The search for therapeutic targets to prevent neurons from dying is ongoing and involves the exploration of a long list of neurotrophic factors. Insulin-like growth factor 2 (IGF2) is a member of the insulin family with known neurotrophic properties. In this study, we used Igf2 knockout (Igf2−/−) neonate mice to determine whether Igf2 deficiency is detrimental to motor neuron survival after axonal injury. Results show that Igf2−/− neonatal mice are more susceptible to motor neuron damage than Igf2+/+ mice, as they have a significantly lower percentage of motor neuron survival after a sciatic nerve transection. Neuronal survival was significantly improved in Igf2−/− mice when IGF2 was administered. These results support the role of IGF2 in neonatal motor neuron survival.

The function of glycogen in the placenta remains controversial. Whether it is used as a source of fuel for placental consumption or by the fetus in times of need has yet to be determined. Two imprinted genes, insulin-like growth factor 2 (Igf2) and H19 are highly expressed in the placenta. We have previously demonstrated that mice with Igf2 deficiency have lower levels of placental glycogen. In this study, we used mice with targeted disruption of the H19 gene (H19(-/-)) to determine the importance of Igf2 overexpression in placental growth and glycogen stores. In addition, since Igf2 mediates most of its functions by signaling through the insulin and/or IGF Type 1 receptors, we determined whether gene deletions to these receptors could affect placental glycogen stores. Our data demonstrate that placentas from H19(-/-) mice are heavier, have higher number of glycogen cells, and contain larger glycogen concentrations than those of H19(+/+) mice. No differences in GSK-3, ERK, or total Akt expression or phosphorylation were found between genotypes; however, Akt1 protein expression levels were significantly increased in H19(-/-) placentas. Results obtained from insulin receptor or IGF Type 1 receptor mutant mice did not show differences in placental glycogen content compared to their wild-type littermates, supporting the notion of a specific placental Igf2 receptor. Taken together, these results support a role for Igf2 and Akt1, but not the insulin nor the IGF Type I receptors, in the regulation of placental growth and glycogen metabolism. (C) 2009 Elsevier Ltd. All rights reserved.

Insulin-like growth factor 2 (Igf2), a member of the insulin gene family, is important for brain development and has known neurotrophic properties. Though Igf2, its receptors, and binding proteins, are expressed in the adult CNS, their role in the adult brain is less well-understood. Here we studied how Igf2 deficiency affects brains of adult Igf2 knockout (Igf2(-/-)) mice following neurotoxic insult produced by the glutamate analog kainic acid (KA). Igf2(-/-) mice exhibited attenuated epileptiform activity in response to KA and were less susceptible to hippocampal neurodegeneration compared with Igf2(+/+) mice. Other brain areas protected by the lack of Igf2 included the amygdala complex, septal nuclei, and thalamic region. Apoptosis, as determined by TUNEL and Hoechst 33342 staining, was accordingly less for Igf2(-/-) mice. Hippocampal slices from Igf2(-/-) mice also were protected against the effects epileptogenic effects of KA compared to Igf2(+/+) mice suggesting that neuroprotection afforded by a lack of Igf2 may be developmental in origin and experiments demonstrating enhanced synaptic inhibition in slices taken from Igf2(-/-) mice support this hypothesis. Taken together, these results suggest that Igf2 may be important for mechanisms and circuits that contribute to neurodegeneration and epilepsy.

