The role of complement in epilepsy: a specific focus on C5ar1 and its role in seizure initiation and disease development

Benson, Melissa (2015). The role of complement in epilepsy: a specific focus on C5ar1 and its role in seizure initiation and disease development PhD Thesis, School of Biomedical Sciences, The University of Queensland. doi:10.14264/uql.2015.628

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Author Benson, Melissa
Thesis Title The role of complement in epilepsy: a specific focus on C5ar1 and its role in seizure initiation and disease development
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
DOI 10.14264/uql.2015.628
Publication date 2015-05-28
Thesis type PhD Thesis
Open Access Status Other
Supervisor Karin Borges
Trent Woodruff
Total pages 140
Language eng
Subjects 111501 Basic Pharmacology
110707 Innate Immunity
1109 Neurosciences
1107 Immunology
1115 Pharmacology and Pharmaceutical Sciences
110903 Central Nervous System
Formatted abstract
Neuroinflammation and its role in seizure initiation and epilepsy disease development is being investigated, specifically the role of innate immune complement activation through the C5a receptor, C5ar1.

C5ar1 expression was found to be significantly upregulated during epilepsy disease progression in several experimental mouse models, in addition to other complement components such as C3, the precursor to C5a. Building upon existing studies supporting the role of inflammation and other complement components in epilepsy, this finding indicates a contribution of C5a and complement activation in this disease. This lead to creation of the primary hypothesis of this thesis; inhibition of C5ar1 activation is potentially neuroprotective and anticonvulsant in epilepsy.

To test this hypothesis a small peptide antagonist of C5ar1, PMX53, was used. It was shown to be anticonvulsant in two acute and two chronic seizure models. PMX53 increased the seizure threshold significantly in both the 6Hz test and the corneal kindling model, as well as reduced EEG seizure power in the pilocarpine-induced SE model, indicating anticonvulsant actions. Studies using the chronic intrahippocampal kainate model combined with EEG, showed that PMX53 was acutely anticonvulsant after a single peripheral administration, significantly reducing total time in seizure by >50% as well as EEG seizure power. In addition, treatment of mice with PMX53 during pilocarpine-SE lead to a vast reduction in EEG seizure power highlighting further anticonvulsant effects. These results suggest inhibition of C5ar1 is beneficial in increasing seizure thresholds in several mouse models of epilepsy.

Further studies using inert structural analogues of PMX53 and C5ar1-deficient mice in the 6Hz model showed that PMX53’s anticonvulsant effect is C5ar1-dependent. This confirms my hypothesis that antagonism of C5ar1 is anticonvulsant. To determine if C5ar1-mediated anticonvulsant effects lead to changes in subsequent cellular damage, neuronal cell death in the hippocampal formation was evaluated acutely after pilocarpine-induced SE. In both PMX53-treated animals and mice lacking the C5ar1 receptor, significant reductions in neuronal degeneration and loss were evident in key hippocampal areas including CA1 and CA3 pyramidal cell layers.

In addition, the explicit role of C5ar1 expression on microglial cells was investigated, given that consistent upregulation of the receptor was shown at varying stages post-epileptic insult in mice. Specifically microglial activation phenotypes were assessed, based on expression patterns of inflammatory and activation markers. M1 phenotype is considered to be more inflammatory in nature and is detected through expression of pro-inflammatory cytokines, i.e. IL-1β, TNFα, IL-6 and classical activation markers, i.e. CD16, CD86. M2 phenotype is defined as being more reparative in nature, with markers such as Arginase1 (Arg1) and Chitinase-like-3 (Ym1) being associated with this phenotype, all linked to different aspects of cell repair, as well as anti-inflammatory cytokines such as IL-4 and IL-10.

Different phenotypes were observed in two disease models; pilocarpine model and intrahippocampal kainate model. The inflammatory M1 phenotype was predominant in the late chronic stage of i.h. kainate model after prolonged and frequent seizure recurrence however was no microglial activation markers were upregulated in chronic pilocarpine SE mice suggesting high seizure frequency may be a factor mediating this activation. Immediately post epileptic insult (SE) differences were also present. A mixed M1/M2 phenotype was seen in pilocarpine SE animals whereas only M1 markers were increased in i.h. kainate SE mice, suggesting that differences in SE may have affected these acute phenotypes, namely the contribution of peripheral inflammation in the pilocarpine model. It is clear from these findings that epilepsy disease progression, potentially dependent on spontaneous seizure frequency, drives a movement toward a recurrent microglial M1 phenotype, showing significant increases in markers such as C3, TNFα and IL-1β. It is proposed that this shift in microglial activation state during epilepsy development drives a more “inflamed” CNS state, leading to a greater chance of hyperexcitability and hence is likely to contribute to further seizure occurrence in a cyclic manner.

In addition, we have shown that C5ar1-deficiency during pilocarpine-SE promoted changes to cytokine expression in immune cells. Namely, C5ar1-deficiency led to ablation of increased TNFα expression in SE microglia acutely post-SE, as well as elevated IL-4 expression on infiltrating immune cells. These changes coupled, suggest a more anti-inflammatory CNS phenotype post-SE which may have putative disease-modifying potential.

The overall findings of this work suggest a role for C5ar1 in epilepsy development and seizure initiation supported by preclinical findings in several experimental epilepsy models showing that C5ar1 antagonism is anticonvulsant. This work also describes a potential new anticonvulsant drug target in C5ar1 which is novel in its mechanism compared to current antiepileptic drugs. In addition, this work has provided evidence that C5ar1 expression on microglia may be linked to a driving shift in microglial activation phenotype throughout disease progression, which leads to a more inflamed and hyper-excitable CNS; a state which promotes seizure initiation and epilepsy disease progression.
Keyword Complement
Status epilepticus

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Created: Mon, 18 May 2015, 13:16:37 EST by Melissa Benson on behalf of Scholarly Communication and Digitisation Service