A novel ligand of calcitonin receptor reveals a potential new sensor that modulates programmed cell death.

We have discovered that the accumulation of an anti-calcitonin receptor (anti-CTR) antibody conjugated to a fluorophore (mAb2C4:AF568) provides a robust signal for cells undergoing apoptotic programmed cell death (PCD). PCD is an absolute requirement for normal development of metazoan organisms. PCD is a hallmark of common diseases such as cardiovascular disease and tissue rejection in graft versus host pathologies, and chemotherapeutics work by increasing PCD. This robust signal or high fluorescent events were verified by confocal microscopy and flow cytometry in several cell lines and a primary culture in which PCD had been induced. In Jurkat cells, GBM-L2 and MG63 cells, the percentage undergoing PCD that were positive for both mAb2C4:AF568 and annexin V ranged between 70 and >90%. In MG63 cells induced for the preapoptotic cell stress response (PACSR), the normal expression of α-tubulin, a key structural component of the cytoskeleton, and accumulation of mAb2C4:AF568 were mutually exclusive. Our data support a model in which CTR is upregulated during PACSR and recycles to the plasma membrane with apoptosis. In cells committed to apoptosis (α-tubulin negative), there is accumulation of the CTR-ligand mAb2C4:AF568 generating a high fluorescent event. The reagent mAb2C4:AF568 effectively identifies a novel event linked to apoptosis.

Adult hippocampal neurogenesis, Rho kinase inhibition and enhancement of neuronal survival.

Adult neurogenesis occurs throughout life; however the majority of new neurons do not survive. Enhancing the survival of these new neurons will increase the likelihood that these neurons could return function following injury. Inhibition of Rho kinase is known to increase neurite outgrowth and regeneration. Previous work in our lab has demonstrated a role for Rho kinase inhibition and survival of new born neurons from the sub-ventricular zone. In this study we examined the role of Rho kinase inhibition on hippocampal neurogenesis. Two concentrations of Rho kinase inhibitor Y27632 (20 and 100 µM) and the proliferative marker EdU were infused in the lateral ventricle for 7 days. Quantification of doublecortin+/EdU+ cells on the 7th day showed that cell numbers were not significantly different, suggesting no effect on neuroblast generation. Following infusion of 100µM Y27632, the number of newborn NeuN+/EdU+ neurons at 35 days in the granular cell layer of the dentate gyrus of the ipsilateral side of the infusion did not display a significant difference; however there was an increase on the contralateral side, suggesting a dose effect. Infusion of a lower dose (20 µM) of Y27632 resulted in an increase in NeuN+/EdU+ cells in the granular cell layer of the ipsilateral side at 35 days. These mice also demonstrated enhanced spatial memory as tested by the Y maze with no significant changes in anxiety or novel object recognition. Rho kinase inhibition enhanced the survival of new born neurons in the dentate gyrus with a specific dosage effect. These results suggest that inhibition of Rho kinase following injury could be beneficial for increasing the survival of new neurons that may aid recovery.

Growth hormone promotes proliferation of adult neurosphere cultures.

OBJECTIVES: Growth hormone (GH) and its receptor (GHR) are widely expressed in the CNS. During development, GH signaling regulates both proliferation of neural progenitor cells as well as their differentiation into neurons and glia. Here we have examined the effect of GH signaling on adult subventricular zone derived neural progenitor cells cultured as neurospheres. DESIGN: GH was added to adult wild-type (WT) neurosphere cultures and neurosphere growth measured using the MTT cell proliferation assay. To examine the influence of endogenous GH production on neural progenitors, neurospheres derived from GH receptor knockout (GHRKO) mice were examined by measuring neurosphere sizes and Ki67 and TUNEL immunoreactivity. In addition, neurosphere growth curves were compared following long term culture. Finally, the differentiation of WT vs. GHRKO neurospheres was compared using immunocytochemistry for betaIII-tubulin and GFAP. RESULTS: While GH alone was insufficient to support neurosphere formation, it enhanced neurosphere growth by 20% in the presence of epidermal growth factor and fibroblast growth factor-2. Compared to wildtype neurospheres, GHRKO neurospheres were smaller, contained fewer proliferating cells and exhibited reduced self-renewal in long term culture. Addition of GH increased STAT5 phosphorylation levels in neurosphere cells. Upon differentiation, GHRKO neurospheres showed accelerated neurogenesis, although over time similar numbers of betaIII-tubulin positive neurons were generated by cells of both genotypes. CONCLUSIONS: GH functions as an autocrine mitogen in adult neurosphere cultures and promotes proliferation of neural progenitor cells as well as self-renewal of neurosphere cultures. In addition, signaling through the GHR appeared to delay neuronal differentiation in adult neurospheres.

