Cell type-specific expression, regulation and compensation of CDKL5 activity in mouse brain
Cell type-specific expression, regulation and compensation of CDKL5 activity in mouse brain
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小鼠脑中 CDKL5 活性的细胞类型特异性表达、调节和补偿
DOI：
10.1038/s41380-024-02434-7
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发表时间：
2024
影响因子：
11
通讯作者：
Silvestre M
中科院分区：
文献类型：
--
作者：
Silvestre M
CDKL5是一种富含大脑的丝氨酸/苏氨酸激酶，与一种称为CDKL5缺乏症(CDD)的严重发育和癫痫脑病有关。为了设计针对CDD的靶向治疗方法，必须确定CDKL5在大脑中的表达和活性，并测试细胞水平是否存在代偿机制。我们在兴奋性神经元、抑制性神经元和星形胶质细胞中产生了条件性CDKL5基因敲除小鼠。为了评估CDKL5的活性，我们使用了一种针对磷酸化EB2的磷酸化抗体，EB2是CDKL5的已知底物。我们发现CDKL5和EB2pS222在兴奋性和抑制性神经元中显著表达，而在星形胶质细胞中未检测到。我们观察到，大约15%-20%的EB2pS222保留在CDKL5基因敲除的脑和初级神经元中。令人惊讶的是，在神经元CDKL5基因敲除培养中，剩余的磷酸化受到NMDA和PP1/PP2A的调节，表明存在代偿性激酶。通过筛选与CDKL5激酶结构域同源性最高的候选蛋白，我们发现CDKL2和ICK可以在HEK293T细胞和原代神经元中磷酸化EB2S222。然后我们产生了CDKL5/CDKL2双基因敲除小鼠来直接测试CDKL2是否在体内磷酸化EB2，发现CDKL2磷酸化了大脑中的CDKL5底物。这项研究是CDKL2有可能取代CDKL5在大脑中的功能的第一个迹象，暗示了新的治疗可能性。
CDKL5 is a brain-enriched serine/threonine kinase, associated with a profound developmental and epileptic encephalopathy called CDKL5 deficiency disorder (CDD). To design targeted therapies for CDD, it is essential to determine where CDKL5 is expressed and is active in the brain and test if compensatory mechanisms exist at cellular level. We generated conditionalCdkl5knockout mice in excitatory neurons, inhibitory neurons and astrocytes. To assess CDKL5 activity, we utilized a phosphospecific antibody for phosphorylated EB2, a well-known substrate of CDKL5. We found that CDKL5 and EB2 pS222 were prominent in excitatory and inhibitory neurons but were not detected in astrocytes. We observed that approximately 15–20% of EB2 pS222 remained inCdkl5knockout brains and primary neurons. Surprisingly, the remaining phosphorylation was modulated by NMDA and PP1/PP2A in neuronal CDKL5 knockout cultures, indicating the presence of a compensating kinase. Using a screen of candidate kinases with highest homology to the CDKL5 kinase domain, we found that CDKL2 and ICK can phosphorylate EB2 S222 in HEK293T cells and in primary neurons. We then generatedCdkl5/Cdkl2dual knockout mice to directly test if CDKL2 phosphorylates EB2 in vivo and found that CDKL2 phosphorylates CDKL5 substrates in the brain. This study is the first indication that CDKL2 could potentially replace CDKL5 functions in the brain, alluding to novel therapeutic possibilities.