A novel anticonvulsant mechanism via inhibition of complement receptor C5ar1 in murine epilepsy models
Introduction
Epilepsy is a debilitating disease, especially if seizures cannot be managed with medication. The global prevalence of treatment-resistance within epileptic populations is approximately 35%, highlighting the need for new anti-epileptic treatments effective for these patients (Kwan et al., 2011).
A new approach different from existing anti-epileptic drug (AED) mechanistic pathways is proposed, stemming from recent findings relating inflammation with epilepsy (Vezzani et al., 2011). Anti-inflammatory approaches to block seizures have already shown to be effective, not only in rodent seizure models (Vezzani and Granata, 2005, Maroso et al., 2011) but also in selected drug-resistant patients (Marchi et al., 2011) advocating inflammation as a key pharmacological target to reduce seizures and modify the disease.
The complement cascade is a major component of the innate immune system, and produces a potent inflammatory response when activated (Amara et al., 2010). Recent evidence has linked complement factor expression and activation with epilepsy and seizure development. For example, gene expression studies demonstrated upregulation of various complement factors in surgically removed tissue from patients with temporal lobe epilepsy (TLE), and in rodent TLE models (Becker et al., 2003, Gorter et al., 2006, Jamali et al., 2006, Aronica et al., 2007). Increased immunoreactivity of multiple complement factors, such as C1q, C3, C4, and the membrane attack complex (MAC), consisting of the C5b-9 complex, are documented on activated microglia and select neurons in brain tissue from TLE patients and rodent TLE models (Aronica et al., 2007, Kharatishvili et al., 2014). The presence of MAC strongly suggests complement cascade activation in “epileptic tissue”, which includes C5a formation. Moreover, in untreated people with epilepsy, significantly increased C3 serum levels were found compared to healthy and epileptic control patients, indicating complement involvement in seizures (Başaran et al., 1994).
There is also functional evidence that complement in the brain may contribute to seizure generation. The presence of specific genetic polymorphisms in the C3 promoter region was significantly greater in mesial TLE patients and correlated with increased febrile seizure susceptibility (Jamali et al., 2010). In addition, sequential infusion of individual C5b-C9 complement proteins into the rat hippocampus resulted in MAC immunoreactivity and induced seizures and neurodegeneration (Xiong et al., 2003). Recently, it was also reported that C5a receptor (C5ar1) expression on microglia was upregulated following excitotoxic kainate insult and is controlled by FosB, a mediator of transcriptional activator AP-1 (Nomaru et al., 2014). Deletion of FosB, and hence lack of C5ar1 upregulation also reduced pro-inflammatory cytokine expression following the insult. These studies provide evidence that complement presence and/or activation in the brain can mediate changes relevant to seizure generation and epilepsy pathophysiology.
In this study, we aimed to identify the therapeutic potential of inhibiting complement-mediated inflammation using several epilepsy models. We focussed on the complement peptide C5a, an inflammatory mediator. It is produced from proteolytic cleavage of its precursor C5, typically via C5 convertases formed from C3 during complement cascade activation. C5a is extremely potent at stimulating immune cell recruitment and subsequent activation through its receptor C5ar1, and induces production of pro-inflammatory cytokines and mediators (Woodruff et al., 2010).
Here, we used the selective cyclic hexapeptide C5ar1 antagonist, PMX53 (Wong et al., 1998, Finch et al., 1999) to assess the role of C5ar1 activation in seizures and epilepsy pathophysiology. Binding studies have demonstrated that PMX53 binds to and inhibits C5ar1 with high affinity (~ 20 nM). Also, PMX53 did not alter the action of closely related inflammatory mediators including fMet-Leu-Phe, leukotriene B4, platelet-activating factor, C3a, or IL-8 (March et al., 2004). Furthermore, it has been shown that PMX53 does not inhibit the binding of C5a to the second, alternative C5a receptor, C5ar2 (Scola et al., 2007). Preclinically PMX53 has been neuroprotective in several neurological disease models, including Huntington's disease, amyotrophic lateral sclerosis, traumatic brain injury, stroke, and spinal cord injury (Sewell et al., 2004, Woodruff et al., 2006, Woodruff et al., 2008, Woodruff et al., 2010, Kim et al., 2008, Li et al., 2014).
We identified the effects of C5ar1 inhibition and absence in several models of experimental epilepsy revealing that it may provide new avenues for AED discovery, specifically for pharmaco-resistant patients.
Section snippets
Mice
8–10 week old male Swiss CD1 mice (Animal Resources Centre, Western Australia) were singly housed with a 12 h light/dark cycle and access to food and water ad libitum. Male C57BL/6 C5ar1-deficient mice were bred into the CD1 background at The University of Queensland (UQ). CD1 mice were obtained from Animal Resource Centre (Perth, Australia). Initial C5ar1-deficient breeding pairs were obtained from Trent Woodruff (UQ) and were originally described by Hollmann et al. (2008). After five
C5ar1 antagonist, PMX53, reached the CNS in levels equivalent to the IC50
LC–MS/MS analysis of plasma and brain tissue after PMX53 injection (3 mg/kg, s.c.) showed that circulating PMX53 levels were highest at the measurement timepoint 15 min post-injection at 432 ng/ml (~ 480 nM) and were rapidly depleted by 90 min (Fig. 1a), with no drug detectable in the plasma or brain after 4 h (data not shown). 15 ng/g of PMX53 was found in perfused brain tissue at 30 min post-injection, equating to approximately 20 nM in brain extracellular fluid, the IC50 for the drug at the C5a
Discussion
In these studies we have shown the therapeutic potential for the inhibition of the innate immune complement receptor C5ar1 as a novel anticonvulsant pathway with possible association to neuroprotective effects.
