Rheb Activation in Subventricular Zone Progenitors Leads to Heterotopia, Ectopic Neuronal Differentiation, and Rapamycin-Sensitive Olfactory Micronodules and Dendrite Hypertrophy of Newborn Neurons

Abstract

mTOR hyperactivity in perinatal neural progenitor cells (NPCs) of Tsc1-heterozygote mice leads to heterotopia and abnormal neuronal morphogenesis as seen in patients with tuberous sclerosis. Considering that pathological hyperactive mTOR also occurs in individuals carrying no genetic mutations, we examined whether increasing mTOR activity in neonatal NPCs of wild-type mice would recapitulate the above phenotypes. Electroporation of a plasmid encoding constitutively active Rheb (RhebCA) into subventricular zone NPCs increased mTOR activity in newborn cells. At 19 days post-electroporation (dpe), heterotopia and ectopic cells with a neuronal morphology were observed along the migratory path (RMS) and in the olfactory bulb (OB). These ectopic cells displayed action potentials and received synaptic inputs identifying them as synaptically integrated neurons. RMS heterotopias contained astrocytes, neurons, and entrapped neuroblasts. Immunostaining at 3 dpe revealed the presence of Mash1+ Olig2− cells in the migratory route accompanied by ectopic neuronal differentiation and altered direction and speed of neuroblast migration at 7 dpe, suggesting a non-cell autonomous disruption of migration. At >19 dpe, newborn RhebCA-expressing neurons displayed altered distribution and formed micronodules in the OB. In addition, they displayed increased dendritic complexity along with altered membrane biophysics and increased frequency of GABAergic synaptic inputs. OB heterotopia, micronodules and dendrite hypertrophy were notably prevented by rapamycin treatment, suggesting their mTOR-dependence. Collectively, these data show that increasing mTOR activity in neonatal NPCs of wild-type mice recapitulate the pathologies observed in Tsc1 mutant mice. In addition, increased mTOR activity in individuals without known mutations could significantly impact neurogenesis and circuit formation.

Rheb in neuronal degeneration, regeneration, and connectivity

“…Interestingly, the activation of Rheb leads to abnormalities in asymmetric cell division in sensory organ precursor cells of Drosophila (Karbowniczek et al, 2010). Also in the mammalian system, insulin signaling plays an important role in age-dependent neuronal growth and differentiation Lafourcade et al, 2013). Constitutive activation of Rheb, in the NPCs and neuroblasts of subventricular zone in mouse may lead to premature neuronal differentiation and disruption in cell migration either though obstruction of the migratory path and/or release of aberrant cues (Lafourcade et al, 2013).…”

“…Also in the mammalian system, insulin signaling plays an important role in age-dependent neuronal growth and differentiation Lafourcade et al, 2013). Constitutive activation of Rheb, in the NPCs and neuroblasts of subventricular zone in mouse may lead to premature neuronal differentiation and disruption in cell migration either though obstruction of the migratory path and/or release of aberrant cues (Lafourcade et al, 2013). In TSC neurons, neuronal migration defects were induced by upregulation of Cul5 expression by the hyperactivated mTORC1, leading to disruption of Reelin-Dab1 signaling (Moon et al, 2015).…”

“…The altered developmental effects in brain of TSC patients is reflective of Rheb overexpression (Lim and Crino, 2013). Rheb activation using constitutively active Rheb, in newborn NPCs and neuroblasts leads to TSC-like lesions, ectopic and premature neuronal differentiation and integration, micronodule formation, and hypertrophic neuronal morphogenesis (Lafourcade et al, 2013). Moreover some of the symptoms of TSC such as seizures are assumed to arise as a consequence of neural cell death and neurodegeneration .…”

“…The embryonic development and differentiation of neural stem cells (NSCs) or NPCs requires mTORC1 activation by Rheb (Bateman and McNeill, 2004;Goorden et al, 2011;Hartman et al, 2013;Lafourcade et al, 2013). The deletion of Rheb in the NSCs leads to impaired postnatal growth and reduced body weight in mice and eventual death (Yang et al, 2014).…”

“…The role of Rheb in neuronal growth, differentiation, aging, axonal regeneration as well as energy homeostasis of the system has gained some popularity (Swiech et al, 2008;Cheng et al, 2011a;Lafourcade et al, 2013;Yang et al, 2014). Interestingly, Rheb over-expression in neurons and in secondary cell lines shows that Rheb changes its function to become an enhancer of apoptosis depending on the cellular state (Wataya-Kaneda et al, 2001;Karassek et al, 2010;Cao et al, 2013;Ehrkamp et al, 2013).…”