C9orf72 expansion within astrocytes reduces metabolic flexibility in amyotrophic lateral sclerosis.

It is important to understand how the disease process affects the metabolic pathways in amyotrophic lateral sclerosis and whether these pathways can be manipulated to ameliorate disease progression. To analyse the basis of the metabolic defect in amyotrophic lateral sclerosis we used a phenotypic metabolic profiling approach. Using fibroblasts and reprogrammed induced astrocytes from C9orf72 and sporadic amyotrophic lateral sclerosis cases we measured the production rate of reduced nicotinamide adenine dinucleotides (NADH) from 91 potential energy substrates simultaneously. Our screening approach identified that C9orf72 and sporadic amyotrophic lateral sclerosis induced astrocytes have distinct metabolic profiles compared to controls and displayed a loss of metabolic flexibility that was not observed in fibroblast models. This loss of metabolic flexibility, involving defects in adenosine, fructose and glycogen metabolism, as well as disruptions in the membrane transport of mitochondrial specific energy substrates, contributed to increased starvation induced toxicity in C9orf72 induced astrocytes. A reduction in glycogen metabolism was attributed to loss of glycogen phosphorylase and phosphoglucomutase at the protein level in both C9orf72 induced astrocytes and induced neurons. In addition, we found alterations in the levels of fructose metabolism enzymes and a reduction in the methylglyoxal removal enzyme GLO1 in both C9orf72 and sporadic models of disease. Our data show that metabolic flexibility is important in the CNS in times of bioenergetic stress.

Directed differentiation of umbilical cord blood stem cells into cortical GABAergic neurons.

Umbilical cord blood contains a population of non-hematopoietic multipotent stem cells that are capable of neuronal differentiation in-vitro. These cells have shown great potential as a therapeutic tool for central nervous system diseases and disorders. However whether these cells are able to produce neurons with similar developmental and functional characteristics to indigenous neurons within the brain remains poorly investigated. In this study, we used purified umbilical cord blood non-hematopoietic stem cells to produced GABAergic neurons with similar developmental and functional characteristics to cortical GABAergic neurons. We analyzed the expression of transcription factors MASH1, DLX1 and DLX2 throughout the 24 days of a sequential neuronal induction protocol and found that their expression patterns resembled those reported in the developing human cortex. The derived neurons also expressed components of GABAergic neurotransmission including GABA regulatory enzymes, GABA receptor subunits and GABA transporters. Thus we have demonstrated that umbilical cord blood stem cells are capable of producing cortical-like GABAergic neurons in vitro. This highlights the potential of umbilical cord blood stem cells as a therapeutic tool for neural injuries and disorders.

In vitro modelling of cortical neurogenesis by sequential induction of human umbilical cord blood stem cells.

Neurogenesis of excitatory neurons in the developing human cerebral neocortex is a complex and dynamic set of processes and the exact mechanisms controlling the specification of human neocortical neuron subtypes are poorly understood due to lack of relevant cell models available. It has been shown that the transcription factors Pax6, Tbr2 and Tbr1, which are sequentially expressed in the rodent neocortex, regulate and define corticogenesis of glutamatergic neocortical neurons. In humans the homologues of these genes are generally expressed in a similar pattern, but with some differences. In this study, we used purified human umbilical cord blood stem cells, expressing pluripotency marker genes (OCT4, SOX2 and NANOG), to model human neocortical neurogenesis in vitro. We analyzed the expression patterns of PAX6, TBR2 and TBR1, at both protein and mRNA levels, throughout the 24 days of a sequential neuronal induction protocol. Their expression patterns correlated with those found in the developing human neocortex where they define different developmental stages of neocortical neurons. The derived cord blood neuron-like cells expressed a number of neuronal markers. They also expressed components of glutamatergic neurotransmission including glutamate receptor subunits and transporters, and generated calcium influxes upon stimulation with glutamate. Thus we have demonstrated that it is possible to model neocortical neurogenesis using cord blood stem cells in vitro. This may allow detailed analysis of the molecular mechanisms regulating neocortical neuronal specification, thus aiding the development of potential therapeutic tools for diseases and injuries of the cerebral cortex.

The corticofugal neuron-associated genes ROBO1, SRGAP1, and CTIP2 exhibit an anterior to posterior gradient of expression in early fetal human neocortex development.