Inhibition of neurosphere proliferation by IFNgamma but not IFNbeta is coupled to neuronal differentiation.

Interferons are produced following neural damage as part of the inflammatory response and may thus affect neural stem cell function. We compared the effects of interferon-gamma and interferon-beta on the proliferation and differentiation of adult murine neural progenitors. Both interferons inhibited neurosphere proliferation due to cell cycle arrest in G1 but only interferon-gamma induced neuronal differentiation. Both interferons induced differential phosphorylation of STAT proteins and a modest and late upregulation of the cell cycle regulator p27 but not several other likely cell cycle regulators. Thus in neural progenitor cells, anti-proliferative effects of interferons are not necessarily linked to differentiation.

Seizures, not hippocampal neuronal death, provoke neurogenesis in a mouse rapid electrical amygdala kindling model of seizures.

PURPOSE: Proliferation of neural precursors adjacent to the granule cell layer of the dentate gyrus has been identified in previous epilepsy models. Convincingly demonstrating that seizure activity is the stimulant for neurogenesis, rather than neuronal death or other insults inherent to seizure models, is difficult. To address this we derived a rapid electrical amygdala kindling model in mice known to be resistant to seizure-induced neuronal death as an experimental model of focal seizures and to analyze subsequent neurogenesis. METHODS: Mice were implanted with bipolar electrodes in the left amygdala and given electrical stimulation (3 s, 100 Hz, 1 ms monophasic square wave pulses every 5 min, 40 in total) while being observed and graded for the development of seizures. Neurogenesis in the hippocampus was assessed by counting bromodeoxyuridine-immunoreactive cells co-labeled for astrocyte (glial fibrillary acidic protein) and neuronal nuclear markers. RESULTS: Bromodeoxyuridine-reactive cell numbers were three-fold higher in stimulated mice compared with controls at 1 week in the subgranular region and at three weeks extensive co-labeling with neuronal nuclear was noted in cells which had migrated into the body of the granule cell layer, while mice receiving stimulation but failing to kindle did not differ significantly from controls. No increase in neuronal death was detected by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling, Fluorojade or fluorescent examination of hematoxylin and eosin-stained sections in any inter-group comparison. CONCLUSIONS: We propose that this kindling paradigm, not previously applied to mice, demonstrates more convincingly than previously the surge in neurogenesis in response to seizures, and the effects of seizures alone in regard to neuronal injury and regeneration.

Analysis of neuronal subpopulations in mice over-expressing suppressor of cytokine signaling-2.

Developing an understanding of factors that regulate development of the nervous system is important if we hope to be able to repair the nervous system after injury or disease. Suppressor of cytokine signaling-2 (SOCS2) is an intracellular regulator of cytokine signaling that blocks the inhibitory effects of growth hormone on neuronal differentiation and promotes neurogenesis. Here we examine the effect of SOCS2 over-expression on brain development by assessing density and soma size of different neuronal populations in the somatosensory cortex and striatum of SOCS2 transgenic mice compared with wildtype C57BL/6 mice. There were no significant differences in brain weight, cortical thickness or striatal area between mice of either genotype. Analysis of NeuN positive neuronal cell density showed a modest but significant 9% increase across layers 2-6 of SOCS2 transgenic cortex, while cortical interneuron subpopulations were variably affected. In the cortex, parvalbumin and somatostatin expressing neuron densities were unaffected, while calretinin and calbindin positive neuronal densities increased by 48% and 45% respectively. There was no apparent difference in glial fibrillary acidic protein positive astrocyte numbers in layers 1 or 6b of cortex. Furthermore, soma sizes of calretinin and calbindin positive cortical neurons were significantly smaller than wildtype, although there was no difference in size of Cresyl Violet-stained layer 5 projection neurons nor of parvalbumin or somatostatin positive cortical neurons. Additionally, synaptic density and dendritic branching were found to be increased in SOCS2 transgenic cortex. These effects on calretinin and calbindin positive cortical neurons and cortical neuronal circuitry were not observed in the striatum of SOCS2-Tg brains. However, striatal cholinergic interneurons were significantly smaller in SOCS2-Tg brains. At embryonic day 14.5, proliferation and apoptosis in the developing telencephalon were similar in each genotype. Therefore, over-expression of SOCS2 variably affects different cortical regions and neuronal populations, with the predominant effect appearing to be on interneurons and neuronal connectivity in the cortex.

