Abstracts

RATIONALE: Topiramate (TPM) has multiple mechanisms of action, which may contribute to its antiepileptic efficacy. For example, it has recently been demonstrated that TPM can interfere with excitatory events by inhibition of AMPA/KA-subtype receptors as assessed by Co⁺-influx in cultured neurons. Glutamatergic neurotransmission is preferentially terminated by the astroglial glutamate transporters (GluTs), GLT-1 and GLAST. These transporters are highly regulated by several factors participating in the neuron-glia crosstalk, including growth factors and glutamate itself acting on the glial KA- and mGluR3 and -5 glutamate receptor subtypes. Moreover, GluT activity is modulated by cell surface-trafficking and the GluT expression level can be altered by downstream effectors of glutamate transporter gene regulation. It is hypothesized that TPM can interfere with the GluT regulatory machinery in astroglia and thus indirectly modulate the excitatory tone by altering the availability of extracellular glutamate.
METHODS: Primary cultures of neocortical astrocytes were established from neonatal mice, and cultured for 3 weeks before use. The last week of culturing 0.25 mM N⁶,2[ssquote]-[italic]O[/italic]-dibutyryladenosine 3[ssquote]:5[ssquote] cyclic monophosphate (dbcAMP) was added to obtain a shift in astrocyte morphology that resembles differentiation. Subsequently, TPM was added directly to the culture medium to final concentrations of 1-100 [mu]M for 48 hours. GluT activity was measured by uptake of the GluT substrate [³H]-D-aspartate. Expression levels of the glutamate transporters GLT-1 and GLAST were measured by Western blotting using specific affinity-purified polyclonal antibodies raised against synthetic peptides corresponding to the amino acid residues 493-508 or amino acid residues 522-541, respectively.
RESULTS: The protein expression level of GLAST was markedly altered by more than 25% after 48 hours exposure to 30 [mu]M TPM. The expression level of GLT-1 did not seem to be influenced by TPM, which is consistent with the fact that GLT-1 and GLAST expression is differentially regulated. In line with the altered expression level, the transport activity was also altered by more than 30% after 48 hours TPM treatment (30 [mu]M). The effect of TPM on the level of expression and the modulation of glutamate uptake were apparently highly susceptible to the general status of the astroglial cell population, since the expression level of GLAST could be both increased and decreased. We are currently investigating whether this effect is dependent on e.g. the phosphorylation status of various kinases that in turn can modulate the GluT regulatory machinery.
CONCLUSIONS: We have found that TPM appears to modulate astroglial glutamate uptake and alter the expression of GLAST in cultures of astrocytes. Since astroglial GluT activity is the major determinant for the termination of glutamatergic neurotransmission, the effect of TPM may indirectly change the availability of extracellular glutamate that in turn could influence neuronal excitation.
[Supported by: The R.W. Johnson Pharmaceutical Research Institute]; (Disclosure: Grant - The R.W. Johnson Research Institute)