ABSTRACT

Macroautophagy (hereafter referred to as autophagy) is a finely tuned process of programmed degradation and recycling of proteins and cellular components, which is crucial in neuronal function and synaptic integrity. Mounting evidence implicates chromatin remodeling in fine-tuning autophagy pathways. However, this epigenetic regulation is poorly understood in neurons. Here, we investigate the role in autophagy of KANSL1, a member of the nonspecific lethal complex, which acetylates histone H4 on lysine 16 (H4K16ac) to facilitate transcriptional activation. Loss-of-function of KANSL1 is strongly associated with the neurodevelopmental disorder Koolen-de Vries Syndrome (KdVS). Starting from KANSL1-deficient human induced-pluripotent stem cells, both from KdVS patients and genome-edited lines, we identified SOD1 (superoxide dismutase 1), an antioxidant enzyme, to be significantly decreased, leading to a subsequent increase in oxidative stress and autophagosome accumulation. In KANSL1-deficient neurons, autophagosome accumulation at excitatory synapses resulted in reduced synaptic density, reduced GRIA/AMPA receptor-mediated transmission and impaired neuronal network activity. Furthermore, we found that increased oxidative stress-mediated autophagosome accumulation leads to increased MTOR activation and decreased lysosome function, further preventing the clearing of autophagosomes. Finally, by pharmacologically reducing oxidative stress, we could rescue the aberrant autophagosome formation as well as synaptic and neuronal network activity in KANSL1-deficient neurons. Our findings thus point toward an important relation between oxidative stress-induced autophagy and synapse function, and demonstrate the importance of H4K16ac-mediated changes in chromatin structure to balance reactive oxygen species- and MTOR-dependent autophagy.

Abbreviations: APO: apocynin; ATG: autophagy related; BAF: bafilomycin A₁; BSO: buthionine sulfoximine; CV: coefficient of variation; DIV: days in vitro; H4K16ac: histone 4 lysine 16 acetylation; iPSC: induced-pluripotent stem cell; KANSL1: KAT8 regulatory NSL complex subunit 1; KdVS: Koolen-de Vries Syndrome; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEA: micro-electrode array; MTOR: mechanistic target of rapamycin kinase; NSL complex: nonspecific lethal complex; 8-oxo-dG: 8-hydroxydesoxyguanosine; RAP: rapamycin; ROS: reactive oxygen species; sEPSCs: spontaneous excitatory postsynaptic currents; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; SYN: synapsin; WRT: wortmannin.

摘要

巨自噬（以下简称自噬）是蛋白质和细胞成分的程序性降解和循环利用的精细调节过程，对神经元功能和突触完整性至关重要。越来越多的证据表明染色质重塑与微调自噬途径有关。然而，这种表观遗传调控在神经元中知之甚少。在这里，我们研究了 KANSL1 在自噬中的作用，KANSL1 是非特异性致死复合物的成员，它乙酰化赖氨酸 16 (H4K16ac) 上的组蛋白 H4 以促进转录激活。KANSL1 的功能丧失与神经发育障碍 Koolen-de Vries 综合征 (KdVS) 密切相关。从来自 KdVS 患者和基因组编辑系的 KANSL1 缺陷型人类诱导多能干细胞开始，我们鉴定了 SOD1（超氧化物歧化酶 1），一种抗氧化酶，显着降低，导致随后的氧化应激和自噬体积累增加。在 KANSL1 缺陷的神经元中，兴奋性突触处的自噬体积累导致突触密度降低、GRIA/AMPA 受体介导的传递降低和神经元网络活动受损。此外，我们发现氧化应激介导的自噬体积累增加导致 MTOR 激活增加和溶酶体功能降低，进一步阻止了自噬体的清除。最后，通过药理学减少氧化应激，我们可以挽救 KANSL1 缺陷神经元中异常的自噬体形成以及突触和神经元网络活动。

缩写： APO：夹竹桃麻素；ATG：自噬相关；BAF：巴弗洛霉素 A ₁；BSO：丁硫氨酸亚砜亚胺；CV：变异系数；DIV：体外天数；H4K16ac：组蛋白 4 赖氨酸 16 乙酰化；iPSC：诱导多能干细胞；KANSL1：KAT8 调节 NSL 复合亚基 1；KdVS：库伦-德弗里斯综合征；LAMP1：溶酶体相关膜蛋白 1；MAP1LC3/LC3：微管相关蛋白1轻链3；MEA：微电极阵列；MTOR：雷帕霉素激酶的机制靶点；NSL复合物：非特异性致死复合物；8-氧代-dG：8-羟基脱氧鸟苷；RAP：雷帕霉素；ROS：活性氧；sEPSCs：自发兴奋性突触后电流；SOD1：超氧化物歧化酶1；SQSTM1/p62：隔离体 1；SYN：突触；WRT：渥曼青霉素。