Genetic and pharmacological targeting of signaling to prevent epileptogenic circuit reorganization, pathological synaptogenesis, and cell death following neonatal cortical insult
Lauren Andresena, Danielle Crokera , David Hamptona and Chris Dullaa a Department of Neuroscience, Tufts University School of Medicine, 136 Harrison Avenue, SC201, Boston, MA, USA b Neuroscience Program, Sackler School of Graduate Biomedical Sciences, Tufts University, 136 Harrison Avenue, SC201, Boston, MA, USA Developmental cortical malformations, such as polymicrogyria, have a high incidence of drugresistant epilepsy, but the underlying mechanisms by which these lesions contribute to the onset of seizure activity remain poorly understood. Using the neonatal freeze-lesion (FL) model in mice, we have shown that FL induces a focal cortical malformation consisting of a microgyrial zone and a hyperexcitable paramicrogryrial zone after a 2-week latent period. FL-cortex also shows an upregulation of reactive astrocytes and the astrocyte-secreted protein thrombospondin (TSP) for one week, prior to the onset of epileptiform activity. TSP is known to induce excitatory synapse formation, which we hypothesize contributes to the pathological reorganization of the FL cortex. The neuronal receptor for TSP is the calcium channel subunit and we have found that is also transiently upregulated during the same time window as increased TSP expression. We hypothesized that TSP and upregulation leads to aberrant excitatory synaptogenesis, pathological network formation, and cell death and that targeting TSP/ signaling will be protective against epileptogenic processes following neonatal cortical insult. To address this hypothesis, we disrupted the interaction of TSP with using a treatment paradigm of once daily, I.P. injections of gabapentin (GBP), an antagonist of TSP/a2d1 signaling, to coincide with the time period of TSP/ upregulation. In vitro hyperexcitability was assessed by recording evoked cortical fields potentials (fEPSP) and by performing extracellular glutamate imaging with a FRET-based biosensor. In vivo seizure susceptibility was further investigated by recording EEG following acute kainate injection. These experiments demonstrated that pharmacologically disrupting TSP/α2δ-1 signaling for one week post-injury, with GBP, prevented the later onset of both in vitro and in vivo hyperexcitability. Importantly, in line with the known mechanism of TSP/ α2δ-1-driven synaptogenesis, GBP treatment also blocked the rise in excitatory synapses following FL. Lastly, we showed that GBP treatment attenuated anatomical cortical reorganization as assessed by changes in GFAP+ reactive astrocytes, cortical layer specific neuronal markers and markers of cell death. Next, we utilized a genetic approach to address the same question but avoid the potential non-specific drug effects of GBP. Using a germ-line, global knockout of α2δ-1, we again performed FL, but now in the absence of α2δ-1. Following injury, KO mice have less epileptiform activity as measured by fEPSP recordings. Interestingly, the FL KO mice have an intermediate phenotype compared to GBP treated wild-type animals, which suggests that KO of during development leads to genetic compensation, perhaps of other pro-synaptogenic pathways, that contribute to pathological network formation following FL. Finally, KO mice appear insensitive to GBP treatment, providing compelling evidence that the therapeutic effects of GBP are specific to its interactions at α2δ-1. These results shed new light on how hyperexcitable networks are formed after injury and suggest the use of GBP, or other modulators of TSP/α2δ-1 signaling, as potential therapeutic agents to minimize epileptogenesis associated with developmental cortical malformations and other post-traumatic epileptic conditions.