Abstracts

During cerebral corticogenesis, radial glia lining the lateral ventricles give rise to cortical neurons and provide the scaffolding along which they migrate. The cortical lesions observed in the brains of individuals heterozygous for the tuberous sclerosis genes, [italic]TSC1[/italic] or [italic]TSC2[/italic], display disrupted lamination and distorted gray-white matter boundaries, suggesting a disregulation in the stereotyped patterns of division and migration that coordinate neuronal production and placement. However, the pathogenesis of these cortical lesions has not been investigated, and the role of the tuberous sclerosis gene products in corticogenesis remains unknown.
Using RNAi-mediated knockdown of [italic]TSC2[/italic] transcript, we have examined the consequences of the loss of function of its gene product tuberin in radial glia and their progeny in embryonic rats [italic]in vivo[/italic]. Oligonucleotides encoding short-hairpin RNAs targeting different unique regions of rat [italic]TSC2[/italic] transcript were annealed and ligated into the GFP-expressing pLL3.7 vector. The construct shT4 was demonstrated by Western blot to knockdown exogenous and endogenous rat [italic]TSC2[/italic].
These vectors were injected intra-ventricularly and electroporated into the developing cortices of embryos of timed pregnant rats with paddle electrodes. Embryonic and postnatal rats were transcardially perfused with PBS and 4% PFA. Dissected brains were post-fixed, dehydrated, embedded in freeze medium and cryosectioned. To investigate the consequences of loss of function of tuberin, shT4 or pLL3.7 was injected [italic]in[/italic] [italic]utero[/italic] and electroporated across the lining of the lateral ventricle of E16 rat embryos. Within 24hrs, similar populations of GFP⁺ cells were evident in the proliferative zones of both control and knockdown conditions. At 72hrs, both control and knockdown cortices contained labeled cells in this region. However, the control included GFP⁺ immature neurons in the upper cortical layers. In contrast, at 72hrs there were no GFP⁺ cells in the cortical plate of knockdown brains, suggesting that the knockdown of tuberin had resulted in a migration defect.
To examine whether the knockdown cells were halted or merely delayed, embryos electroporated with either control or knockdown constructs were examined at the end of the first postnatal week. Control brains demonstrated GFP⁺ pyramidal neurons in the cerebral cortex and very few labeled cells elsewhere. However, in the [italic]TSC2[/italic] knockdown condition, there were few labeled cells in the cortex, the vast majority of GFP⁺ cells having remained within, or deep to, the white matter. Moreover, many of the GFP⁺ cells displayed unusual morphologies, bearing truncated processes and rounded somas. RNAi-mediated knockdown of rat [italic]TSC2[/italic] in neural stem cells [italic]in vivo[/italic] leads to alterations in corticogenesis resulting in cerebral lesions resembling TSC pathology. (Supported by Citizens United for Research in Epilepsy (CURE).)