Astrocytes play a major role in the regulation of brain levels of adenosine, a nucleoside and inhibitory neuromodulator that can act as an endogenous neuroprotectant and anticonvulsant (Boison 2006
, Etherington, et al. 2009
, Boison, et al. 2010
). Mouse models of epilepsy support a role for astrocytic ADK regulation in epileptogenesis (Boison 2008
, Boison 2010
). Until now however it has not yet been established whether dysregulation of ADK is a common mechanism being operative in several forms of epilepsy. We therefore assessed the cellular distribution and expression of ADK in both epileptic tissue of TLE patients and in the TLE rat model.
ADK is known to be rapidly downregulated under different conditions of acute brain injury (Pignataro, et al. 2008
). Accordingly, in a previous micro-array study, which was performed in the electrical post-SE rat model, we observed that the ADK gene is down-regulated 24 hrs after induction of SE in the CA3 region of the hippocampus (Gorter, et al. 2006
). This regulation likely represents an attempt to increase the protective levels of adenosine (Pignataro, et al. 2008
), but may also contribute, in concert with the regulation of glial adenosine receptor (AR) expression, to the development of astrogliosis (Boison 2010
, Hask, et al. 2005
In the post-SE rat model (which resembles the mouse models of epileptogenesis (Boison 2010
), we detected an upregulation of ADK protein, both in hippocampus and temporal cortex, during the latent phase (1 week post SE), which precedes the development of spontaneous electrographic seizures and is characterized by prominent astrogliosis. Immunocytochemical analysis showed ADK expression in reactive astrocytes with both nuclear and cytoplasmic labeling. Two isoforms of ADK have been identified in mammalian organisms and they have a different subcellular localization (ADK-long and –short isoforms with respectively nuclear and cytoplasmic localization) and different functions (Cui, et al. 2009
). Nuclear ADK is likely involved in epigenetic mechanisms, such as methylation reactions, whereas the cytoplasmic isoform is thought to regulate the extracellular levels of adenosine. Accordingly, mice constitutively overexpressing a transgene for the cytoplasmic isoform of ADK are characterized by a reduced adenosine tone, and display spontaneous seizures as well as increased susceptibility to seizure-induced neuronal cell death (Fedele, et al. 2005b
, Pignataro, et al. 2007
, Li, et al. 2008a
, Li, et al. 2008b
). This dual functionality is also suggested by previous developmental studies (Studer, et al. 2006
). In addition, a more recent study confirms the differential role of ADK isoforms, showing that the cytoplasmic, but not the nuclear isoform of ADK is implicated in sleep regulation (Palchykova, et al. 2010
The upregulation of ADK in activated astrocytes was also observed in the temporal cortex and persisted in both hippocampus and cortex into the chronic epileptic phase in rats with a progressive form of epilepsy. ADK expression in rats with a non-progressive form of epilepsy was very similar to control expression. These observations support the implication of glial ADK expression in the progression of epilepsy, increasing seizure severity.
In addition, ADK expression in reactive astrocytes has been confirmed in human MTS specimens of patients undergoing surgery for pharmacologically refractory TLE. Increased ADK in human astrocytes may explain the relatively low adenosine baseline levels detected in microdialysis samples of epileptic patients compared to control human hippocampus (During & Spencer 1992
ADK was predominantly expressed in astrocytes, although neuronal expression was occasionally observed in both rat and human epileptic tissue. Interestingly, ADK is known to be developmentally regulated and ADK expression in neurons has been observed at early postnatal stages in both cerebral cortex and hippocampus (Studer, et al. 2006
). Whether this expression may reflect a return to an earlier developmental state, as part of the process of epileptogenesis, and may contribute to a reduction of neuronal adenosine release, supporting a prolonged excitatory activity, requires further investigation. However, DCX positive neuronal progenitor cells, within the dentate gyrus of adult epileptic hippocampus did not express ADK. This observation is in agreement with the previously reported absence of ADK in neuronal progenitor cells in adult mouse hippocampus (Studer, et al. 2006
We acknowledge limitations to the interpretation of these results. Since we analyzed a relatively small cohort of patients and experimental data were obtained from male rats, we cannot exclude the influence of clinical variables, including gender. Moreover, the expression patterns and regulation of adenosine receptors (A1, A2A, A2B, and A3) deserves further investigation.
We also show that inflammatory molecules, such as LPS and IL-1β may induce increased expression of ADK in human cultured astroglial cells. Inflammatory responses are known to be activated during epileptogenesis and IL-1β is up-regulated in epileptogenic tissue from TLE patients (Ravizza, et al. 2008b
, Vezzani, et al. 2008
). Thus, this cytokine could play a role in the regulation of ADK levels in astrocytes, providing a potential additional layer of modulatory crosstalk between the astrocyte-based adenosine cycle and inflammation. Further more detailed studies are needed to explore the relevance of this interaction.
In conclusion, the critical role of astroglial ADK in epileptic tissue is supported by these findings, providing additional evidence of ADK regulation in astrocytes during epileptogenesis, as well as during chronic epilepsy in both rat and human TLE. Our findings suggest that overexpression of ADK is a common pathological hallmark of medically intractable chronic epilepsy