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Disorders of the brain and spinal cord are common worldwide problems but have remained very difficult to treat. As a group they have diverse etiologies and can be due to trauma, infection, tumors, genetic mutations and environmental insults. Though distinct in etiology, neurological disorders share an overall intractability as current therapies are largely limited to treatment of symptoms. Improved outcomes are further constrained by the minimal endogenous capacity of the brain and spinal cord for repair. Spectacular recent scientific advances, however, suggest that new stem cell-based approaches may change this undesirable situation. In this review, I will broadly outline the challenges of studying and treating disorders of the brain and spinal cord. I will review ongoing attempts to use stem cell-based therapies to both model and treat neurological disorders. While this field is in its infancy, expected advances and needed breakthroughs point to a future where patient-derived stem cells will be the basis for the emerging discipline of regenerative neurology.

Tuberous sclerosis complex (TSC) is a multisystem genetic disorder with severe neurologic manifestations, including epilepsy, autism, anxiety and attention deficit hyperactivity disorder. TSC is caused by the loss of either the TSC1 or TSC2 genes that normally regulate the mammalian target of rapamycin (mTOR) kinase. mTOR exists within two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Loss of either TSC gene leads to increased mTORC1 but decreased mTORC2 signaling. As the contribution of decreased mTORC2 signaling to neural development and homeostasis has not been well studied, we generated a conditional knockout (CKO) of Rictor, a key component of mTORC2. mTORC2 signaling is impaired in the brain, whereas mTORC1 signaling is unchanged. Rictor CKO mice have small brains and bodies, normal lifespan and are fertile. Cortical layering is normal, but neurons are smaller than those in control brains. Seizures were not observed, although excessive slow activity was seen on electroencephalography. Rictor CKO mice are hyperactive and have reduced anxiety-like behavior. Finally, there is decreased white matter and increased levels of monoamine neurotransmitters in the cerebral cortex. Loss of mTORC2 signaling in the cortex independent of mTORC1 can disrupt normal brain development and function and may contribute to some of the neurologic manifestations seen in TSC.

Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death., Competing Interests: The authors have declared that no competing interests exist.

Manganese (Mn) is an environmental risk factor for Parkinson's disease (PD). Recessive inheritance of PARK2 mutations is strongly associated with early onset PD (EOPD). It is widely assumed that the influence of PD environmental risk factors may be enhanced by the presence of PD genetic risk factors in the genetic background of individuals. However, such interactions may be difficult to predict owing to the complexities of genetic and environmental interactions. Here we examine the potential of human induced pluripotent stem (iPS) cell-derived early neural progenitor cells (NPCs) to model differences in Mn neurotoxicity between a control subject (CA) with no known PD genetic risk factors and a subject (SM) with biallelic loss-of-function mutations in PARK2 and family history of PD but no evidence of PD by neurological exam. Human iPS cells were generated from primary dermal fibroblasts of both subjects. We assessed several outcome measures associated with Mn toxicity and PD. No difference in sensitivity to Mn cytotoxicity or mitochondrial fragmentation was observed between SM and CA NPCs. However, we found that Mn exposure was associated with significantly higher reactive oxygen species (ROS) generation in SM compared to CA NPCs despite significantly less intracellular Mn accumulation. Thus, this report offers the first example of human subject-specific differences in PD-relevant environmental health related phenotypes that are consistent with pathogenic interactions between known genetic and environmental risk factors for PD., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Tuberous sclerosis complex (TSC) is a genetic disease with severe neurologic and psychiatric manifestations including epilepsy, developmental delay, and autism. Despite much progress in defining abnormal signaling pathways including the contribution of increased mTORC1 signaling, specific abnormalities that underlie the severe neurologic features in TSC remain poorly understood. We hypothesized that epilepsy and autism in TSC result from abnormalities of γ-aminobutyric acidergic (GABAergic) interneurons. To test this hypothesis, we generated conditional knockout mice with selective deletion of the Tsc1 gene in GABAergic interneuron progenitor cells. These interneuron-specific Tsc1 conditional knockout (CKO) mice have impaired growth and decreased survival. Cortical and hippocampal GABAergic interneurons of CKO mice are enlarged and show increased mTORC1 signaling. Total numbers of GABAergic cells are reduced in the cortex with differential reduction of specific GABAergic subtypes. Ectopic clusters of cells with increased mTORC1 signaling are also seen suggesting impaired interneuron migration. The functional consequences of these cellular changes are evident in the decreased seizure threshold on exposure to the proconvulsant flurothyl. These findings support an important role for the Tsc1 gene during GABAergic interneuron development, function, and possibly migration.

