Overexpression of death-associated protein kinase is a component in glutamate-induced neuronal apoptosis cascade

Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603

Our data show that CNPase distribution is related to the MAPK/ERK pathway and suggest that T3 might act by this pathway corresponding to a non-genomic effect of the hormone.

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Keywords: Human brain; Schizophrenia; Myelin basic protein; Glial fibrillary acidic protein doi:10.1016/j.ijdevneu.2006.09.227

Keywords: Thyroid hormones; CNPase; Oligodendrocytes; MAPK/ERK pathway doi:10.1016/j.ijdevneu.2006.09.226 [P167] Altered levels of GFAP and MBP in brain of patients with schizophrenia—An evidence for impaired myelinization process G. Burbaeva ∗ , I. Boksha, O. Savushkina Mental Health Research Center RAMS, Russia Neurodevelopmental hypothesis of schizophrenia (SCH) occupies a high position today. We tested a supposition on altered glial function, including oligodendroglial dysfunction, and its consequences for myelinisation in adult brain in this disorder. Previously, we have found significant anomalies in glutamate metabolism in SCH (Burbaeva et al., 2003) implicating substantial alterations in glutamate/glutamine cycle, energy metabolism (Burbaeva et al., 2002) and, hence, neuronal-glial relationships in this pathology. Now we have quantified levels of myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in prefrontal cortex of patients with SCH (n = 23) and compared them with the control group (n = 22). Brain extracts have been prepared with 1% sodium dodecyl sulfate (SDS) under heating. GFAP and MBP were quantified by Western-immunoblotting with commercially available monoclonal antibodies (Biogenesis, UK). Significant elevation in GFAP level (P = 0.009) and significant decrease in MBP (P = 0.026) have been observed in SCH group as compared with controls. In addition, a significant correlation between amounts of GFAP, MBP and glutamate metabolizing enzymes has been observed. In controls, GFAP amount correlated negatively with the amounts of glutamine synthetase (R = −0.46, P = 0.03) and creatine kinase BB (R = −0.43, P = 0.04), whereas MBP amount correlated negatively with the amounts of glutamine synthetase (R = −0.42, P = 0.049) and glutamate dehydrogenase (particulate associated isoform, (Burbaeva et al., 2003)) (R = −0.47, P = 0.03). In SCH group, all the correlative links typical for control brain have disappeared, but significant positive correlation (R = 0.41, P = 0.049) between GFAP and glutamine synthetase-like protein (Boksha et al., 2000) was found, wherein GSLP level was found significantly elevated in SCH group as compared with controls, P = 0.027. Thus, direct neurochemical confirmation has been observed that GFAP and MBP amounts are altered in prefrontal cortex of chronic patients with SCH, in addition, altered pattern of correlative links suggests interrelationships with glutamate and energy metabolism deviations found in this mental pathology.

SESSION 3 [P168] Overexpression of death-associated protein kinase is a component in glutamate-induced neuronal apoptosis cascade X.Y. Yu ∗ , L. Sun, C. Liou, H. Zhao, Z.Q. Xu Dalian University, China Death-associated protein kinase (DAPK) is a Ca2+ /calmodulin-regulated serine/threonine kinase that acts as a positive mediator of apoptosis in various death signaling pathways (Inbal et al., 1997). In the developing central nervous system, DAPK is widely expressed in proliferative and postmitotic regions in brain; and the expression is gradually decreased postnatally but remains high in a number of areas (Yamamoto et al., 1999). This temporal and spatial regulation of DAPK expression suggests that it may be involved in various neuronal functions. Increasing evidence indicate that the catalytic activity and expression of DAPK are also involved in neuronal death induced by cerebral ischemia and seizure (Schumacher et al., 2002; Henshall et al., 2003). Glutamate, the principal excitatory amino acid neurotransmitter in the central nervous system, is also a potential toxin leading to excitotoxicity that has been implicated in various neurological disorders. To date, several members of protein kinases and proteases including PKC, MAPK, CaMK, PTK, ERK, Caspase and Calpain, have been shown to be involved in glutamate-induced neuronal apoptosis cascade in which these enzymes are activated, or are in an elevated level of expression. However, the relationship between DAPK expression and glutamate excitotoxicity is not known. We investigated the characteristics of DAPK expression using Western blot in glutamate-induced neuronal apoptosis in primary cultured cortical neurons. A significant increase in DAPK expression was found in glutamate-treated cultures exhibiting a prevailing form of neuronal apoptosis in a delayed time course, compared to the non glutamate-treated cultures. This result suggest that DAPK overexpression may have a previously unappreciated role in glutamate-induced neuronal apoptosis cascade, and that blocking DAPK expression may be as a potential therapeutic target for excitotoxicity. Keywords: Death-associated protein kinase; Excitotoxicity; Neuronal apoptosis References Henshall, D.C., Araki, T., Schindler, C.K., et al., 2003. Expression of deathassociated protein kinase and recruitment to the tumor necrosis factor signaling pathway following brief seizures. J. Neurochem. 86, 1260–1270.

