Data_Sheet_1_Complement C4-deficient mice have a high mortality rate during PTZ-induced epileptic seizures, which correlates with cognitive problems and the deficiency in the expression of Egr1 and other immediate early genes.PDF

Complement system plays an important role in the immune defense against pathogens; however, recent studies demonstrated an important role of complement subunits C1q, C4, and C3 in normal functions of the central nervous system (CNS) such as non-functional synapse elimination (synapse pruning), and during various neurologic pathologies. Humans have two forms of C4 protein encoded by C4A and C4B genes that share 99.5% homology, while mice have only one C4B gene that is functionally active in the complement cascade. Overexpression of the human C4A gene was shown to contribute to the development of schizophrenia by mediating extensive synapse pruning through the activation C1q-C4-C3 pathway, while C4B deficiency or low levels of C4B expression were shown to relate to the development of schizophrenia and autism spectrum disorders possibly via other mechanisms not related to synapse elimination. To investigate the potential role of C4B in neuronal functions not related to synapse pruning, we compared wildtype (WT) mice with C3- and C4B- deficient animals for their susceptibility to pentylenetetrazole (PTZ)- induced epileptic seizures. We found that C4B (but not C3)–deficient mice were highly susceptible to convulsant and subconvulsant doses of PTZ when compared to WT controls. Further gene expression analysis revealed that in contrast to WT or C3-deficient animals, C4B-deficient mice failed to upregulate expressions of multiple immediate early genes (IEGs) Egrs1-4, c-Fos, c-Jus, FosB, Npas4, and Nur77 during epileptic seizures. Moreover, C4B-deficient mice had low levels of baseline expression of Egr1 on mRNA and protein levels, which was correlated with the cognitive problems of these animals. C4-deficient animals also failed to upregulate several genes downstream of IEGs such as BDNF and pro-inflammatory cytokines IL-1β, IL-6, and TNF. Taken together, our study demonstrates a new role of C4B in the regulation of expression of IEGs and their downstream targets during CNS insults such as epileptic seizures.

补体系统在抵御病原体入侵的免疫防御中扮演着至关重要的角色；然而，近期研究揭示了补体亚单位C1q、C4和C3在维持中枢神经系统（CNS）正常功能，如非功能突触消除（突触修剪）以及各种神经病理学过程中的重要作用。人类有两种形式的C4蛋白，分别由C4A和C4B基因编码，两者之间具有99.5%的同源性，而小鼠则仅有一个功能活跃的C4B基因参与补体级联反应。研究表明，人类C4A基因的过表达可通过激活C1q-C4-C3途径，导致广泛的突触修剪，从而参与精神分裂症的发展。另一方面，C4B的缺乏或表达水平低下与精神分裂症和自闭症谱系障碍的发生可能通过其他与突触消除无关的机制相关。为了探究C4B在神经元功能（非突触修剪相关）中的潜在作用，我们比较了野生型（WT）小鼠与C3和C4B缺陷型动物对戊四氮（PTZ）诱导的癫痫发作的易感性。我们发现，与野生型对照组相比，C4B缺陷型小鼠对PTZ的惊厥和亚惊厥剂量表现出高度易感性。进一步的基因表达分析显示，与野生型或C3缺陷型动物不同，C4B缺陷型小鼠在癫痫发作期间未能上调多种即刻早期基因（IEGs）的表达，如Egrs1-4、c-Fos、c-Jus、FosB、Npas4和Nur77。此外，C4B缺陷型小鼠的Egr1在mRNA和蛋白质水平上的基线表达水平较低，这与这些动物的认知问题相关。C4缺陷型动物同样未能上调IEGs下游的多个基因，如BDNF和促炎细胞因子IL-1β、IL-6和TNF。综合以上研究结果，我们的研究揭示了C4B在调节中枢神经系统损伤（如癫痫发作）过程中IEGs及其下游靶基因表达的新作用。