Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, 23298, USA.
Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA, 23298, USA.
J Mol Neurosci. 2022 Sep;72(9):1965-1976. doi: 10.1007/s12031-022-01988-x. Epub 2022 Jul 19.
Altered amine transporter function, phosphorylation, and association with interacting proteins are evident in animals with a history of psychostimulant exposure. Our previous studies have shown that the Thr258/Ser259 motif in the norepinephrine transporter (NET) is involved in amphetamine (AMPH)-mediated NET regulation and behavior. However, the neurobiological consequences of in vivo Thr258/Ser259-dependent NET regulation in an intact animal model are unclear. Therefore, we generated a viable construct-valid NET-Thr258Ala/Ser259Ala (NET-T258A/S259A) mouse model using CRISPR/Cas9 technology by replacing Thr258/Ser259 motif with Ala258/Ala259 motif. NET-T258A/S259A mice have a birth rate consistent with Mendelian inheritance ratios. Both male and female homozygous NET-T258A/S259A mice are viable, display normal growth and general health, and exhibit normal body weight (sex-dependent) and total activity in the open field similar to their wild-type (WT) littermates. NET-T258A/S259A mice showed reduced NET function in the prefrontal cortex (PFC) compared to WT mice while NET function in the nucleus accumbens (NAc) remained unchanged. Compared to respective WT counterparts, NET-T258A/S259A males but not females showed significantly reduced locomotor activation in response to acute AMPH administration and significantly reduced AMPH-induced conditioned place preference (CPP). When tested in the males only, acute AMPH administration inhibited NET function and surface expression in the WT NAc but not in the NET-T258A/S259A NAc while AMPH administration inhibited DAT function and surface expression in the NAc of both WT and NET-T258A/S259A mice. Collectively, our findings reveal that the mice carrying the T258A/S259A mutation in NET gene display brain region-specific differences in NET functional expression and blunted response to AMPH.
改变胺转运蛋白的功能、磷酸化和与相互作用蛋白的关联在有精神兴奋剂暴露史的动物中很明显。我们之前的研究表明，去甲肾上腺素转运体（NET）中的 Thr258/Ser259 基序参与了安非他命（AMPH）介导的 NET 调节和行为。然而，在完整动物模型中，体内 Thr258/Ser259 依赖性 NET 调节的神经生物学后果尚不清楚。因此，我们使用 CRISPR/Cas9 技术生成了一个可行的构建体验证 NET-Thr258Ala/Ser259Ala（NET-T258A/S259A）小鼠模型，通过用 Ala258/Ala259 取代 Thr258/Ser259 基序。NET-T258A/S259A 小鼠的出生率符合孟德尔遗传比例。雄性和雌性纯合 NET-T258A/S259A 小鼠均具有活力，表现出正常的生长和整体健康状况，并且其体重（性别依赖性）和旷场中的总活动与野生型（WT）同窝仔相似。与 WT 小鼠相比，NET-T258A/S259A 小鼠的前额叶皮质（PFC）中 NET 功能降低，而伏隔核（NAc）中的 NET 功能保持不变。与各自的 WT 对应物相比，NET-T258A/S259A 雄性但不是雌性在急性 AMPH 给药后表现出运动激活明显减少，并且 AMPH 诱导的条件位置偏好（CPP）明显减少。仅在雄性中进行测试时，急性 AMPH 给药抑制了 WT NAc 中的 NET 功能和表面表达，但不抑制 NET-T258A/S259A NAc 中的 NET 功能和表面表达，而 AMPH 给药抑制了 WT 和 NET-T258A/S259A 小鼠 NAc 中的 DAT 功能和表面表达。总的来说，我们的研究结果表明，携带 NET 基因中 T258A/S259A 突变的小鼠在 NET 功能表达上表现出脑区特异性差异，并且对 AMPH 的反应迟钝。