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Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603

Inbal, B., Cohen, O., Polak-Charcon, S., et al., 1997. DAP kinase links the control of apoptosis to metastasis. Nature 390, 180–184. Schumacher, A.M., Velentza, A.V., Watterson, D.M., et al., 2002. DAPK catalytic activity in the hippocampus increases during the recovery phase in an animal model of brain hypoxic-ischemic injury. Biochim. Biophys. Acta 4, 128–137. Yamamoto, M., Takahashi, H., Nakamura, T., et al., 1999. Developmental changes in distribution of death-associated protein kinase mRNA. J. Neurosci. Res. 58, 674–683.

doi:10.1016/j.ijdevneu.2006.09.228 [P169] Expression of phospho-Akt and phospho-MAPK in the adult dorsal root ganglia following axotomy M. Naidu 1,∗ , R. Asher 2 1 University

of Malaya, Malaysia;

2 University

Homozygous mouse mutants (Xt/Xt) die perinatally, display severe polydactyly and syndactyly and are frequently exencephalic. Non-exencephalic Xt/Xt embryos exhibit diverse brain malformations, including absence of olfactory bulbs and midline telencephalic structures, as well as an abnormal di-telencephalic junction. Here we analyze the developing Xt/Xt telencephalon at early stages of neurogenesis by using region-specific markers, cell birth dating techniques and by studying cell death. We have found that there is an enlargement of the ventral compared to the dorsal telencephalon in the Xt/Xt embryos. No significant differences were observed in cell proliferation and cell death between the wild type and the mutants that could account for this enlargement. Thus, we purpose that this disproportion might be the result of an early patterning defect in the mutants. Our results suggest that Gli3 is essential for the correct allocation of telencephalic tissue to dorsal versus ventral fate.

of Cambridge,

UK

Keywords: Gli3; Mouse; Telencephalon

Neurons require neurotrophic factors for their survival and neurite-outgrowth, during development. Two major signalling mechanisms that were implicated in neuronal survival and neurite-outgrowth are the phosphatidylinositol-3kinase-Akt (PI3K-Akt) pathway, and Mitogen-activated protein kinase (MAPK)/Ras-extracellular signal-regulated kinase (RasMAPK) pathway. Even though the details of these two signalling cascades remains to be fully understood, PI3-Akt cascade has been suggested to play a major role in mediating neurotrophinpromoted cell survival, and MAPK cascade is thought to be involved in mediating neurite-outgrowth. By using the rodent nerve-muscle paradigm, we investigated if PI3-Akt pathway and Ras-MAPK pathways are also activated during peripheral axon regeneration following axotomy in the adult. Our immunohistochemical and biochemical methods of investigations showed that both phospho-Akt and phospho-MAPK are expressed in the normal and is up-regulated in response to injury in the dorsal root ganglia. We conclude that this phenomenon may be vital in ensuring cell survival and successful axon regeneration even in the adult peripheral nervous system.

doi:10.1016/j.ijdevneu.2006.09.230

Keywords: Axon; Axotomy; Dorsal root ganglia; Regeneration doi:10.1016/j.ijdevneu.2006.09.229 [P170] Gli3 is essential for the formation of the developing forebrain T. Yu ∗ , V. Fotaki, D.J. Price University of Edinburgh, UK The zinc-finger transcription factor Gli3 is homologous to the Drosophila segment polarity gene cubitus interruptus and has been shown to be crucial in the development of mammalian brain and limbs. Mutation in the Gli3 gene results in the human autosomal dominant disorder Greig’s cephalopolysyndactyly syndrome and the extra-toe (Xt) phenotype in mouse.

[P171] Functional role of parkin and a related E3 gene during neuronal phenotypic maturation H. Chun Chosun University, South Korea The locus coeruleus (LC), the most prominent noradrenergic (NA) nucleus in the brain, projects NA fibers throughout the brain. This system plays key roles in many brain functions, such as motor output, memory, attention and vestibular reflexes. In particular, a marked loss of LC–NA neurons was reported in autosomal recessive juvenile parkinsonism (AR-JP), which is resulted from the loss of function mutation in the parkin gene. To assess the functional role of parkin in NA neurons in LC, we have characterized cell type-specific physiological changes produced by the expression of parkin and a related RING-finger E3 gene. To identify gene-specific mRNA and protein in tissue level, the in situ hybridization and immunohistochemistry were performed with parkin and several E3 gene cDNA probes or antibodies. NA neuronal progenitor cell line (LC3541) was used for the elucidation of molecular mechanisms underlying the cell type-specific functional role of parkin during phenotypic maturation. When we tested the expression level of parkin and related E3 genes such as BRCA1, siah-1, TRC8, NF-X1, kf-1 and praja1 in brain, only parkin and praja1 were specifically expressed in the NA neurons in LC. In the undifferentiated NA progenitor cells, the expression of parkin and praja1 was barely detectable. However, the parkin and praja1, together with a representative NA phenotype marker, dopamine-beta-hydroxylase (DBH), were increased by in vitro phenotypic differentiation. Overexpression either parkin or praja1 in the undifferentiated NA progenitor cells enhanced the expression of characteristic NA neu-