Imbalanced autophagy causes synaptic deficits in a human model for neurodevelopmental disorders

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. <b>Abbreviations</b>: 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.

巨自噬（Macroautophagy，下称自噬）是一种受到精密调控的蛋白质与细胞组分程序性降解及循环利用过程，对神经元功能与突触完整性至关重要。越来越多的证据表明，染色质重塑（chromatin remodeling）参与自噬通路的精细调控，但目前学界对神经元中这一表观遗传调控机制的认知仍较为匮乏。本研究聚焦于KAT8调控的非特异性致死复合物（nonspecific lethal complex，NSL复合物）成员KANSL1（KAT8 regulatory NSL complex subunit 1）在自噬中的作用——该复合物可在组蛋白H4的赖氨酸16位进行乙酰化修饰（H4K16ac，histone 4 lysine 16 acetylation），以促进转录激活。KANSL1功能缺失与神经发育障碍库伦-德弗里斯综合征（Koolen-de Vries Syndrome，KdVS）密切相关。
本研究以KdVS患者及基因组编辑获得的KANSL1缺陷型人诱导多能干细胞（induced-pluripotent stem cell，iPSC）为模型，发现抗氧化酶超氧化物歧化酶1（superoxide dismutase 1，SOD1）的表达水平显著下调，进而引发氧化应激升高与自噬体堆积。在KANSL1缺陷的神经元中，兴奋性突触处的自噬体堆积会导致突触密度降低、GRIA/AMPA受体介导的突触传递减弱，以及神经元网络活动受损。
进一步研究发现，氧化应激介导的自噬体堆积会激活MTOR（mechanistic target of rapamycin kinase，雷帕霉素机制性靶激酶）通路并削弱溶酶体功能，进一步阻碍自噬体的清除。最后，通过药理学手段降低氧化应激水平，可挽救KANSL1缺陷神经元中异常的自噬体形成，以及突触与神经元网络活动缺陷。综上，本研究揭示了氧化应激诱导的自噬与突触功能之间的重要关联，并证实了H4K16ac介导的染色质结构改变在平衡活性氧（reactive oxygen species，ROS）依赖与MTOR依赖的自噬过程中的关键作用。
<b>缩略语说明</b>：APO：夹竹桃麻素（apocynin）；ATG：自噬相关蛋白（autophagy related）；BAF：巴弗洛霉素A₁（bafilomycin A₁）；BSO：丁胱亚磺酰亚胺（buthionine sulfoximine）；CV：变异系数（coefficient of variation）；DIV：体外培养天数（days in vitro）；H4K16ac：组蛋白H4赖氨酸16乙酰化；iPSC：诱导多能干细胞；KANSL1：KAT8调控NSL复合物亚基1；KdVS：库伦-德弗里斯综合征；LAMP1：溶酶体相关膜蛋白1（lysosomal associated membrane protein 1）；MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）；MEA：微电极阵列（micro-electrode array）；MTOR：雷帕霉素机制性靶激酶；NSL复合物：非特异性致死复合物；8-oxo-dG：8-羟基脱氧鸟苷（8-hydroxydesoxyguanosine）；RAP：雷帕霉素（rapamycin）；ROS：活性氧；sEPSCs：自发性兴奋性突触后电流（spontaneous excitatory postsynaptic currents）；SOD1：超氧化物歧化酶1；SQSTM1/p62：自噬衔接蛋白p62（sequestosome 1）；SYN：突触素（synapsin）；WRT：渥曼青霉素（wortmannin）。