Insulin-like growth factor-2 (IGF2) is a member of the insulin gene family with known neurotrophic properties. The actions of IGF2 are mediated via the IGF type 1 and type 2 receptors as well as through the insulin receptors, all of which are widely expressed throughout the brain. Since IGF2 is up-regulated in the brain after injury, we wanted to determine whether the absence of IGF2 can lead to any alteration on brain morphology and/or in the response of its receptor binding sites following a neurotoxic insult. No morphological differences were observed between the brains of IGF2 knockout (IGF2−/−) and wild-type control (IGF2+/+) mice. However, our in vitro receptor autoradiography results indicate that IGF2−/− mice had lower endogenous levels of [125I]IGF1 and [125I]insulin receptor binding sites in the hippocampus and cerebellum as compared to IGF2+/+ mice, while endogenous [125I]IGF2 receptor binding showed a decrease only in the cerebellum. Seven days after kainic acid administration, the [125I]insulin receptor binding sites were significantly decreased in all brain regions of the IGF2+/+ mice, while the levels of [125I]IGF1 and [125I]IGF2 binding sites were decreased only in select brain areas. The IGF2−/− mice, on the other hand, showed increased [125I]IGF1 and [125I]IGF2 and [125I]insulin receptor binding sites in selected regions such as the hippocampus and cerebellum. These results, taken together, suggest that deletion of IGF2 gene does not affect gross morphology of the brain but does selectively alter endogenous [125I]IGF1, [125I]IGF2 and [125I]insulin receptor binding sites and their response to neurotoxicity.

Following its discovery 20 years ago, corticotropin-releasing hormone (CRH) has been postulated to mediate both hormonal and behavioural responses to stressors. Here, we characterize and describe a behavioural role for the murine gene, UcnIII, which encodes a recently discovered CRH-related neuropeptide, urocortin III. We found that mouse UcnIII is expressed predominantly in regions of the brain known to be involved in stress-related behaviours, and its expression in the hypothalamus increases following restraint. In addition, we found that intracerebroventricular administration of mUcnIII stimulates behaviours that are associated with reduced anxiety, including exploration of an open field and decreased latency to enter the lit compartment of a dark-light chamber, but has no effect on the elevated-plus maze. Finally, we found that mUcnIII does not exert any effects on the hormonal stress response. Based upon our findings, UcnIII may be an endogenous brain neuropeptide that is modulated by stress and stimulates behaviours associated with reduced anxiety. In this capacity, UcnIII may attenuate stress-related behaviours, which may be useful both to help cope with stressful situations as well as to avoid pathology associated with excessive reaction to stressors.

Cyclin-dependent kinase 5 (Cdk5) is activated on binding of activator proteins p35 and p39. A N-terminally truncated p35, termed p25, is generated through cleavage by the Ca2+-dependent protease calpain after induction of ischemia in rat brain. p25 has been shown to accumulate in brains of patients with Alzheimer's disease and may contribute to A-β peptide-mediated toxicity. Studies from transfected neurons as well as p35 and p25 transgenic mice have indicated that Cdk5, when activated by p25, gains some toxic function compared with p35/Cdk5. It remains unclear, however, whether p25/Cdk5 signaling additionally channels into pathways usually used by p35/Cdk5 and whether p25 is associated with a loss of p35 function. To clarify these issues, we have generated p25-transgenic mice in a p35-null background. We find that low levels of p25 during development induce a partial rescue of the p35−/− phenotype in several brain regions analyzed, including a rescue of cell positioning of a subset of neurons in the neocortex. In accordance with the partial rescue of brain anatomy, phosphorylation of the Cdk5 substrate mouse disabled 1 is partially restored during development. Besides this, p25/Cdk5 fails to phosphorylate other substrates that are normally phosphorylated by p35/Cdk5. Our results show that p25 can substitute for p35/Cdk5 under certain circumstances during development. In addition, they suggest that p25 may have lost some functions of p35.

Insulin resistance and diabetes might promote neurodegenerative disease, but a molecular link between these disorders is unknown. Many factors are responsible for brain growth, patterning, and survival, including the insulin-insulin-like growth factor (IGF)-signaling cascades that are mediated by tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. Irs2 signaling mediates peripheral insulin action and pancreatic beta-cell function, and its failure causes diabetes in mice. In this study, we reveal two important roles for Irs2 signaling in the mouse brain. First, disruption of the Irs2 gene reduced neuronal proliferation during development by 50%, which dissociated brain growth from Irs1-dependent body growth. Second, neurofibrillary tangles containing phosphorylated tau accumulated in the hippocampus of old Irs2 knock-out mice, suggesting that Irs2 signaling is neuroprotective. Thus, dysregulation of the Irs2 branch of the insulin-Igf-signaling cascade reveals a molecular link between diabetes and neurodegenerative disease.

