Neuronal and glia abnormalities in Tsc1-deficient forebrain and partial rescue by rapamycin
Highlights
► In this study we inactivate the mouse Tsc1 gene in dorsal neural progenitor cells. ► We found increased mTORC1 and decreased mTORC2 signaling in the cerebral cortex. ► Tsc1Emx1–Cre CKO mice have abnormalities in cortical neurons as well as glia. ► Tsc1Emx1–Cre CKO mice are smaller than littermates and die by postnatal day 25. ► Postnatal rapamycin treatment rescues mortality and reverses glial abnormalities.
Introduction
Tuberous Sclerosis Complex (TSC) is a relatively common genetic disorder that affects approximately 1:6,000 individuals worldwide (Crino et al., 2006). While inheritable as an autosomal dominant disorder, the majority of patients with TSC have spontaneous mutations in either the TSC1 gene (encodes hamartin) or TSC2 gene (encodes tuberin). Loss of function of either gene can lead to the full clinical spectrum of TSC though individuals with TSC2 inactivation tend to have more severe disease manifestations (Dabora et al., 2001, Ess, 2009, Jones et al., 1999, Sancak et al., 2005). While TSC impacts multiple organs; brain manifestations are especially debilitating due to a very high prevalence of epilepsy, developmental delay, autism and mental retardation (Rosser et al., 2006). These clinical features are generally ascribed to the presence of extensive focal brain malformations (tubers) that are found predominantly in the cortex of these patients. Pathological analyses of tubers show severe disruption of cortical layers and prominent “giant” cells that express markers of both neuronal and glia lineages (Ess et al., 2005, Mizuguchi and Takashima, 2001). These findings suggest that they may result from abnormal differentiation of neural progenitor cells. Much progress has been made during the last decade in determining the function(s) of hamartin and tuberin, including roles in cellular proliferation, cell size determination and control of apoptosis. Specific actions of hamartin are not well understood but may regulate cellular adhesion through control of the GTPase Rho (Lamb et al., 2000). In contrast, tuberin function has been extensively studied with the identification of multiple upstream and downstream signaling pathways that converge upon this protein. These include inactivation by Akt and activation by AMPK (Inoki et al., 2003, Kwiatkowski, 2003). The most highly conserved region in tuberin is the GTPase activating protein (GAP) domain. This domain is required for inactivation of the small G protein Rheb (Ras homolog enriched in brain) that in turn is an activator of the serine/threonine kinase mTOR. mTOR functions within two distinct multiprotein complexes, mTORC1 and mTORC2. mTORC1 contains mTOR, raptor and mLST8 and controls mRNA translation by phosphorylation of ribosomal S6-kinase and 4E-BP1 (Inoki et al., 2005). In contrast, mTORC2 contains mTOR, rictor and mLST8. mTORC2 function is much less understood but appears to be involved in cytoskeleton organization as well as activation of Akt, Protein Kinase C and SGK1 (Guertin et al., 2006, Huang and Manning, 2009, Huang et al., 2009). The mTORC1 and mTORC2 complexes also have extensive feedback mechanisms and interactions (Dibble et al., 2009). In addition, while mTORC1 is potently inhibited by rapamycin, mTORC2 is largely insensitive to this agent though prolonged rapamycin exposure may prevent mTORC2 formation (Sarbassov et al., 2004, Sarbassov et al., 2006). Cells from multiple tissues deficient for either Hamartin or Tuberin exhibit increased constitutive activity of mTORC1 with greatly augmented levels of phospho-S6-kinase, phospho-S6 and phospho-4E-BP1. These findings quickly led to an appreciation that mTORC1 inhibitors such as rapamycin may be novel and rational therapeutics for TSC. In fact, clinical trials using rapamycin for patients with TSC have been undertaken with very promising results to date (Bissler et al., 2008, Franz et al., 2006). Initial attempts to model TSC using conventional knockout of the mouse Tsc1 or Tsc2 genes revealed the requirement of each gene during embryonic development as Tsc1−/− and Tsc2−/− embryos die by embryonic day 12 (Kobayashi et al., 1999, Kobayashi et al., 2001, Kwiatkowski et al., 2002, Onda et al., 1999). Alternative