Data_Sheet_1_PKN1 Is a Novel Regulator of Hippocampal GluA1 Levels.pdf

Alterations in the processes that control α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression, assembly and trafficking are closely linked to psychiatric and neurodegenerative disorders. We have recently shown that the serine/threonine kinase Protein kinase N1 (PKN1) is a developmentally active regulator of cerebellar synaptic maturation by inhibiting AKT and the neurogenic transcription factor neurogenic differentiation factor-2 (NeuroD2). NeuroD2 is involved in glutamatergic synaptic maturation by regulating expression levels of various synaptic proteins. Here we aimed to study the effect of Pkn1 knockout on AKT phosphorylation and NeuroD2 levels in the hippocampus and the subsequent expression levels of the NeuroD2 targets and AMPAR subunits: glutamate receptor 1 (GluA1) and GluA2/3. We show that PKN1 is expressed throughout the hippocampus. Interestingly, not only postnatal but also adult hippocampal phospho-AKT and NeuroD2 levels were significantly elevated upon Pkn1 knockout. Postnatal and adult Pkn1–/– hippocampi showed enhanced expression of the AMPAR subunit GluA1, particularly in area CA1. Surprisingly, GluA2/3 levels were not different between both genotypes. In addition to higher protein levels, we also found an enhanced GluA1 content in the membrane fraction of postnatal and adult Pkn1–/– animals, while GluA2/3 levels remained unchanged. This points toward a very specific regulation of GluA1 expression and/or trafficking by the novel PKN1-AKT-NeuroD2 axis. Considering the important role of GluA1 in hippocampal development as well as the pathophysiology of several disorders, ranging from Alzheimer’s, to depression and schizophrenia, our results validate PKN1 for future studies into neurological disorders related to altered AMPAR subunit expression in the hippocampus.

调控α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor，AMPAR）表达、组装与转运过程的异常，与多种精神疾病及神经退行性疾病密切相关。本课题组近期研究发现，丝氨酸/苏氨酸激酶蛋白激酶N1（Protein kinase N1，PKN1）可通过抑制AKT及神经源性转录因子神经分化因子2（Neurogenic differentiation factor-2，NeuroD2），在发育阶段调控小脑突触成熟。NeuroD2可通过调控多种突触蛋白的表达水平，参与谷氨酸能突触的成熟过程。本研究旨在探究Pkn1基因敲除对海马体中AKT磷酸化水平及NeuroD2表达量的影响，以及由此引发的NeuroD2靶标蛋白与AMPAR亚基——谷氨酸受体1（Glutamate receptor 1，GluA1）与GluA2/3的表达变化。研究表明，PKN1在整个海马体中均有表达。值得注意的是，Pkn1基因敲除后，不仅幼年期海马体的磷酸化AKT与NeuroD2水平显著升高，成年海马体中二者的表达量同样显著提升。幼年期与成年期的Pkn1敲除（Pkn1–/–）海马体中，AMPAR亚基GluA1的表达均出现增强，尤其在CA1区更为显著。令人意外的是，两种基因型小鼠的GluA2/3水平并无差异。除总蛋白水平升高外，我们还发现幼年期与成年期Pkn1–/–小鼠的膜组分中GluA1含量显著提升，而GluA2/3的水平未发生改变。这表明，全新的PKN1-AKT-NeuroD2信号轴可特异性调控GluA1的表达与/或转运过程。鉴于GluA1在海马体发育以及阿尔茨海默病、抑郁症、精神分裂症等多种疾病的病理生理过程中发挥关键作用，本研究结果证实，PKN1可作为未来研究海马体AMPAR亚基表达异常相关神经系统疾病的潜在靶点。