Here we studied the role of signaling through ErbB-family receptors in interactions between unmyelinated axons and non-myelinating Schwann cells in adult nerves. We generated transgenic mice that postnatally express a dominant-negative ErbB receptor in non-myelinating but not in myelinating Schwann cells. These mutant mice present a progressive peripheral neuropathy characterized by extensive Schwann cell proliferation and death, loss of unmyelinated axons and marked heat and cold pain insensitivity. At later stages, C-fiber sensory neurons die by apoptosis, a process that may result from reduced GDNF (glial cell line–derived neurotrophic factor) expression in the sciatic nerve. Neuregulin 1 (NRG1)-ErbB signaling mediates, therefore, reciprocal interactions between non-myelinating Schwann cells and unmyelinated sensory neuron axons that are critical for Schwann cell and C-fiber sensory neuron survival. This study provides new insights into ErbB signaling in adult Schwann cells, the contribution of non-myelinating Schwann cells in maintaining trophic support of sensory neurons, and the possible role of disrupted ErbB signaling in peripheral sensory neuropathies.

Growth factor suppression of apoptosis correlates with the phosphorylation and inactivation of multiple proapoptotic proteins, including the BCL-2 family member BAD. However, the physiological events required for growth factors to block cell death are not well characterized. To assess the contribution of BAD inactivation to cell survival, we generated mice with point mutations in the BAD gene that abolish BAD phosphorylation at specific sites. We show that BAD phosphorylation protects cells from the deleterious effects of apoptotic stimuli and attenuates death pathway signaling by raising the threshold at which mitochondria release cytochrome c to induce cell death. These findings establish a function for endogenous BAD phosphorylation, and elucidate a mechanism by which survival kinases block apoptosis in vivo.

BACKGROUND:This study was conducted to investigate, in vivo, the dose and duration effects of ketamine administration on neuronal degeneration in the developing rat brain.METHODS:Seven-day-old (P7) Sprague-Dawley rats were treated with intraperitoneal injections of ketamine, a noncompetitive N-methyl-D-aspartate receptor antagonist. Degenerating neurones were identified by the cupric-silver stain from 10 brain regions using the stereological disector method.RESULTS:A single dose of ketamine (25, 50 and 75 mg.kg-1) did not increase neuronal degeneration compared with the saline-treated control. However, repeated doses of ketamine (25 mg.kg-1) at 90-min intervals over 9 h increased degenerating neurones in seven out of 10 brain regions.CONCLUSIONS:These findings suggest that the duration of ketamine exposure correlates with increased neuronal degeneration in the developing rat brain.

Angelman syndrome (AS), characterized by motor dysfunction, mental retardation, and seizures, is caused by several genetic etiologies involving chromosome 15q11-q13, including mutations of the UBE3A gene. UBE3A encodes UBE3A/E6-AP, a ubiquitin-protein ligase, and shows brain-specific imprinting, with brain expression predominantly from the maternal allele. Lack of a functional maternal allele of UBE3A causes AS. In order to understand the causal relationship between maternal UBE3A mutations and AS, we have constructed a mouse model with targeted inactivation of Ube3a. The inactive allele contains a lacZ reporter gene for analysis of brain-specific imprinting. Maternal, but not paternal, transmission of the targeted allele leads to beta-galactosidase activity in hippocampal and cerebellar neurons. Maternal inheritance of the Ube3a mutant allele also causes impaired performance in tests of motor function and spatial learning, as well as abnormal hippocampal EEG recordings. As predicted from the dependence of UBE3A-mediated ubiquitination of p53 on HPV E6 protein, our maternal-deficient mice show normal brain p53 levels.