Tuberous sclerosis complex (TSC) is a multiorgan hamartomatous disease caused by loss of function mutations of either the TSC1 or TSC2 genes. Neurological symptoms of TSC predominate in younger patients, but renal pathologies are a serious aspect of the disease in older children and adults. To study TSC pathogenesis in the kidney, we inactivated the mouse Tsc1 gene in the distal convoluted tubules (DCT). At young ages, Tsc1 conditional knockout (CKO) mice have enlarged kidneys and mild cystogenesis with increased mammalian target of rapamycin complex (mTORC)1 but decreased mTORC2 signaling. Treatment with the mTORC1 inhibitor rapamycin reduces kidney size and cystogenesis. Rapamycin withdrawal led to massive cystogenesis involving both distal as well as proximal tubules. To assess the contribution of decreased mTORC2 signaling in kidney pathogenesis, we also generated Rictor CKO mice. These animals did not have any detectable kidney pathology. Finally, we examined primary cilia in the DCT. Cilia were longer in Tsc1 CKO mice, and rapamycin treatment returned cilia length to normal. Rictor CKO mice had normal cilia in the DCT. Overall, our findings suggest that loss of the Tsc1 gene in the DCT is sufficient for renal cystogenesis. This cytogenesis appears to be mTORC1 but not mTORC2 dependent. Intriguingly, the mechanism may be cell autonomous as well as non-cell autonomous and possibly involves the length and function of primary cilia.

Recent successes in deriving human-induced pluripotent stem cells (hiPSCs) allow for the possibility of studying human neurons derived from patients with neurological diseases. Concomitant inhibition of the BMP and TGF-β1 branches of the TGF-β signaling pathways by the endogenous antagonist, Noggin, and the small molecule SB431542, respectively, induces efficient neuralization of hiPSCs, a method known as dual-SMAD inhibition. The use of small molecule inhibitors instead of their endogenous counterparts has several advantages including lower cost, consistent activity, and the maintenance of xeno-free culture conditions. We tested the efficacy of DMH1, a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. We compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration, we could selectively modulate the number of SOX1 expressing cells, whereas PAX6, another neural precursor marker, remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations, therefore, suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. We further demonstrate that DMH1-induced neural progenitors can be differentiated into β3-tubulin expressing neurons, a subset of which also express tyrosine hydroxylase. Thus, the combined use of DMH1, a highly specific BMP-pathway inhibitor, and SB431542, a TGF-β1-pathway specific inhibitor, provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors.

Tuberous Sclerosis Complex (TSC) is a multiorgan genetic disease that prominently features brain malformations (tubers) with many patients suffering from epilepsy and autism. These malformations typically exhibit neuronal as well as glial cell abnormalities and likely underlie much of the neurological morbidity seen in TSC. Tuber pathogenesis remains poorly understood though upregulation of the mTORC1 signaling pathway in TSC has been consistently demonstrated. Here we address abnormal brain development in TSC by inactivating the mouse Tsc1 gene in embryonic neural progenitor cells. This strategy permits evaluation of the role of the Tsc1 gene in both neuronal as well as glial cell lineages. Tsc1(Emx1-Cre) conditional knockout (CKO) animals die by 25 days of life. Their brains have increased size and contain prominent large cells within the cerebral cortex that have greatly increased mTORC1 signaling and decreased mTORC2 signaling. Severe defects of cortical lamination, enlarged dysmorphic astrocytes and decreased myelination were also found. Tsc1(Emx1-Cre) CKO mice were then treated with rapamycin to see if the premature death and brain abnormalities can be rescued. Postnatal rapamycin treatment completely prevented premature death and largely reversed the glia pathology but not abnormal neuronal lamination. These findings support a model that loss of function of the TSC genes in embryonic neural progenitor cells causes cortical malformations in patients with TSC. The dramatic effect of rapamycin suggests that even with extensive multi-lineage abnormalities, a postnatal therapeutic window may exist for patients with TSC., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin) or TSC2 (encodes tuberin) genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242) in the tsc2 gene. This tsc2(vu242) allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2(vu242) is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2(vu242/vu242) homozygous zebrafish, and is moderately increased in tsc2(vu242/+) heterozygotes. Forebrain neurons are poorly organized in tsc2(vu242/vu242) homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2(vu242/vu242) mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

