Abstracts

Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder caused by inactivating mutations one of two tumor suppressor genes: TSC1 or TSC2. In the central nervous system the disease manifests as seizures and developmental delay, a major cause of morbidity in TSC patients. Using a conditional, [apos]floxed[apos], allele of Tsc1, in combination with a cre recombinase allele under the control of the Synapsin promoter, we have generated a mouse brain model of TSC. Mice at postnatal day 21-23 (P21-23) were collected and protein lysates were prepared from Tsc1c/cSyn-Cre+ mice and control littermates for immunoblot analysis of the TSC proteins, phospho-S6 and other components of the mTOR pathway. A second cohort of animals was collected at P21-23 and an immunohistochemical analysis of the neuronal markers NeuN, SMI311, and the glial cell markers vimentin and GFAP was performed. Co-expression of these specific cell markers with phospho-S6 was also assessed. This analysis was done on 50um frozen brain sentions using the floating method. Tsc1c/cSyn-Cre+ mice are expected to have loss of Tsc1 in most neurons starting at embryonic day 13.5. They develop normally immediatley after birth but fail to gain weight at the same rate as control littermates beginning at day 5. In the first 2 weeks of life they are somewhat hyperactive and develop a constant tremor however as they get older they become less active and appear vacant. They have a median survival of 28 days and death appears to be due to failure to thrive with a potential contribution from seizure activity.
A marked reduction in Tsc1 levels is seen by immunoblot analysis of whole brain lysates, with activation of the mTOR cascade assessed by increased phosphorylation of the ribosomal protein S6 (pS6). pS6 levels are also markedly increased in enlarged neurons seen in the neocortex, the white matter and the hippocampus, by immunohistochemistry. These cytomegalic cells are positive for the neuronal markers NeuN and non-phosphorylated neurofilament SMI311 however lack expression of the glial markers GFAP and Vimentin.
Rapamycin was given to Tsc1c/cSyn-Cre+ pups at 1 or 3mg/kg IP 3 times per week starting at age 14d. Treatment led to a 2.3-fold increase in median survival, with improved activity however there was still a progressive increase in neuronal size. These results indicate the importance of continued Tsc1 expression even after neuronal differentiation is initiated during brain development. We have produced a model in which there are several features similar to those seen in TSC patients, including enlarged dysplastic SMI311+ neurons similar to those seen in human cortical tubers. The results also suggest a potential therapeutic value of rapamycin for treatment of TSC brain disease. (Supported by NIH.)