Abstracts

Rationale: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder due to inactivating mutations in either TSC1 or TSC2 genes that leads to cellular dysplasia and the development of hamartomas in various organs. The most common neurological symptoms of TSC are early onset epilepsy, cognitive dysfunction and autism. We have demonstrated that in human TSC cortical tubers the expression of specific glutamate receptor subunits is altered, possibly contributing to seizure development [Ann Neurol 63(4): 454]. Here we hypothesized that in a mouse model in which Tsc1 gene is selectively inactivated in neurons [J Neurosci 27(21): 5547], the expression of critical glutamatergic synaptic proteins is altered and that rapamycin, a specific mTOR inhibitor, may reverse these changes. Methods: The expression of NMDA receptor (NMDAR) subunits, together with the post-synaptic density scaffolding protein PSD-95, the pre-synaptic protein synapsin and the mTOR activation marker phospho-S6 (pS6) were analyzed by double label immunocytochemistry and western blotting. Mice (n=21) were sacrificed at P21-P29. Brains were cut at 50 μm and double labeled with neuronal markers and NR1 (1:100), NR2A (1:200), NR2B (1:500), PSD-95 (1:200) or synapsin (1:500) antibodies. For western blotting, whole brains were used to separate the membrane fraction. Blots were incubated with NR1 (1:200), NR2A (1:500), NR2B (1:1000), PSD-95 (1: 500), synapsin (1:200) and pS6 (1:1000). Rapamycin (3mg/kg, i.p.) was administered from P7 to P28 every other day. Results: Double labeling of Tsc1 mutant and control tissue demonstrated that, relative to control neurons, there was a marked increase in NR2B and PSD-95 expression in most enlarged dysplastic neurons in the mutant, which was associated with no apparent differences in NR1, NR2A and synapsin expression. Similarly, western blot quantification demonstrated that while NR1 and NR2A levels were not significantly different in the mutant mice (p>0.05) when compared to controls (95.5% of controls for NR1 and 136% for NR2A), NR2B expression was significantly higher in the mutant mice (213%, p<0.01). PSD-95 levels were also significantly higher in the Tsc1 mutants (249% of control, p<0.05), while there was no significant difference in synapsin expression between genotypes (112% of control). Rapamycin treatment effectively suppressed mTOR activation in the mutant mice by decreasing pS6 expression to only 34% of control levels. The same treatment significantly decreased NR2B and PSD-95 expression to a level comparable to controls (104% of control for NR2B and 99% of control for PSD-95; p<0.05). Conclusions: Our results suggest a potential role for altered NMDAR function in TSC-associated epilepsy. Rapamycin effect on synaptic protein expression supports the role of mTOR activation in seizure pathogenesis in TSC. Therapy focused at abnormal NMDAR function or altered mTOR signaling might be beneficial for TSC patients. Supported by: TS Alliance (DMT, FEJ, DJK), Epilepsy Fdn (DMT), Lombroso Trust (DMT), CURE (DMT), NIH/ NINDS: NS31718 (FEJ), DP1 0D003347 (FEJ), P01NS24279 (DJK).