Yoo and Lubec show that the amount of p25 is decreased in the brains they studied from patients with Alzheimer's disease or Down's syndrome. Their results persuaded us to conduct a more extensive survey of the p25/p35 ratio in AD brains (to be published elsewhere), as the number of samples was small in both of our studies (eight AD brains in our original study and six in theirs). After analysing a further 25 AD brains and those from 25 age-matched controls, we found that p25 levels are consistently higher in AD brains and that the difference is statistically significant (Student's t-test). This is in agreement with our original observations, as well as being consistent with earlier reports of increased Cdk5 kinase activity in AD brain and of increased amounts of p25 in an animal model of neurodegeneration.

Yoo and Lubec show that the amount of p25 is decreased in the brains they studied from patients with Alzheimer's disease or Down's syndrome. Their results persuaded us to conduct a more extensive survey of the p25/p35 ratio in AD brains (to be published elsewhere), as the number of samples was small in both of our studies (eight AD brains in our original study and six in theirs). After analysing a further 25 AD brains and those from 25 age-matched controls, we found that p25 levels are consistently higher in AD brains and that the difference is statistically significant (Student's t -test). This is in agreement with our original observations 1 , as well as being consistent with earlier reports of increased Cdk5 kinase activity in AD brain 2 and of increased amounts of p25 in an animal model of neurodegeneration 3 .

Stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by proinflammatory cytokines results in increased release of glucocorticoid that restrains further development of the inflammatory process. IL-6 has been suggested to stimulate the HPA axis during immune activation independent of the input of hypothalamic corticotropin-releasing hormone (CRH). We used the corticotropin-releasing hormone-deficient (Crh(-/-)) mouse to elucidate the effect of CRH deficiency on IL-6 expression and IL-6-induced HPA axis activation during turpentine-induced inflammation. We demonstrate that during inflammation CRH is required for a normal adrenocorticotropin hormone (ACTH) increase but not for adrenal corticosterone rise. The paradoxical increase of plasma IL-6 associated with CRH deficiency suggests that IL-6 release during inflammation is CRH-dependent. We also demonstrate that adrenal IL-6 expression is CRH-dependent, as its basal and inflammation-induced expression is blocked by CRH deficiency. Our findings suggest that during inflammation, IL-6 most likely compensates for the effects of CRH deficiency on food intake. Finally, we confirm that the HPA axis response is defective in Crh(-/-)/IL-6(-/-) mice. These findings, along with the regulation of IL-6 by CRH, support the importance of the interaction between the immune system and the HPA axis in the pathophysiology of inflammatory diseases.

cGMP has been implicated in the regulation of many essential functions in the brain, such as synaptic plasticity, phototransduction, olfaction, and behavioral state. Cyclic nucleotide phosphodiesterase (PDE) hydrolysis of cGMP is the major mechanism underlying the clearance of cGMP and is likely to be important in any process that depends on intracellular cGMP. PDE9A has the highest affinity for cGMP of any PDE, and here we studied the localization of this enzyme in the rat brain using in situ hybridization. PDE9A mRNA is widely distributed throughout the brain with varying regional expression. The pattern of PDE9A mRNA expression closely resembles that of soluble guanylyl cyclase (sGC) in the rat brain, suggesting a possible functional association or coupling of these two enzymes in the regulation of cGMP levels. Most of the brain areas expressing PDE9A mRNA also contain neuronal nitric oxide synthase (NOS), the enzymatic source of NO and the principal activator of sGC. PDE9A is the only cGMP-specific PDE with significant expression in the forebrain, and as such is likely to play an important role in NO–cGMP signaling.

