(A) selective HDAC6 inhibitor improves behavioral and pathological phenotypes in mouse models of Alzheimer’s and Huntington’s disease

국문 초록 (Abstract)

HDAC6 저해제, 축삭수송, 알츠하이머 병, Histone deacetylase 6 (HDAC6) is a cytoplasmic deacetylase that regulates microtubule dynamics, axonal transport, and protein quality control. Dysregulation of HDAC6 activity is implicated in neurodegenerative diseases characterized by axonal dysfunction and protein aggregation, including Alzheimer’s disease (AD) and Huntington’s disease (HD).
The pharmacological profile and therapeutic efficacy of a brain-penetrant and selective HDAC6 inhibitor, HD6i-01, were evaluated in preclinical models of AD (PS19 mice) and HD (YAC128 mice). in vitro and in vivo assays were conducted to assess HDAC6 selectivity, pharmacokinetics, axonal transport, oxidative stress, and behavioral outcomes. Tau and mutant huntingtin burden were analyzed by western blotting, and retrograde tracing was used to visualize projection-specific transport in disease-relevant circuits.
HD6i-01 exhibited potent and selective HDAC6 inhibition, with favorable brain exposure and in vivo target engagement. In cell-based and iPSC-derived neuron assays, HD6i-01 restored mitochondrial motility and reduced ROS levels. In PS19 and YAC128 mice, long-term treatment improved cognitive and motor function, reduced total and phosphorylated tau, and decreased polyQ and mutant huntingtin accumulation. Retrograde tracing revealed restoration of axonal transport in the septohippocampal and nigrostriatal circuits. HDAC6 inhibition also attenuated oxidative stress and rescued molecular markers such as DARPP-32 and tyrosine hydroxylase in the striatum.
Selective HDAC6 inhibition by HD6i-01 restores axonal transport, alleviates protein pathology, and improves behavioral outcomes in mouse models of AD and HD, supporting its prospect as a therapeutic strategy for neurodegenerative disorders.타우병증, 헌팅턴 병

국문 초록 (Abstract)

히스톤 디아세틸화효소 6 (HDAC6)은 α-tubulin, HSP90, peroxiredoxin 등 다양한 비히스톤 기질의 탈아세틸화를 통해 축삭수송 조절, 단백질 품질 관리, 산화 스트레스 반응 등에 관여하는 세포질 특이 탈아세틸화효소이다. 최근 HDAC6의 과활성이 알츠하이머 병, 헌팅턴 병과 같은 신경퇴행성 질환의 병리 진행에 관여한다는 보고가 증가하고 있으며, 이에 따라 HDAC6를 선택적으로 저해하는 치료 전략이 새로운 질병 조절 접근법으로 주목받고 있다. 본 연구에서는 선택적 HDAC6 저해제인 HD6i-01의 약리학적 특성 및 약효를 알츠하이머 병과 헌팅턴 병 모델에서 평가하였다.
HD6i-01은 효소 기반 분석에서 HDAC6에 대해 높은 선택성을 나타냈으며, 세포 실험에서도 α-tubulin 아세틸화를 농도의존적으로 증가시키는 반면 histone H3의 아세틸화는 변화시키지 않았다. 이와 상관성을 보이며 HD6i-01 처리에 의해 mutant tau 과발현 신경세포 또는 헌팅턴 병 환자 유래 iPSC에서 미토콘드리아 이동성이 회복되어, in vitro 수준에서 축삭수송 기능이 개선됨을 시사하였다.
PS19 알츠하이머 병 마우스에서는 3개월의 투여로 인지기능 평가에서 유의한 행동 회복이 관찰되었으며, total tau와 phospho-tau (T231) 단백질 발현 수준이 감소하였다. 헌팅턴 병 마우스 모델인 YAC128에서도 3개월의 투여로 운동 협응성과 근력 향상이 확인되었으며, 대뇌 피질에서 polyQ 및 mutant huntingtin 단백질의 발현이 유의하게 감소하였다. YAC128 마우스의 선조체에서는 DARPP-32 및 tyrosine hydroxylase (TH)의 발현이 부분적으로 회복되어 도파민성 기능 유지 가능성을 보여주었다. 급성 독성 모델인 3-nitropropionic acid 처리 마우스에서도 운동 기능 회복, 산화 스트레스 완화, TH 발현 회복 등 복합적인 신경 보호 효과가 확인되었다. 또한, 세 모델 모두에서 약물 투여에 의해 뇌 조직의 acetylated α-tubulin의 증가가 관찰되어 in vivo 표적 결합을 확인할 수 있었다. Retrograde tracing 분석을 통해 PS19 및 YAC128 마우스에서 HD6i-01 투여 시 각각 septohippocampal 및 nigrostriatal 회로의 축삭수송이 회복됨이 확인되었으며, 이는 in vitro에서 관찰된 축삭수송 기능 회복과의 연속성을 보여준다.
이러한 다중 기전 기반의 효과는 HD6i-01이 단백질 응집, 축삭수송 장애, 산화 스트레스 등 다양한 병태생리적 기전을 동시에 조절할 수 있는 잠재력을 지님을 보여주며, 알츠하이머 병과 헌팅턴 병의 질병 조절 치료제로 개발될 수 있는 가능성을 시사한다.