SOCS1 regulates interferon-gamma mediated sensory neuron survival.

Differentiation and survival of sensory neurons is regulated by factors such as NGF and LIF. Regulation of signal transduction pathways downstream of such factor signalling by suppressor of cytokine signalling (SOCS) proteins, which negatively regulate the JAK/STAT pathway, may modulate biological outcome. In this study, SOCS1 regulation of growth factor mediated sensory neuron survival was examined. SOCS1 expression by sensory neurons was up-regulated by IFNgamma. Survival of sensory neurons from SOCS1 null mice in NGF or LIF was similar to wildtype mice. IFNgamma partially supported survival of wildtype neurons but supported survival of SOCS1 null neurons as effectively as NGF or LIF. Thus it appears that SOCS1 is a major regulator of sensory neuron responses to the inflammatory cytokine, IFNgamma.

Interactions between fibroblast growth factors and Notch regulate neuronal differentiation.

The differentiation of precursor cells into neurons has been shown to be influenced by both the Notch signaling pathway and growth factor stimulation. In this study, the regulation of neuronal differentiation by these mechanisms was examined in the embryonic day 10 neuroepithelial precursor (NEP) population. By downregulating Notch1 expression and by the addition of a Delta1 fusion protein (Delta Fc), it was shown that signaling via the Notch pathway inhibited neuron differentiation in the NEP cells, in vitro. The expression of two of the Notch receptor homologs, Notch1 and Notch3, and the ligand Delta1 in these NEP cells was found to be influenced by a number of different growth factors, indicating a potential interaction between growth factors and Notch signaling. Interestingly, none of the growth factors examined promoted neuron differentiation; however, the fibroblast growth factors (FGFs) 1 and 2 potently inhibited differentiation. FGF1 and FGF2 upregulated the expression of Notch and decreased expression of Delta1 in the NEP cells. In addition, the inhibitory response of the cells to the FGFs could be overcome by downregulating Notch1 expression and by disrupting Notch cleavage and signaling by the ablation of the Presenilin1 gene. These results indicate that FGF1 and FGF2 act via the Notch pathway, either directly or indirectly, to inhibit differentiation. Thus, signaling through the Notch receptor may be a common regulator of neuronal differentiation within the developing forebrain.

Expression of "suppressor of cytokine signalling" (SOCS) genes in the developing and adult mouse nervous system.

Growth factor and cytokine signalling in the developing nervous system has multiple effects, ranging from cell differentiation and cell survival to modulation of cell phenotype. Molecules that can regulate growth factor signalling pathways will therefore be of importance in determining the cellular response to factor stimulation. Members of a recently described gene family, the suppressor of cytokine signalling (SOCS) family, can regulate signalling events downstream of predominantly cytokine stimulation and may have important roles in the nervous system. We have examined the temporal and spatial expression of SOCS-1, SOCS-2, and SOCS-3 in the developing and adult nervous system by use of Northern analysis and in situ hybridisation. All three genes were expressed in the brain, with maximal expression from embryonic day 14 to postnatal day 8 and declining thereafter, with SOCS-2 being the most highly expressed. In situ hybridisation analysis showed that SOCS-1 and SOCS-3 had a low and widespread pattern of expression, whereas SOCS-2 expression was higher and tightly regulated. Its expression pattern indicated that SOCS-2 was expressed exclusively in neurons and that it was switched on developmentally at the time of neuronal differentiation.

Cytokines that signal through the leukemia inhibitory factor receptor-beta complex in the nervous system.

Cytokines that signal through the leukemia inhibitory factor (LIF) receptor, such as LIF and ciliary neuronotrophic factor, have a wide range of roles within both the developing and mature nervous system. They play a vital role in the differentiation of neural precursor cells into astrocytes and can prevent or promote neuronal differentiation. One of the conundrums regarding signalling through the LIF receptor is how it can have multiple, often conflicting roles in different cell types, such as enhancing the differentiation of astrocytes while inhibiting the differentiation of some neuronal cells. Factors that can modulate signal transduction downstream of cytokine signalling, such as "suppressor of cytokine signalling" proteins, which inhibit the JAK/STAT but not the mitogen-activated protein kinase pathway, may therefore play an important role in determining how a given cell will respond to cytokine signalling. This review discusses the general effects of cytokine signalling within the nervous system. Special emphasis is placed on differentiation of neural precursor cells and the role that regulation of cytokine signalling may play in how a given precursor cell responds to cytokine stimulation.