The intermediate filament glial fibrillary acidic protein (GFAP) is a classic marker for several types of glial cells, including astrocytes and nonmyelinating Schwann cells. The pattern of expression of GFAP in the postnatal murine inner ear, from postnatal day 3 (P3) to P38, was studied by anti‐GFAP immunostaining in wild‐type mice as well as in two lines of transgenic mice expressing either β‐galactosidase (LacZ) or green fluorescent protein (GFP) under the control of the GFAP promoter. Analysis of protein and promoter activity shows that several classes of supporting cells in the sensory epithelia, as well as Schwann cells and satellite cells express GFAP. Early after birth, all cochlear supporting cells express GFAP, in a gradient decreasing in intensity from base to apex. After P15, GFAP expression in the organ of Corti is mostly restricted to supporting cells of the inner hair cell area (i.e., inner border and inner phalangeal cells) and outer hair cell area (i.e., Deiters' cells). A small population of limbic cells also showed expression in a base‐to‐apex gradient. In the vestibular organs, high expression was detected in supporting cells in extrastriolar regions of the utricular macula and in the canal ampullae, with weaker staining in the saccular macula. These results suggest that supporting cells of the inner ear have important similarities to glial cells and may play roles similar to those of astrocytes or Schwann cells in supporting the normal development and maintenance of neurons and sensory cells of the inner ear. J. Comp. Neurol. 442:156–162,2002. © 2002 Wiley‐Liss, Inc.

Corticotropin-releasing hormone (CRH)-deficient (KO) mice provide a unique system to define the role of CRH in regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Despite several manifestations of chronic glucocorticoid insufficiency, basal pituitary proopiomelanocortin (POMC) mRNA, adrenocorticotrophic hormone (ACTH) peptide content within the pituitary, and plasma ACTH concentrations are not elevated in CRH KO mice. The normal POMC mRNA content in KO mice is dependent upon residual glucocorticoid secretion, as it increases in both KO and WT mice after adrenalectomy; this increase is reversed by glucocorticoid, but not aldosterone, replacement. However, the normal plasma levels of ACTH in CRH KO mice are not dependent upon residual glucocorticoid secretion, because, after adrenalectomy, these levels do not undergo the normal increase seen in KO mice despite the increase in POMC mRNA content. Administration of CRH restores ACTH secretion to its expected high level in adrenalectomized CRH KO mice. Thus, in adrenal insufficiency, loss of glucocorticoid feedback by itself can increase POMC gene expression in the pituitary; but CRH action is essential for this to result in increased secretion of ACTH. This may explain why, after withdrawal of chronic glucocorticoid treatment, reactivation of CRH secretion is a necessary prerequisite for recovery from suppression of the HPA axis.

The hypothalamic-pituitary-adrenal (HPA) axis, including hypothalamic corticotropin-releasing hormone (CRH) and pituitary corticotropin, is one of the first endocrine systems to develop during fetal life, probably because glucocorticoid secretion is necessary for the maturation of many essential fetal organs. Consistent with this, pregnant mice with an inactivating mutation in the Crh gene deliver CRH-deficient offspring that die at birth with dysplastic lungs, which can be prevented by prenatal maternal glucocorticoid treatment. But children lacking the ability to synthesize cortisol (because of various genetic defects in adrenal gland development or steroidogenesis) are not born with respiratory insufficiency or abnormal lung development, suggesting that the transfer of maternal glucocorticoid across the placenta might promote fetal organ maturation in the absence of fetal glucocorticoid production. We used pregnant mice with a normal HPA axis carrying fetuses with CRH deficiency to characterize the relative contributions of the fetal and maternal adrenal to the activity of the fetal HPA axis, and related these findings to fetal lung development. We found that in the presence of fetal adrenal insufficiency, normal fetal lung development is maintained by the transfer of maternal glucocorticoid to the fetus, specifically during the circadian peak in maternal glucocorticoid secretion.

Mammalian nonhomologous DNA end joining employs Ku70, Ku80, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, and DNA ligase IV (Lig4). Herein, we show that Ku70 and Ku80 deficiency but not DNA-PKcs deficiency results in dramatically increased death of developing embryonic neurons in mice. The Ku-deficient phenotype is qualitatively similar to, but less severe than, that associated with XRCC4 and Lig4 deficiency. The lack of a neuronal death phenotype in DNA-PKcs-deficient embryos and the milder phenotype of Ku-deficient versus XRCC4- or Lig4-deficient embryos correlate with relative leakiness of residual end joining in these mutant backgrounds as assayed by a V(D)J recombination end joining assay. We conclude that normal development of the nervous system depends on the four evolutionarily conserved nonhomologous DNA end joining factors.