Human Variants in the Neuronal Basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) Transcription Factor Complex NPAS4/ARNT2 Disrupt Function

Neuronal Per-Arnt-Sim homology (PAS) Factor 4 (NPAS4) is a neuronal activity-dependent transcription factor which heterodimerises with ARNT2 to regulate genes involved in inhibitory synapse formation. NPAS4 functions to maintain excitatory/inhibitory balance in neurons, while mouse models have shown it to play roles in memory formation, social interaction and neurodegeneration. NPAS4 has therefore been implicated in a number of neuropsychiatric or neurodegenerative diseases which are underpinned by defects in excitatory/inhibitory balance. Here we have explored a broad set of non-synonymous human variants in NPAS4 and ARNT2 for disruption of NPAS4 function. We found two variants in NPAS4 (F147S and E257K) and two variants in ARNT2 (R46W and R107H) which significantly reduced transcriptional activity of the heterodimer on a luciferase reporter gene. Furthermore, we found that NPAS4.F147S was unable to activate expression of the NPAS4 target gene BDNF due to reduced dimerisation with ARNT2. Homology modelling predicts F147 in NPAS4 to lie at the dimer interface, where it appears to directly contribute to protein/protein interaction. We also found that reduced transcriptional activation by ARNT2 R46W was due to disruption of nuclear localisation. These results provide insight into the mechanisms of NPAS4/ARNT dimerisation and transcriptional activation and have potential implications for cognitive phenotypic variation and diseases such as autism, schizophrenia and dementia.

神经元Per-Arnt-Sim同源结构域（PAS）因子4（NPAS4）是一类依赖神经元活动的转录因子，可与ARNT2形成异二聚体，调控参与抑制性突触形成的靶基因。NPAS4的核心功能是维持神经元内的兴奋/抑制平衡，现有小鼠模型研究证实其在记忆形成、社交互动及神经退行性病变过程中发挥关键调控作用。鉴于此，NPAS4被认为与多种以兴奋/抑制平衡缺陷为病理基础的神经精神疾病或神经退行性疾病密切相关。本研究针对NPAS4与ARNT2中的多个人类非同义变异，系统性探究了其对NPAS4功能的破坏效应。研究发现，NPAS4中的2个变异位点（F147S与E257K）以及ARNT2中的2个变异位点（R46W与R107H），可显著降低该异二聚体对荧光素酶报告基因的转录活性。进一步机制研究显示，NPAS4.F147S因与ARNT2的二聚化能力显著下降，无法正常激活其靶基因脑源性神经营养因子（Brain-Derived Neurotrophic Factor，BDNF）的表达。同源建模预测结果表明，NPAS4中的F147位点位于蛋白质二聚体界面，可直接参与蛋白质-蛋白质相互作用。此外，ARNT2 R46W所介导的转录激活能力降低，源于其核定位过程遭到破坏。本研究结果阐明了NPAS4/ARNT异二聚化及转录激活的分子机制，对认知表型变异及孤独症、精神分裂症、痴呆等疾病的相关研究具有潜在的参考价值。