Abstract
Hypersynchronous neuronal excitation manifests clinically as seizure (ictogenesis), and may recur spontaneously and repetitively after a variable latency period (epileptogenesis). Despite tremendous research efforts to describe molecular pathways and signatures of epileptogenesis, molecular pathomechanisms leading to chronic epilepsy remain to be clarified. We hypothesized that epigenetic modifications may form the basis for a cellular memory of epileptogenesis, and used a primary neuronal cell culture model of the rat hippocampus to study the translation of massive neuronal excitation into persisting changes of epigenetic signatures and pro-epileptogenic target gene expression. Increased spontaneous activation of cultured neurons was detected 3 and 7 days after stimulation with 10 μM glutamate when compared to sham-treated time-matched controls using calcium-imaging in vitro. Chromatin-immunoprecipitation experiments revealed short-term (3 h, 7 h, and 24 h) and long-term (3 d and 2 weeks) changes in histone modifications, which were directly linked to decreased expression of two selected epilepsy target genes, e.g. excitatory glutamate receptor genes Gria2 and Grin2a. Increased promoter methylation observed 4 weeks after glutamate stimulation at respective genes suggested long-term repression of Gria2 and Grin2a genes. Inhibition of glutamatergic activation or blocking the propagation of action potentials in cultured neurons rescued altered gene expression and regulatory epigenetic modifications. Our data support the concept of a cellular memory of epileptogenesis and persisting epigenetic modifications of epilepsy target genes, which are able to turn normal into pro-epileptic neurons and circuits.
Highlights
Temporal lobe epilepsy (TLE) is the most frequent focal epilepsy in humans and often associated with an initial precipitating injury, such as brain trauma, inflammation or prolonged febrile seizures, followed by a clinically silent latency period before onset of chronic recurrent seizures [1, 2]
Over time we detected a significant increase in amplitude and frequency of spikes, synchronization of neuronal firing over large networks of neurons as well as a shift from single spikes towards bursts of spikes following glutamate stimulation compared to shamtreated time-matched controls (Fig. 3c)
We studied epigenetic gene expression of ionotropic α-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA) receptor subunit Gria2 and N-methyl-D-aspartate (NMDA) receptor subunit Grin2a at 5 different time points after glutamate stimulation (i.e. 3 h, 7 h, 24 h, 3 d, and 2 w)
Summary
Temporal lobe epilepsy (TLE) is the most frequent focal epilepsy in humans and often associated with an initial precipitating injury, such as brain trauma, inflammation or prolonged febrile seizures, followed by a clinically silent latency period before onset of chronic recurrent seizures [1, 2]. We asked if neuronal hyperexcitation alters the epigenetic machinery of hippocampal neurons towards the previously described pro-epileptogenic cellular signature. Well-characterized epilepsy genes were investigated as potential targets of a proepileptogenic cellular signature in our simplistic cell culture model. Blockage of glutamatergic signaling by D,L-AP5 and NBQX and of the propagation of action potentials by TTX was performed to provide evidence for the principal role of neuronal excitation as trigger of the epigenetic machinery. This experimental strategy was designed to help answering the question if synchronized neuronal hyperexcitation is capable of inducing long-lasting epigenetic signatures and facilitating a cellular memory of epileptogenesis (CME)
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