Developing neocortical progenitors express transcription factors in gradients that induce programs of region-specific gene expression. Our previous work identified anteriorly upregulated expression gradients of a number of corticofugal neuron-associated gene probe sets along the anterior-posterior axis of the human neocortex (8-12 postconceptional weeks [PCW]). Here, we demonstrate by real-time polymerase chain reaction, in situ hybridization and immunohistochemistry that 3 such genes, ROBO1, SRGAP1, and CTIP2 are highly expressed anteriorly between 8-12 PCW, in comparison with other genes (FEZF2, SOX5) expressed by Layer V, VI, and subplate neurons. All 3 were prominently expressed by early postmitotic neurons in the subventricular zone, intermediate zone, and cortical plate (CP) from 8 to 10 PCW. Between 12 and 15 PCW expression patterns for ER81 and SATB2 (Layer V), TBR1 (Layer V/VI) and NURR1 (Layer VI) revealed Layer V forming. By 15 PCW, ROBO1 and SRGAP1 expression was confined to Layer V, whereas CTIP2 was expressed throughout the CP anteriorly. We observed ROBO1 and SRGAP1 immunoreactivity in medullary corticospinal axons from 11 PCW onward. Thus, we propose that the coexpression of these 3 markers in the anterior neocortex may mark the early location of the human motor cortex, including its corticospinal projection neurons, allowing further study of their early differentiation.

Investigating gradients of gene expression involved in early human cortical development.

The division of the neocortex into functional areas (the cortical map) differs little between individuals, although brain lesions in development can lead to substantial re-organization of regional identity. We are studying how the cortical map is established in the human brain as a first step towards understanding this plasticity. Previous work on rodent development has identified certain transcription factors (e.g. Pax6, Emx2) expressed in gradients across the neocortex that appear to control regional expression of cell adhesion molecules and organization of area-specific thalamocortical afferent projections. Although mechanisms may be shared, the human neocortex is composed of different and more complex local area identities. Using Affymetrix gene chips of human foetal brain tissue from 8 to 12.5 post-conceptional weeks [PCW, equivalent to Carnegie stage (CS) 23, to Foetal stage (F) 4], human material obtained from the MRC-Wellcome Trust Human Developmental Biology Resource (http://www.hdbr.org), we have identified a number of genes that exhibit gradients along the anterior-posterior axis of the neocortex. Gene probe sets that were found to be upregulated posteriorally compared to anteriorally, included EMX2, COUPTFI and FGF receptor 3, and those upregulated anteriorally included cell adhesion molecules such as cadherins and protocadherins, as well as potential motor cortex markers and frontal markers (e.g. CNTNAP2, PCDH17, ROBO1, and CTIP2). Confirmation of graded expression for a subset of these genes was carried out using real-time PCR. Furthermore, we have established a dissociation cell culture model utilizing tissue dissected from anteriorally or posteriorally derived developing human neocortex that exhibits similar gradients of expression of these genes for at least 72 h in culture.

Caveolin and GLT-1 gene expression is reciprocally regulated in primary astrocytes: association of GLT-1 with non-caveolar lipid rafts.

Caveolae represent membrane microdomains acting as integrators of cellular signaling and functional processes. Caveolins are involved in the biogenesis of caveolae and regulate the activity of caveolae-associated proteins. Although caveolin proteins are found in the CNS, the regulation of caveolins in neural cells is poorly described. In the present study, we investigated different modes and mechanisms of caveolin gene regulation in primary rat astrocytes. We demonstrated that activation of cAMP-dependent signaling pathways led to a marked reduction in protein levels of caveolin-1/-2 in cortical astrocytes. Application of transforming growth factor-alpha (TGF-alpha) also resulted in a decrease of caveolin-1/-2 expression. Decreased caveolin protein levels were mirrored by diminished caveolin gene transcription. The repressive effect of TGF-alpha on caveolin-1 expression was MAP kinase-independent and partly mediated through the PI3-kinase pathway. Further downstream, inhibition of histone deacetylases abrogated TGF-alpha effects, suggesting that chromatin remodeling processes could contribute to caveolin-1 repression. Intriguingly, alterations of caveolin gene expression in response to cAMP or TGF-alpha coincided with reciprocal and brain-region specific changes in glial glutamate transporter GLT-1 expression. The reciprocal regulation of caveolin-1 and GLT-1 expression might be gated through a common PI3-kinase dependent pathway triggered by TGF-alpha. Finally, we showed that GLT-1 is located in non-caveolar lipid rafts of cortical astrocytes. In conclusion, this study highlights the occurrence of the reciprocal regulation of caveolin and GLT-1 expression during processes such as astrocyte differentiation via common signaling pathways. We also provide strong evidence that GLT-1 itself is concentrated in lipid rafts, inferring an important role for glial glutamate transporter function.