Nerve growth factor modulates myelin-associated glycoprotein binding to sensory neurons.

Myelin-associated glycoprotein (MAG) is a molecule expressed by myelinating cells at the myelin/axon interface, which binds to an as yet unidentified molecule on neurons. We have used a MAG-immunoglobulin Fc fusion protein to examine the expression and regulation of the MAG binding molecule on sensory neurons in culture. Binding of the MAG-Fc reached a maximum at 24-48 h and was higher on neurons which expressed high levels of neurofilament. Nerve growth factor (NGF) upregulated expression of the MAG binding molecule in a dose dependent manner. Schwann cells co-cultured with sensory neurons in serum-free medium stimulated maximal expression of the MAG binding molecule, which was decreased by addition of anti-NGF to the co-cultures. This indicated that Schwann cells can modulate expression of the MAG binding molecule via production of NGF and may represent a physiological mechanism for regulation of MAG-MAG binding molecule interactions during myelination and remyelination.

Cellular distribution and developmental expression of AMP-activated protein kinase isoforms in mouse central nervous system.

The mammalian AMP-activated protein kinase is a heterotrimeric serine/threonine protein kinase with multiple isoforms for each subunit (alpha, beta, and gamma) and is activated under conditions of metabolic stress. It is widely expressed in many tissues, including the brain, although its expression pattern throughout the CNS is unknown. We show that brain mRNA levels for the alpha2 and beta2 subunits were increased between embryonic days 10 and 14, whereas expression of alpha1, beta1, and gamma1 subunits was consistent at all ages examined. Immunostaining revealed a mainly neuronal distribution of all isoforms. The alpha2 catalytic subunit was highly expressed in neurons and activated astrocytes, whereas the alpha1 catalytic subunit showed low expression in neuropil. The gamma1 noncatalytic subunit was highly expressed by neurons, but not by astrocytes. Expression of the beta1 and beta2 noncatalytic subunits varied, but some neurons, such as granule cells of olfactory bulb, did not express detectable levels of either beta isoform. Preferential nuclear localization of the alpha2, beta1, and gamma1 subunits suggests new functions of the AMP-activated protein kinase, and the different expression patterns and cellular localization between the two catalytic subunits alpha1 and alpha2 point to different physiological roles.

MAG and MOG enhance neurite outgrowth of embryonic mouse spinal cord neurons.

Myelin-associated glycoprotein (MAG) inhibits neurite outgrowth of postnatal spinal cord neurons, but its effect on embryonic neurons is unknown. The effect on neurite outgrowth of another myelin protein, myelin-oligodendrocyte glycoprotein (MOG) is also unknown. We determined the effect of MAG and MOG on embryonic day 17 spinal cord neurons, which were cultured on MAG, MOG or control transfected CHO cells. Neurite outgrowth was examined and both total neurite length and longest neurite length were significantly enhanced by both MAG and MOG. These findings show that, in contrast to postnatal spinal cord neurons, MAG can enhance neurite outgrowth of embryonic spinal cord neurons. In addition, another myelin protein, MOG, can also modulate neurite outgrowth.

Neural precursor differentiation into astrocytes requires signaling through the leukemia inhibitory factor receptor.

The differentiation of precursor cells into neurons or astrocytes in the developing brain has been thought to be regulated in part by growth factors. We show here that neural precursors isolated from the developing forebrain of mice that are deficient in the gene for the low-affinity leukemia inhibitory factor receptor (LIFR-/-) fail to generate astrocytes expressing glial fibrillary acidic protein (GFAP) when cultured in vitro. Precursors from mice heterozygous for the null allele show normal levels of GFAP expression. These findings support the in vivo findings that show extremely low levels of GFAP mRNA in brains of embryonic day 19 LIFR-/- mice. In addition, monolayers of neural cells from LIFR-/- mice are far less able to support the neuronal differentiation of normal neural precursors than are monolayers from heterozygous or wild-type animals, indicating that endogenous signaling through the LIFR is required for the expression of both functional and phenotypic markers of astrocyte differentiation. LIFR-/- precursors are not irreversibly blocked from differentiating into astrocytes: they express GFAP after long-term passaging or stimulation with bone morphogenetic protein-2. These findings strongly implicate the LIF family of cytokines in the regulation of astrocyte differentiation and indeed the LIF-deficient animals show a significant reduction in the number of GFAP cells in the hippocampus. However, because this reduction is only partial it suggests that LIF may not be the predominant endogenous ligand signaling through the LIFR.