The ability of PMX53 to significantly increase seizure thresholds and reduce spontaneous recurrent seizures in several murine seizure models suggests that C5ar1 has an important influence on seizures and that C5ar1 inhibition may be a viable strategy to reduce seizure burden in several
Conclusions
In conclusion, this work has demonstrated a pathogenic role of complement C5ar1 signalling in seizure initiation and severity as well as SE-induced neuronal degeneration. From these studies the definitive mechanisms of anticonvulsant action for PMX53 are still unclear. However, this work has outlined that inflammatory cytokine expression and modulation of microglial potassium channels are affected by C5ar1 absence or inhibition by PMX53 and may be part of a larger mechanism of action (Fig. 7).
Acknowledgments
This work was supported by grants 63145 and 1044407 (KB) from the Australian National Health and Medical Research Council and also funding from Australian Rotary Health and the Rotary Club of Koo Wee Rup—Lang Lang. We thank David She for kindling of the mice, Kah Ni Tan for assistance with qPCR, Dr. Detlev Boison and Dr. Panos Theofilas for teaching of the i.h. kainate model and EEG and finally Dr. Silvia Manzanero for her assistance with flow cytometry experiments. All authors declare they
References (64)
- et al.
Complement activation in experimental and human temporal lobe epilepsy
Neurobiol. Dis.
(2007) - et al.
The dual role of TNF-α and its receptors in seizures
Exp. Neurol.
(2013) - et al.
Humoral and cellular immune parameters in untreated and phenytoin- or carbamazepine-treated epileptic patients
Int. J. Immunopharmacol.
(1994) - et al.
Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model
Exp. Neurol.
(2003) - et al.
Disruption of the C5a receptor gene increases resistance to acute gram-negative bacteremia and endotoxic shock: opposing roles of C3a and C5a
Mol. Immunol.
(2008) - et al.
Complement factor C5a and epidermal growth factor trigger the activation of outward potassium currents in cultured murine microglia
Neuroscience
(1996) - et al.
Arthritis critically dependent on innate immune system players
Immunity
(2002) - et al.
The complement component C5a induces the expression of plasminogen activator inhibitor-1 in human macrophages via NF-κB activation
J. Thromb. Haemost.
(2006) - et al.
Protective effect of C5a receptor inhibition after murine reperfused stroke
Neurosurgery
(2008) - et al.
Evidence for a unique profile of levetiracetam in rodent models of seizures and epilepsy
Eur. J. Pharmacol.
(1998)
Complement C5a is detrimental to histological and functional locomotor recovery after spinal cord injury in mice
Neurobiol. Dis.
Interleukin-1β biosynthesis inhibition reduces acute seizures and drug resistant chronic epileptic activity in mice
Neurotherapeutics
The C5a complement activation peptide increases IL-1β and IL-6 release from amyloid-β primed human monocytes: implications for Alzheimer's disease
J. Neuroimmunol.
Expression of C5a receptor in mouse brain: role in signal transduction and neurodegeneration
Neuroscience
Anticonvulsant profile of a balanced ketogenic diet in acute mouse seizure models
Epilepsy Res.
Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration
Brain Res.
The role of the N-terminal domain of the complement fragment receptor C5L2 in ligand binding
J. Biol. Chem.
Complement C3 and C5 play critical roles in traumatic brain cryoinjury: blocking effects on neutrophil extravasation by C5a receptor antagonist
J. Neuroimmunol.
Triheptanoin in acute mouse seizure models
Epilepsy Res.
Anticonvulsant effects of a triheptanoin diet in two mouse chronic seizure models
Neurobiol. Dis.
Molecular intercommunication between the complement and coagulation systems
J. Immunol.
A novel non-transcriptional pathway mediates the proconvulsive effects of interleukin-1beta
Brain
Control of synaptic strength by glial TNFα
Science
Correlated stage- and subfield-associated hippocampal gene expression patterns in experimental and human temporal lobe epilepsy
Eur. J. Neurosci.
CXCR4-activated astrocyte glutamate release via TNFα: amplification by microglia triggers neurotoxicity
Nat. Neurosci.
A role for leukocyte-endothelial adhesion mechanisms in epilepsy
Nat. Med.
Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a
J. Med. Chem.
Potential new antiepileptogenic targets indicated by microarray analysis in a rat model for temporal lobe epilepsy
J. Neurosci.
Seizure suppression by adenosine A1 receptor activation in a mouse model of pharmacoresistant epilepsy
Epilepsia
The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria
N. Engl. J. Med.
An approach for the development and selection of chromatographic methods for high-throughput metabolomic screening of urine by ultra pressure LC–ESI-ToF-MS
Metabolomics
Cytokine effects on glutamate uptake by human astrocytes
Neuroimmunomodulation
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