Corticotropin-releasing hormone-mediated induction of intracellular signaling pathways and brain-derived neurotrophic factor expression is inhibited by the activation of the endocannabinoid system.

CRH receptor (CRHR) 1 and the cannabinoid receptor 1 (CB1) are both G protein-coupled receptors. Activation of CRHR1 leads to increases in cAMP production and phosphorylation of the transcription factor cAMP response element-binding protein (CREB). In contrast, CB1 is negatively coupled to the cAMP signaling cascade. In this study, we analyzed a putative interaction between these two systems focusing on the regulation of the expression of brain-derived neurotrophic factor (BDNF), a CREB-regulated gene. In situ hybridization revealed coexpression of CRHR1 and CB1 receptors in the granular layer of the cerebellum. Therefore, we analyzed the effects of CRH and the CB1 agonist WIN-55,212-2 on BDNF expression in primary cerebellar neurons from rats and mice. We observed that application of CRH for 48 h led to an increase in BDNF mRNA and protein levels. This effect was inhibited by WIN-55,212-2. At the level of intracellular signaling, short-term application of WIN-55,212-2 inhibited CRH-induced cAMP accumulation and CREB phosphorylation. Pharmacological analysis demonstrated that the CRHR1 antagonist R121919, the protein kinase A inhibitor H89, and the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester inhibited CRH-mediated BDNF expression. Moreover, depolarization-induced BDNF synthesis was also inhibited by long-term application of WIN-55,212-2 in wild-type mice but not in CB1-deficient mice. Thus, these data highlight an interaction between the CRH and the cannabinoid system in the regulation of BDNF expression by influencing cAMP and Ca2+ signaling pathways.

Regulation of glial glutamate transporter expression by growth factors.

Injuries to the brain result in the decline of glial glutamate transporter expression within hours and a recovery after several days. One consequence of this disturbed expression seems to consist in the temporary accumulation of toxic extracellular glutamate levels followed by secondary neuronal cell death. Whereas evidence exists that the decline in glutamate transporter expression results from a loss of neuronal PACAP influences on astroglia, the mechanism(s) inducing the reexpression of glial glutamate transporters is presently unknown. We now demonstrate that the injury-induced growth factors EGF, TGFalpha, FGF-2, and PDGF all promote the expression of the glutamate transporters GLT-1 and/or GLAST in cultured cortical astroglia. In contrast, similar stimulatory influences were absent with GDNF and BDNF, growth factors not affected by brain injuries. The effects of EGF, TGFalpha, FGF-2, and PDGF on glial glutamate transport were only partly redundant and involved distinctly different signaling pathways. Unlike EGF, TGFalpha, and FGF-2, PDGF promoted GLT-1, but not GLAST expression and further failed to increase the maximal velocity of sodium-dependent glutamate uptake. Moreover, FGF-2 only affected glial glutamate transport when the RAF-MEK-ERK signaling pathway was concomitantly inhibited with PD98059. Depending on the extracellular growth factor and glutamate transporter subtype, the observed stimulatory effects required the activation of PKA, PKC, and/or AKT. We suggest that after brain injury, reactive processes may limit secondary neuronal cell death by promoting glial glutamate transport. The detailed knowledge of these compensatory mechanisms will eventually allow us to therapeutically interfere with glutamate-associated neuronal cell death in the brain.

Brain region-specific neuroprotective action and signaling of corticotropin-releasing hormone in primary neurons.