Multiple roles for endothelin in melanocyte development: regulation of progenitor number and stimulation of differentiation.

Melanocytes in the skin are derived from the embryonic neural crest. Recently, mutations in endothelin 3 and the endothelin receptor B genes have been shown to result in gross pigment defects, indicating that this signalling pathway is required for melanocyte development. We have examined the effects of endothelins on melanocyte progenitors in cultures of mouse neural crest. Firstly, they stimulate an increase in progenitor number and act synergistically with another factor, Steel factor, in the survival and proliferation of the progenitors. These findings are consistent with findings from mice with natural mutations in the endothelin receptor B gene, which show an early loss of melanocyte progenitors. Secondly, endothelins induce differentiation of the progenitors into fully mature pigmented melanocytes. This finding is consistent with the expression of endothelins in the skin of mice at the initiation of pigmentation. The melanocytes generated in endothelin-treated cultures also become responsive to alpha melanocyte-stimulating hormone, which then acts to regulate the activity of the pigmentation pathway. These findings indicate two key roles for endothelin in melanocyte development: regulation of expansion of the progenitor pool and differentiation of progenitors into mature melanocytes.

Dysmyelination in class I MHC transgenic mice.

Why is it that oligodendrocytes do not normally express major histocompatibility complex (MHC) molecules? To examine the effect of aberrant MHC expression in oligodendrocytes, transgenic mice have been produced which expressed the class I MHC gene, H-2Kb, under direction of the MBP promoter [Turnley et al. (1991b) Nature, 353:566-569; Yoshioka et al. (1991) Mol. Cell. Biol., 11:5479-5486]. A proportion of these mice exhibited a shivering phenotype, with tonic seizures and early death. Oligodendrocyte function and viability was shown to be affected, resulting in severe dysmyelination of the CNS. Is this phenomenon of cell damage due to aberrant expression of MHC molecules restricted to oligodendrocytes, and could other, non-MHC molecules, when aberrantly expressed, result in similar cell damage? This paper discusses these questions and examines possible mechanisms for the oligodendrocyte damage and hypomyelination observed in these transgenic mice. Finally, the implications of aberrant MHC expression in oligodendrocytes for demyelinating diseases such as multiple sclerosis are discussed.

Dysmyelination in transgenic mice resulting from expression of class I histocompatibility molecules in oligodendrocytes.

Major histocompatibility complex (MHC) molecules are not normally expressed in the central nervous system (CNS). However, aberrant expression has been observed in multiple sclerosis lesions and could contribute to the destruction of myelin or the myelinating cells known as oligodendrocytes. The mechanism of cell damage associated with aberrant MHC molecule expression is unclear: for example, overexpression of class I and class II MHC molecules in pancreatic beta cells in transgenic mice leads to nonimmune destruction of the cells and insulin-dependent diabetes mellitus. We have generated transgenic mice that express class I H-2Kb MHC molecules, under the control of the myelin basic protein promoter, specifically in oligodendrocytes. Homozygous transgenic mice have a shivering phenotype, develop tonic seizures and die at 15-22 days. This phenotype, which we term 'wonky', is due to hypomyelination in the CNS, and not to involvement of the immune system. The primary defect appears to be a shortage of myelinating oligodendrocytes resulting from overexpression of the class I MHC molecules.

Regulation of MHC molecules on MBP positive oligodendrocytes in mice by IFN-gamma and TNF-alpha.

The expression of class I and class II histocompatibility antigens by myelin basic protein (MBP)-positive oligodendrocytes, in response to exogenous cytokines, has been investigated in vitro. It has been found that interferon-gamma (IFN-gamma), although capable of class I induction, does not induce class II on oligodendrocytes. Furthermore, tumour necrosis factor-alpha (TNF-alpha), which was shown to induce class I MHC on other neural cells, failed to induce class I on oligodendrocytes. A combination of IFN-gamma and TNF-alpha also failed to facilitate the expression of class II antigens on oligodendrocytes, nor did it amplify the expression of class I seen with IFN-gamma alone. Thus it appears that MBP+ murine oligodendrocytes are refractory to class II induction, and express class I in response to IFN-gamma but not TNF-alpha. The differential regulation and class of MHC expression may have implications in terms of the initiation and targeting of immune responses directed toward the oligodendrocyte.