목차 (Table of Contents)

• I. Introduction 1
• 1. Neurodegenerative diseases 1
• 1) Alzheimer's disease (AD) 1
• 2) Huntington's disease (HD) 3
• 2. Histone deacetylase 6 (HDAC6) 4
• 1) Structure of HDAC6 4
• 2) Functions of HDAC6 5
• 3. HDAC6 dysregulation in AD and HD 9
• 4. Aims of this study 11
• II. Materials and Methods 12
• 1. Animals 12
• 2. Drugs and chemicals 13
• 3. HDAC1/6 enzyme assay 16
• 4. HDAC panel assay 17
• 5. Cell line culture 17
• 6. Mouse primary cortical neuron culture 18
• 7. Human derived iPSC differentiation 19
• 8. Transfection (P301S tau) 20
• 9. Western blot assay (cell) 21
• 10. Glutamate induced ROS assay 23
• 11. 3-NP induced ROS assay 23
• 12. Mitochondrial trafficking assay 24
• 13. in vivo drug administration schedule (PS19) 27
• 14. in vivo drug administration schedule (YAC128) 27
• 15. in vivo drug administration schedule (3-NP) 28
• 16. Behavioral experiments 30
• 1) Y-maze test 30
• 2) Morris water maze 30
• 3) Passive avoidance test 31
• 4) Acceleration rotarod test 32
• 5) Grip strength test 33
• 17. Western blot assay (tissue) 34
• 18. Retrograde tracing 34
• 1) Hippocampal projections to the medial septum 34
• 2) Substantia nigra projections to the striatum 36
• 19. Data anaysis 38
• III. Results 39
• 1. HD6i-01 exhibits high selectivity and potency for HDAC6 inhibition 39
• 1) HD6i-01 shows improved HDAC6/HDAC1 selectivity relative to reference inhibitors 39
• 2) HD6i-01 exhibits superior HDAC6 selectivity over pan-HDAC inhibitor LBH589 42
• 3) HD6i-01 shows greater HDAC6 selectivity than pan-HDAC inhibitor LBH589 in SH-SY5Y cells 44
• 2. HD6i-01 rescues mitochondrial axonal transport in neurodegenerative disease models 46
• 1) Improvement of axonal transport in Tau-overexpressing neurons by HD6i-01 46
• 2) Rescue of mitochondrial transport deficits in HD iPSC-derived neurons by HD6i-01 49
• 3. HD6i-01 mitigates intracellular ROS in neurotoxic conditions 51
• 1) HD6i-01 suppresses glutamate-induced ROS generation 51
• 2) HD6i-01 attenuates 3-NP-induced oxidative stress 54
• 4. HD6i-01 reduces total human tau levels in P301S tau-expressing neurons 56
• 5. HD6i-01 improves cognitive function in the PS19 tauopathy model 58
• 1) HD6i-01 restores memory impairment in PS19 mice in the Y-maze test 58
• 2) HD6i-01 improves spatial learning and memory of PS19 mice in the MWM 61
• 3) HD6i-01 restores memory retention in the passive avoidance test in PS19 mice 65
• 6. HD6i-01 ameliorates motor deficits in the YAC128 Huntington's disease model 67
• 1) HD6i-01 improves motor coordination in YAC128 mice in the accelerating rotarod test 67
• 2) HD6i-01 restores muscle strength in YAC128 mice in the grip strength test 70
• 7. HD6i-01 alleviates motor and clinical deficits in the 3-NP Huntington's disease model 72
• 8. HD6i-01 increases acetylated α-tubulin in brain and PBMC as a pharmacodynamic marker 75
• 9. HD6i-01 alleviates tau accumulation and phosphorylation in a tauopathy model 79
• 10. HD6i-01 ameliorates molecular and cellular pathology in the brains of YAC128 mice 83
• 1) HD6i-01 reduces mutant huntingtin and polyQ protein levels in the cortex of YAC128 mice 83
• 2) HD6i-01 restores striatal acetylation and partially recovers neuronal markers in YAC128 mice 86
• 11. HD6i-01 engages HDAC6 and restores dopaminergic input in the 3-NP mouse model 90
• 12. HD6i-01 restores long-range axonal transport in tauopathy and Huntington's disease models 93
• 1) HD6i-01 restores hippocampalseptal axonal transport in PS19 mice via retrograde tracing 93
• 2) HD6i-01 rescues disrupted nigrostriatal axonal transport in a Huntington's disease mouse model 96
• 14. Pharmacokinetic properties and brain penetration of HD6i-01 98
• IV. Discussion 100
• 1. HDAC6 as a therapeutic target in neurodegenerative diseases 100
• 2. Pharmacological profile and target engagement of HD6i-01 101
• 3. Axonal transport restoration as a central mechanism of action 102
• 4. Functional rescue and molecular pathology across models 104
• 5. Therapeutic implications and prospects for clinical translation 107
• V. Conclusion 109
• References 111