CRH regulates the body's response to stressful stimuli by modulating the activity of the hypothalamic pituitary axis. In primary cultures and cell lines, CRH also acts as a potent neuroprotective factor in response to a number of toxins. Using primary neuronal cultures from the cerebellum, cerebral cortex, and hippocampus, we demonstrate that CRH exerts a brain region-specific neuroprotective effect on amyloid beta 25-35 toxicity. At low CRH concentrations (10(-8) M), neuroprotective effects can be observed only in cerebellar and hippocampal cultures, but a higher CRH concentration (10(-7) M) additionally led to the protection of cortical neurons. These neuroprotective effects were inhibited by H89, a specific protein kinase A inhibitor. Western blot analysis, carried out using phospho-specific antibodies directed against MAPK, cAMP response element-binding protein (CREB), and glycogen synthase kinase (GSK)3 beta also resulted in brain legion-specific differences regarding intracellular signaling. Correlating with cell survival, low CRH concentrations resulted in activation of the CREB pathway and inactivation of GSK3 beta in cerebellar and hippocampal cultures, but higher concentrations additionally resulted in activated CREB and inactivated GSK3 beta in cortical cultures. In contrast, MAPK activation occurred only in cortical neurons. Differences in signaling were found to be independent of receptor expression levels because RT-PCR analysis indicated no region-specific differences in CRHR1 mRNA expression.

Functional interaction of estrogen receptor alpha and caveolin isoforms in neuronal SK-N-MC cells.

Estrogen receptors (ERs) are expressed in neuronal cells and exhibit a wide variety of activities in the central nervous system. The actions of ERs are regulated in a hormone-dependent manner as well as by a number of co-activators and -repressors. A recently identified co-activator of ERalpha is caveolin-1 which has been shown to mediate the ligand-independent activation of this steroid receptor. In the present study we have demonstrated that neuronal SK-N-MC cells lacking functional ERalpha show high levels of caveolin-1/-2 specific transcripts and proteins. Ectopic expression of ERalpha in SK-N-MC cells leads to the transcriptional suppression of caveolin-1 and -2 genes. This silencing event is accompanied by changes in the methylation pattern of the caveolin-1 promoter. Certain CpG dinucleotides were methylated in the caveolin-1 promoter region of the SK-ERalpha cells whereas the same sites were non-methylated in control SK-N-MC cells, implicating a gene silencing mechanism including hypermethylation of DNA. In addition, inhibitors of methyltransferases or histone deacetylases, enzymes involved in the establishment and maintenance of silenced chromatin status, partially restored caveolin transcription in SK-ERalpha cells. In conclusion, our observations provide a possible mechanism of negative feedback regulation of ERalpha co-activator caveolin by the steroid receptor itself in this cellular model.

Estrogen receptor alpha-mediated silencing of caveolin gene expression in neuronal cells.

Estrogen receptors (ER alpha/ER beta) are expressed in neuronal cells and exhibit a variety of activities in the central nervous system. ER activity is regulated in a ligand-dependent manner and by co-regulatory factors. Caveolin-1 is a recently identified co-activator of ER alpha mediating the ligand-independent activation of this steroid receptor. Here the influence of ERs on caveolin expression in human neuroblastoma SK-N-MC cells as well as in rodent brain was investigated. We found that ectopic expression of ER alpha in SK-N-MC cells (SK-ER alpha) leads to a ligand-independent transcriptional suppression of caveolin-1/-2 genes. This suppression is specifically mediated by ER alpha and not ER beta because ER beta counteracts the observed caveolin-silencing process. Interestingly, decreased caveolin expression in SK-ER alpha is accompanied by changes in the methylation pattern of caveolin promoters. The analysis of selected promoter regions of the human caveolin-1 gene showed that certain CpG dinucleotides were hypermethylated in SK-ER alpha cells, whereas the same sites were unmethylated in control, ER beta-, and ER alpha/beta co-expressing SK-N-MC cells. Inhibition of DNA methylation or histone deacetylation led to partial re-expression of caveolin-1/-2 genes in SK-ER alpha. In vivo analysis revealed a down-regulation of caveolin-1 expression after long term estrogen exposure in certain regions of the mouse brain. In conclusion, we have shown for the first time that ER alpha and not ER beta silences caveolin-1/-2 expression in an epigenetic fashion in neuronal cells. The observed mechanism of gene silencing by ER alpha may have implications for the transcriptional regulation of further ER alpha target genes.