strategies are thus needed to address the role of Tsc1 and Tsc2 during brain development and function. To this end, conditional knockout mouse models of TSC have been generated using a floxed allele of the Tsc1 gene (Uhlmann et al., 2002). This approach has been successfully employed to target the Tsc1 gene in astrocytes as well as post-mitotic neurons (Meikle et al., 2007, Uhlmann et al., 2002). Furthermore, mTORC1 inhibitors were able to reverse much of the phenotypes seen in these mouse models of TSC (Meikle et al., 2008, Meikle et al., 2007, Zeng et al., 2008). While providing new information about the pathogenesis of TSC including the development of epilepsy and the role of either astrocytes or post-mitotic neurons, these models have not fully recapitulated the CNS abnormalities seen in TSC. This is likely due to the relatively late onset of Cre expression during development and restriction of Tsc1 gene inactivation to astrocytes or post-mitotic neurons. Recently, another model of TSC was reported using a newly developed floxed Tsc2 allele whose inactivation was driven by the human GFAP promoter (Way et al., 2009). These mice have neuronal and glia abnormalities with increased mTORC1 signaling though abnormalities of mTORC2 or the effect of mTORC1 inhibitors were not studied.
Based on our previous work using human surgical samples from patients with TSC, we hypothesized that the CNS abnormalities in TSC result from TSC1 or TSC2-deficient neural progenitor cells. To test our hypothesis, we have now deleted the Tsc1 gene in early embryonic neural progenitor cells to study neuronal as well as glia differentiation. These conditional knockout mice (Tsc1Emx1–Cre CKO) have a greatly shortened lifespan and extensive neuronal as well as glial cell pathology. Postnatal treatment with rapamycin prevented the premature death and largely reversed glia but not the extensive neuronal pathology. These results support a critical role of the Tsc1 gene in neural progenitor cells during the generation of the cerebral cortex and suggest that hamartin is necessary for the proper differentiation of neurons, astrocytes and oligodendrocytes. While speculative, a postnatal window may exist for mTORC1 inhibitors to preferentially target glia pathology in patients with TSC.
Section snippets
Animals
Mice with Tsc1 floxed alleles were obtained from Dr. David Gutmann (Washington University, St. Louis, MO) and originally generated by Dr. David Kwiatkowski (Harvard University, Boston, MA) (Uhlmann et al., 2002). Homozygous Tsc1 floxed animals have been maintained on a mixed SV129-C57/Bl6 genetic background for over four years in our colony, appear completely normal and breed without difficulty. Emx1–Cre mice were obtained from Jackson Laboratories (Strain #005638, Bar Harbor, ME) and
Tsc1 Gene inactivation, shortened lifespan and decreased weight
To inactivate the Tsc1 gene in neural progenitor cells of the developing forebrain, we crossed Tsc1 floxed mice to Emx1–Cre animals to generate mice homozygous for the floxed allele and heterozygous for Emx1–Cre. These Tsc1Emx1–Cre conditional knockout (CKO) mice express Cre recombinase by embryonic day 10.5 (E10.5) in dorsal neural progenitor cells (Gorski et al., 2002). Dorsal neural progenitor cells give rise to almost all excitatory neurons of the cortex as well as astrocytes and a subset
Discussion
TSC is a vital model system for the study of neurologic disease in children given the extraordinarily high prevalence of epilepsy and autism that is seen in these patients. Tubers are generally thought to underlie most of the severe neurological features in TSC though this issue is not completely resolved (Wong, 2007). Human studies have demonstrated abnormalities of both neuronal and glial cell differentiation in tubers as well as subependymal giant cell astrocytomas (SEGAs) resected from
Acknowledgments
We thank Drs. David Gutmann and David Kwiatkowski for gift of Tsc1 floxed mice, encouragement and helpful discussions. This work was supported by NIH grant 5K08NS050484 to K.C.E. Support was also provided by the Vanderbilt Kennedy Center for Research on Human Development and the Tuberous Sclerosis Alliance.
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The first two authors contributed equally to this work.