Abstract
Wyatt SK, Witt T, Barbaro NM, Cohen-Gadol AA, Brewster AL. Exp Neurol 2017;295:184–193.
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.
Commentary
The immune system has become increasingly associated with disorders of the central nervous system (1,2). The classical complement pathway is part of the innate immune system and is comprised of a series of stepwise proteolytic reactions initiated by the binding of an antigen-antibody complex to C1q. The intermediates along this pathway, such as C3a and C3b, can recruit microglia, coat cells in an “eat-me” signal (in a process known as opsonization), and trigger inflammatory responses. Exciting recent work has implicated the classical complement pathway in the control of synaptic density in both physiologic and pathologic brain states. In the developing visual system, physiologic synaptic pruning via microglial phagocytosis is driven by complement (3). On the other hand, in neurodegenerative diseases such as Alzheimer's, overactivation of the complement system has been implicated in pathologic synapse loss (4). As we learn more about how microglia shape synaptic connectivity, the excitement about this avenue of exploration has begun to spread to the epilepsy field.
In a recent Experimental Neurology study from the Brewster group at Purdue, Wyatt and co-authors show enhanced classical complement pathway activation in human epileptic brain samples. Human brain tissue was collected from patients with medically refractory epilepsy (RE) associated with focal cortical dysplasia. On pathologic examination, these samples exhibited neuronal loss, mesial temporal sclerosis, and cortical and subpial gliosis. The authors show an increase in complement protein levels (C1q and iC3b, a fragment of C3b) in brain samples from RE brains, as compared with control samples (peritumoral and other brain tissues from patients with no history of seizures). This finding is in line with previous work from Aronica et al. (5), which showed increased C1q and C3 mRNA levels in the hippocampus of human temporal lobe epilepsy (TLE) patients. However, Wyatt et al. further investigated the molecular mediators of microglial phagocytosis downstream of the complement pathway. They found decreased levels of Trem2 and Pros1 (both “eat-me” signals that can trigger phagocytosis) in the RE brain relative to controls.
While the authors find no change in the overall cleaved caspase-3 levels (a marker for apoptotic cell death) or IBA1 (a marker for microglia) in the RE brain samples, they describe a marked change in the morphology of microglia in the RE brains. Microglia in the nonepileptic brain samples had punctate labeling, suggesting a ramified architecture. Microglia from the RE brains, on the other hand, were “hypertrophied/bushy and amoeboid” suggesting an activated phagocytic state. They also show close physical proximity between dendrites and microglia in the RE brains. In striking images, the microglia appear to be reaching out to neuronal processes in the RE samples, perhaps chewing away at important neuronal structures. These exciting new findings squarely position the complement system as a potential player in synaptic reorganization associated with epilepsy. They also suggest that even though complement cascade signals are elevated, downstream phagocytotic signaling may be compromised.
A follow-up study examines the complement pathway during epileptogenesis following experimental status epilepticus in rodents (6). The authors find increased hippocampal C1q, C3, and iC3b protein levels in the weeks following status epilepticus. They also report a significant correlation between hippocampal iC3b levels and behavioral seizure frequency. This subsequent study adds nicely to their human work and brings up a number of interesting questions. Does the delay between seizure induction and complement upregulation suggest that increased complement is a response to seizure activity, rather than a component of epileptogenesis? Perhaps not, as the increased complement following status epilepticus could play a crucial role in the development of chronic epilepsy during the latent seizure-free period. Conversely, is enhanced complement activity following seizures an attempt to degrade excitatory connections and stabilize circuit function? These are important questions that will need to be answered before we can fully understand the role of the complement pathway in epilepsy. Pharmacologic inhibition and genetic knockout studies will help elucidate whether the complement cascade contributes to epileptogenesis, or is a response to it. Interestingly, a C1q-null mouse has already been developed. This mouse exhibits seizure activity associated with increased density of dendritic spines and increased pyramidal cell branching relative to controls (7, 8). These studies are consistent with complement serving to constrain excitation and prevent overramification of neuronal processes, but at this point these results remain purely correlational. Additional work will be required to fully understand how complement signaling is upregulated and microglial phagocytosis of neuronal structures is altered in epilepsy.
Another limitation of these studies is that the source of elevated complement in the epileptic brain is unknown. Systemic complement proteins are mostly produced in the liver, and cannot generally cross the blood–brain barrier. Seizure activity, normal aging, and other pathophysiologic states, however, can compromise the blood–brain barrier and allow for circulating proteins or cells to enter the brain parenchyma. Thus, the increased complement activation observed in the epileptic brain may be the result of infiltration from the systemic immune system. On the other hand, if the C1q observed in the brain is generated solely from resident CNS cells, it would suggest focal disruption of endogenous C1q signaling. Perhaps the changes in complement and microglial activation described by Wyatt et al. represent a divergence from normal neuroimmune function, resulting in additional pathologic changes in the epileptic brain. Another clue to help us understand the role of complement may lie in the brain regions investigated. Both of the studies from the Brewster lab examined brain areas associated with a seizure focus (the site of focal cortical dysplasia in the human study, and the hippocampus in an animal model of TLE). A possible future direction would be to determine if complement signaling is elevated in generalized or multifocal epilepsies.
While evidence of microglial activation in epilepsy has been probed in multiple studies and has been reviewed elsewhere (9), there is no consensus on whether microglia serve a purely protective or pathologic role in epilepsy. Perhaps the microglial activation state, driven by complement or other inflammatory mediators, underlies a pathologic role for these cells in epilepsy. If this is true, a potential therapeutic approach would be to return the innate CNS immune system (including its main cellular component, microglia) to its normal state. Thus, microglia could return to their physiologic network functions, such as surveillance of the neuropil (10), synaptic pruning (11), and clearing of apoptotic cell fragments (12), instead of existing in their “epileptic” state described by Brewster and others in the field. Conversely, enhanced complement and microglial/neuronal interaction could be indicative of a compensatory mechanism to eliminate excitatory synapses and restore the normal network function. In this case, disrupting microglial function could be detrimental. Thus, a very specific modulation of immune activation (instead of a purely ablative or anti-inflammatory approach) may have increased success in treating epilepsy and other CNS disorders. Collaboration with our colleagues in immunology, and continued work on the molecular underpinnings of neuroimmune mechanisms of epilepsy (13), will guide us to novel understanding of how microglia participate in epileptogenesis.