Tuberous Sclerosis Complex (TSC) is a multiorgan genetic disease caused by loss of function of either the TSC1 (encodes hamartin) or TSC2 (encodes tuberin) genes. Patients with TSC have benign tumors (hamartomas) in multiple organs though brain involvement is typically the most disabling aspect of the disease as very high rates of neurodevelopmental disorders are seen. While first described well over 120 years ago, recent advances have transformed TSC into a prototypical disorder that exemplifies the methods and potential of molecular medicine. This review will detail historical aspects of TSC and its strong associations with neurodevelopmental disorders focusing on epilepsy and autism. Finally, promising new approaches for the treatment of epilepsy and autism in patients with TSC as well as those in the general population will be discussed.

This commentary discusses a retrospective study by Muzykewicz et al. that details EEG results from children with infantile spasms in the setting of tuberous sclerosis complex (TSC). In this study, several factors were identified as being predictive of poor cognitive outcome, including worsening degree of hypsarrhythmia, abnormalities in EEG background activity, and incomplete response to vigabatrin. Notably, the majority of patients had a poor outcome, experiencing cognitive impairment and intractable epilepsy irrespective of whether they were treated with adrenocorticotropic hormone (corticotropin) or vigabatrin, despite the fact that vigabatrin has shown promise in previous studies. However, among the entire cohort a third of patients enjoyed greater than 1 year of seizure freedom and about a quarter had either mild or no cognitive impairment at follow-up. Overall, these findings underscore the great challenges that are faced in the treatment of infantile spasms in patients with TSC. I suggest that, while vigabatrin has been shown to have impressive short-term efficacy, the intractable seizures and cognitive impairment in many patients with TSC strongly indicate that new therapies and treatment strategies are urgently needed.

Mice with inactivation of the Tuberous sclerosis complex-1 (Tsc1) gene in glia (Tsc1 GFAP CKO mice) have deficient astrocyte glutamate transporters and develop seizures, suggesting that abnormal glutamate homeostasis contributes to neurological abnormalities in these mice. We examined the hypothesis that Tsc1 GFAP CKO mice have elevated extracellular brain glutamate levels that may cause neuronal death, abnormal glutamatergic synaptic function, and associated impairments in behavioral learning. In vivo microdialysis documented elevated glutamate levels in hippocampi of Tsc1 GFAP CKO mice and several cell death assays demonstrated neuronal death in hippocampus and neocortex. Impairment of long-term potentiation (LTP) with tetanic stimulation was observed in hippocampal slices from Tsc1 GFAP CKO mice and was reversed by low concentrations of NMDA antagonist, indicating that excessive synaptic glutamate directly inhibited LTP. Finally, Tsc1 GFAP CKO mice exhibited deficits in two hippocampal-dependent learning paradigms. These results suggest that abnormal glutamate homeostasis predisposes to excitotoxic cell death, impaired synaptic plasticity and learning deficits in Tsc1 GFAP CKO mice.

Tuberous sclerosis complex (TSC) is a multiorgan genetic disease caused by inactivation of either the TSC1 or TSC2 genes. The disorder typically has profound neurologic involvement and often presents early in life with epilepsy, developmental delay, mental retardation, and autism. These features are generally accepted to result from structural brain abnormalities that are found in patients with TSC. Although much progress has recently been made in discerning the function(s) of the TSC genes, many questions remain as to the role of these genes in brain development and homeostasis. This review will summarize recent progress and suggest future avenues of basic science research.

