotoxicity in vivo 88 5.3. Conclusions 91 6. Translational collaborations: development of improved HDAC6 inhibitors 93 6.1. In vitro screening using neuronal cell types 93 6.1.1. Identification of potent HDAC6-selective inhibitors in N2a cells 95 6.1.2. Conclusions 95 6.2. In vivo screening using our mouse model for VIPN 97 6.2.1. Article #1 partially protects against VIPN at the electrophysiological and behavioural level 97 6.2.2. Article #1 increased α-tubulin acetylation in vivo. 99 6.2.3. Conclusions 99 CHAPTER 5: GENERAL DISCUSSION 101 CHAPTER 6: SUMMARY 107 CHAPTER 7: NEDERLANDSTALIGE SAMENVATTING 109 FUNDING & SCIENTIFIC ACKNOWLEDGEMENTS 112 REFERENCES 113 CURICULUM VITAE 137nrpages: 139status: publishe