Department of Neurochemistry, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Department of Neuroscience I, Research Institute of Environmental Medicine, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan.
J Neurosci. 2018 Jun 13;38(24):5551-5566. doi: 10.1523/JNEUROSCI.2357-17.2018. Epub 2018 May 17.
Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCC during brain development remain unclear. Calcium signaling has been shown to be essential for neurodevelopmental processes such as sculpting of neurites, functional wiring, and fine tuning of growing networks. To investigate this relationship, we performed submembraneous calcium imaging using a membrane-tethered genetically encoded calcium indicator (GECI) Lck-G-CaMP7. We successfully recorded pontaneous regenerative calcium transients (SRCaTs) in developing mouse excitatory cortical neurons prepared from both sexes before synapse formation. SRCaTs originated locally in immature neurites independently of somatic calcium rises and were significantly more elevated in the axons than in dendrites. SRCaTs were not blocked by tetrodoxin, a Na channel blocker, but were strongly inhibited by hyperpolarization, suggesting a voltage-dependent source. Pharmacological and genetic manipulations revealed the critical importance of the Ca1.2 (CACNA1C) pore-forming subunit of L-type VGCCs, which were indeed expressed in immature mouse brains. Consistently, knocking out Ca1.2 resulted in significant alterations of neurite outgrowth. Furthermore, expression of a gain-of-function Ca1.2 mutant found in Timothy syndrome, an autosomal dominant multisystem disorder exhibiting syndromic autism, resulted in impaired radial migration of layer 2/3 excitatory neurons, whereas postnatal abrogation of Ca1.2 enhancement could rescue cortical malformation. Together, these lines of evidence suggest a critical role for spontaneous opening of L-type VGCCs in neural development and corticogenesis and indicate that L-type VGCCs might constitute a perinatal therapeutic target for neuropsychiatric calciochannelopathies. Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCCs during brain development remain unclear. We here combined the latest Ca indicator technology, quantitative pharmacology, and electroporation and found a hitherto unsuspected role for L-type VGCCs in determining the Ca signaling landscape of mouse immature neurons. We found that malfunctional L-type VGCCs in immature neurons before birth might cause errors in neuritic growth and cortical migration. Interestingly, the retarded corticogenesis phenotype was rescued by postnatal correction of L-type VGCC signal aberration. These findings suggest that L-type VGCCs might constitute a perinatal therapeutic target for neurodevelopment-associated psychiatric disorders.
尽管许多关联研究将神经发育障碍（如自闭症和精神分裂症）中的 L 型电压门控钙通道（VGCC）的基因多态性和突变与这些疾病联系起来，但特定 L 型 VGCC 在大脑发育过程中的作用仍不清楚。钙信号已被证明对于神经发育过程至关重要，例如神经突的塑造、功能连接和生长网络的微调。为了研究这种关系，我们使用膜结合的遗传编码钙指示剂（GECI）Lck-G-CaMP7 进行了亚膜钙成像。我们成功地记录了来自两性的发育中的小鼠兴奋性皮质神经元在突触形成之前自发产生的再生钙瞬变（SRCaTs）。SRCaTs 局部起源于不成熟的神经突中，与体细胞钙升高无关，并且在轴突中明显高于树突。河豚毒素（一种钠通道阻断剂）不能阻断 SRCaTs，但强烈抑制超极化，表明其为电压依赖性来源。药理学和遗传学操作表明，L 型 VGCC 的钙通道形成亚基 Ca1.2（CACNA1C）的重要性，该亚基确实在不成熟的小鼠脑中表达。一致地，敲除 Ca1.2 导致神经突生长的显著改变。此外，表达在常染色体显性多系统疾病 Timothy 综合征中发现的一种功能获得性 Ca1.2 突变体，导致层 2/3 兴奋性神经元的放射状迁移受损，而产后 Ca1.2 增强的消除可以挽救皮质畸形。总之，这些证据表明，L 型 VGCC 的自发开放在神经发育和皮质发生中起着关键作用，并表明 L 型 VGCC 可能成为围产期神经精神钙通道病的治疗靶点。尽管许多关联研究将神经发育障碍（如自闭症和精神分裂症）中的 L 型电压门控钙通道（VGCC）的基因多态性和突变与这些疾病联系起来，但特定 L 型 VGCC 在大脑发育过程中的作用仍不清楚。我们在这里结合了最新的钙指示剂技术、定量药理学和电穿孔，并发现 L 型 VGCC 在决定小鼠不成熟神经元的钙信号景观方面发挥了意想不到的作用。我们发现，出生前不成熟神经元中功能失调的 L 型 VGCC 可能导致神经突生长和皮质迁移错误。有趣的是，出生后 L 型 VGCC 信号异常的校正挽救了皮质发生延迟的表型。这些发现表明，L 型 VGCC 可能成为围产期治疗与神经发育相关的精神障碍的靶点。
