Wyatt Season K, Witt Thomas, Barbaro Nicholas M, Cohen-Gadol Aaron A, Brewster Amy L
Department of Psychological Sciences, Purdue University, West Lafayette, IN, USA.
Goodman Campbell Brain and Spine, Department of Neurological Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.
Exp Neurol. 2017 Sep;295:184-193. doi: 10.1016/j.expneurol.2017.06.009. Epub 2017 Jun 7.
Microglia-mediated neuroinflammation is widely associated with seizures and epilepsy. Although microglial cells are professional phagocytes, less is known about the status of this phenotype in epilepsy. Recent evidence supports that phagocytosis-associated molecules from the classical complement (C1q-C3) play novel roles in microglia-mediated synaptic pruning. Interestingly, in human and experimental epilepsy, altered mRNA levels of complement molecules were reported. Therefore, to identify a potential role for complement and microglia in the synaptodendritic pathology of epilepsy, we determined the protein levels of classical complement proteins (C1q-C3) along with other phagocytosis signaling molecules in human epilepsy. Cortical brain samples surgically resected from patients with refractory epilepsy (RE) and non-epileptic lesions (NE) were examined. Western blotting was used to determine the levels of phagocytosis signaling proteins such as the complements C1q and C3, MerTK, Trem2, and Pros1 along with cleaved-caspase 3. In addition, immunostaining was used to determine the distribution of C1q and co-localization to microglia and dendrites. We found that the RE samples had significantly increased protein levels of C1q (p=0.034) along with those of its downstream activation product iC3b (p=0.027), and decreased levels of Trem2 (p=0.045) and Pros1 (p=0.005) when compared to the NE group. Protein levels of cleaved-caspase 3 were not different between the groups (p=0.695). In parallel, we found C1q localization to microglia and dendrites in both NE and RE samples, and also observed substantial microglia-dendritic interactions in the RE tissue. These data suggest that aberrant phagocytic signaling occurs in human refractory epilepsy. It is likely that alteration of phagocytic pathways may contribute to unwanted elimination of cells/synapses and/or impaired clearance of dead cells. Future studies will investigate whether altered complement signaling contributes to the hyperexcitability that result in epilepsy.
小胶质细胞介导的神经炎症与癫痫发作和癫痫密切相关。尽管小胶质细胞是专职吞噬细胞，但对于该表型在癫痫中的状态了解较少。最近的证据支持经典补体（C1q - C3）中的吞噬相关分子在小胶质细胞介导的突触修剪中发挥新作用。有趣的是，在人类和实验性癫痫中，有报道称补体分子的mRNA水平发生了改变。因此，为了确定补体和小胶质细胞在癫痫突触树突病理中的潜在作用，我们测定了人类癫痫中经典补体蛋白（C1q - C3）以及其他吞噬信号分子的蛋白水平。对难治性癫痫（RE）患者和非癫痫性病变（NE）患者手术切除的皮质脑样本进行了检查。采用蛋白质印迹法测定吞噬信号蛋白的水平，如补体C1q和C3、MerTK、Trem2、Pros1以及裂解的半胱天冬酶3。此外，采用免疫染色法确定C1q的分布以及与小胶质细胞和树突的共定位。我们发现，与NE组相比，RE样本中C1q的蛋白水平显著升高（p = 0.034），其下游激活产物iC3b的蛋白水平也显著升高（p = 0.027），而Trem2（p = 0.045）和Pros1（p = 0.005）的水平降低。两组之间裂解的半胱天冬酶3的蛋白水平没有差异（p = 0.695）。同时，我们在NE和RE样本中均发现C1q定位于小胶质细胞和树突，并且在RE组织中还观察到大量小胶质细胞与树突的相互作用。这些数据表明，在人类难治性癫痫中存在异常的吞噬信号。吞噬途径的改变可能导致细胞/突触的不必要清除和/或死细胞清除受损。未来的研究将探讨补体信号改变是否导致了引发癫痫的兴奋性过高。
