Table_2_CLK2 in GABAergic neurons is critical in regulating energy balance and anxiety-like behavior in a gender-specific fashion.docx

IntroductionCdc2-like kinase (CLK2) is a member of CLK kinases expressed in hypothalamic neurons and is activated in response to refeeding, leptin, or insulin. Diet-induced obesity and leptin receptor-deficient db/db mice lack CLK2 signal in the hypothalamic neurons. The neurotransmiter gamma-aminobutyric acid (GABA) is among the most prevalent in the central nervous system (CNS), particularly in the hypothalamus. Given the abundance of GABA-expressing neurons and their potential influence on regulating energy and behavioral homeostasis, we aimed to explore whether the deletion of CLK2 in GABAergic neurons alters energy homeostasis and behavioral and cognitive functions in both genders of mice lacking CLK2 in Vgat-expressing neurons (Vgat-Cre; Clk2loxP/loxP) on chow diet.
MethodsWe generated mice lacking Clk2 in Vgat-expressing neurons (Vgat-Cre; Clk2loxP/loxP) by mating Clk2loxP/loxP mice with Vgat-IRES-Cre transgenic mice and employed behavior, and physiological tests, and molecular approaches to investigate energy metabolism and behavior phenotype of both genders.
Results and discussionWe showed that deletion of CLK2 in GABAergic neurons increased adiposity and food intake in females. The mechanisms behind these effects were likely due, at least in part, to hypothalamic insulin resistance and upregulation of hypothalamic Npy and Agrp expression. Besides normal insulin and pyruvate sensitivity, Vgat-Cre; Clk2loxP/loxP females were glucose intolerant. Male Vgat-Cre; Clk2loxP/loxP mice showed an increased energy expenditure (EE). Risen EE may account for avoiding weight and fat mass gain in male Vgat-Cre; Clk2loxP/loxP mice. Vgat-Cre; Clk2loxP/loxP mice had no alteration in cognition or memory functions in both genders. Interestingly, deleting CLK2 in GABAergic neurons changed anxiety-like behavior only in females, not males. These findings suggest that CLK2 in GABAergic neurons is critical in regulating energy balance and anxiety-like behavior in a gender-specific fashion and could be a molecular therapeutic target for combating obesity associated with psychological disorders in females.

引言
Cdc2样激酶（CLK2）属于CLK激酶家族，在下丘脑神经元中表达，可被再喂养、瘦素或胰岛素激活。饮食诱导肥胖小鼠及瘦素受体缺陷型db/db小鼠的下丘脑神经元中无法检测到CLK2信号。神经递质γ-氨基丁酸（GABA）是中枢神经系统（CNS）中分布最为广泛的神经递质之一，在下丘脑中尤为丰富。鉴于表达GABA的神经元数量众多，且其对能量与行为稳态具有潜在调控作用，本研究旨在探究在饲喂标准饲料的条件下，Vgat阳性神经元中敲除CLK2的小鼠（Vgat-Cre; Clk2loxP/loxP）的雌雄个体，其GABA能神经元中CLK2缺失是否会改变能量稳态以及行为与认知功能。
方法
本研究通过将Clk2loxP/loxP小鼠与Vgat-IRES-Cre转基因小鼠杂交，构建了在Vgat阳性神经元中敲除Clk2的小鼠（Vgat-Cre; Clk2loxP/loxP），并采用行为学、生理学检测及分子生物学手段，探究该基因型雌雄小鼠的能量代谢与行为表型。
结果与讨论
本研究发现，GABA能神经元中敲除CLK2会使雌性小鼠的体脂含量与摄食量增加，其潜在机制至少部分与下丘脑胰岛素抵抗及下丘脑Npy、Agrp基因表达上调有关。此外，Vgat-Cre; Clk2loxP/loxP雌性小鼠虽胰岛素与丙酮酸敏感性正常，但出现了糖耐量受损。雄性Vgat-Cre; Clk2loxP/loxP小鼠的能量消耗（EE）升高，这一变化可避免其体重与脂肪量增加。雌雄两性的Vgat-Cre; Clk2loxP/loxP小鼠的认知与记忆功能均未发生改变。值得注意的是，GABA能神经元中敲除CLK2仅会改变雌性小鼠的焦虑样行为，对雄性小鼠无此影响。上述结果表明，GABA能神经元中的CLK2以性别特异性方式调控能量平衡与焦虑样行为，或可作为治疗女性伴随心理障碍的肥胖症的分子靶点。