Individuals with tuberous sclerosis complex (TSC) exhibit a variety of neurologic abnormalities, including mental retardation, epilepsy, and autism. Examination of human TSC brains demonstrate dysplastic astrocytes and neurons, areas of abnormal neuronal migration (tubers), and hamartomatous growths, termed subependymal nodules, which can progress to subependymal giant cell astrocytomas (SEGA). Previous studies have suggested that these neuropathologic features may result from abnormal neuroglial cell differentiation. In an effort to provide support for this hypothesis and to identify specific markers of aberrant neuroglial cell differentiation in TSC, we employed gene expression profiling on Tsc1 conditional knockout (Tsc1(GFAP)CKO) mouse astrocytes. We identified several transcripts implicated in central nervous system development that are differentially expressed in Tsc1(-/-) astrocytes compared to wild-type astrocytes. We validated the differential expression of select transcripts on the protein level both in primary cultures of Tsc1(-/-) astrocytes and in Tsc1(GFAP)CKO mouse brains. Moreover, we show that these markers are also differentially expressed within cortical tubers, but not in adjacent normal tissue from TSC patient brains. This study provides supportive evidence for a developmental defect in neuroglial cell differentiation relevant to the genesis of TSC nervous system pathology and underscores the utility of mouse modeling for understanding the molecular pathogenesis of human disease., (Copyright 2004 Wiley-Liss, Inc.)

Excessive astrocytosis in cortical tubers in tuberous sclerosis complex (TSC) suggests that astrocytes may be important for epileptogenesis in TSC. We previously demonstrated that astrocyte-specific Tsc1 gene inactivation in mice (Tsc1 cKO mice) results in progressive epilepsy. Here, we report that glutamate transporter expression and function is impaired in Tsc1 cKO astrocytes. Tsc1 cKO mice exhibit decreased GLT-1 and GLAST protein expression. Electrophysiological assays demonstrate a functional decrease in glutamate transport currents of Tsc1 cKO astrocytes in hippocampal slices and astrocyte cultures. These findings suggest that Tsc1 inactivation in astrocytes causes dysfunctional glutamate homeostasis, leading to seizure development in TSC.

The c-myb gene encodes a sequence specific transactivator that is required for fetal hematopoiesis, but its potential role in other tissues is less clear because of the early fetal demise of mice with targeted deletions of the c-myb gene and incomplete of knowledge about c-myb's expression pattern. In the hematopoietic system, c-Myb protein acts on target genes whose expression is restricted to individual lineages, despite Myb's presence and role in multiple immature lineages. This suggests that c-Myb actions within different cell type-specific contexts are strongly affected by combinatorial interactions. To consider the possibility of similar c-Myb actions could extend into non-hematopoietic systems in other cell and tissue compartments, we characterized c-myb expression in developing and adult mice using in situ hybridization and correlated this with stage-specific differentiation and mitotic activity. Diverse tissues exhibited strong c-myb expression during development, notably tooth buds, the thyroid primordium, developing trachea and proximal branching airway epithelium, hair follicles, hematopoietic cells, and gastrointestinal crypt epithelial cells. The latter three of these all maintained high expression into adulthood, but with characteristic restriction to immature cell lineages prior to their terminal differentiation. In all sites, during fetal and adult stages, loss of c-Myb expression correlated strikingly with the initiation of terminal differentiation, but not the loss of mitotic activity. Based on these data, we hypothesize that c-Myb's function during cellular differentiation is both an activator of immature gene expression and a suppressor of terminal differentiation in diverse lineages.

Locus control regions (LCRs) are powerful assemblies of cis elements that organize the actions of cell-type-specific trans-acting factors. A 2.3-kb LCR in the human adenosine deaminase (ADA) gene first intron, which controls expression in thymocytes, is composed of a 200-bp enhancer domain and extended flanking sequences that facilitate activation from within chromatin. Prior analyses have demonstrated that the enhancer contains a 28-bp core region and local adjacent augmentative cis elements. We now show that the core contains a single critical c-Myb binding site. In both transiently cotransfected human cells and stable chromatin-integrated yeast cells, c-Myb strongly transactivated reporter constructs that contained polymerized core sequences. c-Myb protein was strongly evident in T lymphoblasts in which the enhancer was active and was localized within discrete nuclear structures. Fetal murine thymus exhibited a striking concordance of endogenous c-myb expression with that of mouse ADA and human ADA LCR-directed transgene expression. Point mutation of the c-Myb site within the intact 2.3-kb LCR severely attenuated enhancer activity in transfections and LCR activity in transgenic thymocytes. Within the context of a complex enhancer and LCR, c-Myb can act as an organizer of thymocyte-specific gene expression via a single binding site.