The magnetism responsive gene Ntan1 in mouse brain

We have previously identified Ntan1 as a magnetism response gene by differential display screening in cultured rat hippocampal neurons. Ntan1 mRNA was ubiquitously expressed in all the mouse tissues examined but relatively abundant in brain, retina and testis. Ntan1 mRNA expression was detectable in the embryonic 12-day mouse brain and gradually increased with ageing. In situ hybridization analysis showed high localization of Ntan1 mRNA in pyramidal cell layer of CA region and granular cell layer of dentate gyrus in the hippocampus, and Purkinje and granular cell layers in the cerebellum, respectively. Ntan1 mRNA expression was significantly increased about two-fold 12 h after brief exposure for 15 min to magnetism at 100 mT with a gradual decrease thereafter in cultured mouse hippocampal neurons. When embryonic 12-day-old or newborn mice were successively exposed to magnetic fields at 100 mT for 2 h, four times per day until the postnatal seventh day, Ntan1 mRNA was significantly increased about 1.5-2-fold in the hippocampus in vivo. The mice exposed to magnetic fields under the same condition showed significantly decreased locomotor activity. These results suggest that magnetic exposure affects higher order neural functions through modulation of genes expression.     

Tissue inhibitor of metalloproteinases-2 is expressed in the interstitial matrix in adult mouse organs and during embryonic development.

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a member of a family of inhibitors of matrix-degrading metalloproteinases. A better insight into the role of this inhibitor during development and in organ function was obtained by examining the temporospatial expression of TIMP-2 in mice. Northern blot analysis indicated high levels of TIMP-2 mRNA in the lung, skin, reproductive organs, and brain. Lower levels of expression were found in all other organs with the exception of the liver and gastrointestinal tissue, which were negative of these tissues with complete absence of TIMP-2 mRNA in the epithelium. In the testis, TIMP-2 was present in the Leydig cells, and in the brain, it was expressed in pia matter and in neuronal tissues. TIMP-2 expression in the placenta increased during late gestation and was particularly abundant in spongiotrophoblasts In mouse embryo (day 10.5-18.5), TIMP-2 mRNA was abundant in mesenchymal tissues that surrounded developing epithelia and maturing skeleton. The pattern of expression significantly differs from that observed with TIMP-1 and TIMP-3, therefore, suggesting specific roles for each inhibitor during tissue remodeling and development.     

Neuronal expression of myeloperoxidase is increased in Alzheimer's disease

Myeloperoxidase, a heme protein expressed by professional phagocytic cells, generates an array of oxidants which are proposed to contribute to tissue damage during inflammation. We now report that enzymatically active myeloperoxidase and its characteristic amino acid oxidation products are present in human brain. Further, expression of myeloperoxidase is increased in brain tissue showing Alzheimer's neuropathology. Consistent with expression in phagocytic cells, myeloperoxidase immunoreactivity was present in some activated microglia in Alzheimer brains. However, the majority of immunoreactive material in brain localized with amyloid plaques and, surprisingly, neurons including granule and pyramidal neurons of the hippocampus. Confirming neuronal localization of the enzyme, several neuronal cell lines as well as primary neuronal cultures expressed myeloperoxidase protein. Myeloperoxidase mRNA was also detected in neuronal cell lines. These results reveal the unexpected presence of myeloperoxidase in neurons. The increase in neuronal myeloperoxidase expression we observed in Alzheimer disease brains raises the possibility that the enzyme contributes to the oxidative stress implicated in the pathogenesis of the neurodegenerative disorder.     

Notch2 expression negatively correlates with glial differentiation in the postnatal mouse brain

Notch family molecules are thought to be negative regulators of neuronal differentiation in early brain development. After expression in the embryonic period, Notch2 continues to be expressed postnatally in the specific regions in the rodent brain. Here, we examined Notch2 expression in the postnatal mouse brain using lacZ knockin animals at the Notch2 locus. Notch2 expression was observed in the developing cerebellum and hippocampus, characteristic regions where neurogenesis persists after birth. Double staining of sections revealed that Notch2 was expressed by Bergmann glia in the cerebellum, radial glia in the hippocampus, and some astrocytes in both regions. Notch2 expression by glial cells was clearly confirmed in dissociated cell cultures. Interestingly, neocortical glia, many of which did not express Notch2 in vivo, did express Notch2 in a dissociated culture condition. The triple staining of dissociated cell cultures revealed that stronger Notch2 expression correlated with the immature type of glial gene expressions: stronger vimentin and weaker glial fibrillary acidic protein expressions. In addition, Notch2 expression correlated with the incorporation of bromodeoxyuridine both in vivo and in vitro. Thus, these findings demonstrate that Notch2 is expressed not only by neuronal cells in the embryonic brain, but also by glial cells in the postnatal brain, and that its expression negatively correlates with glial differentiation, proposing its novel function as a negative regulator of glial differentiation in mammalian brain development.     

Enhanced amyloidogenic metabolism of the amyloid beta-protein precursor in the X11L-deficient mouse brain

X11L, a neuronal adaptor protein, associates with the cytoplasmic domain of APP and suppresses APP cellular metabolism. APP is the precursor of Abeta, whose metabolism is strongly implicated in Alzheimer disease pathogenesis. To examine the roles of X11L function in APP metabolism, including the generation of Abeta in the brain, we produced X11L-deficient mutant mice on the C57BL/6 background. The mutant mice did not exhibit histopathological alterations or compensatory changes in the expression of other X11 family proteins, X11 and X11L2. The expression level and distribution of APP in the brain of mutant mice were also identical to those in wild-type mice. However, in the hippocampus, where substantial levels of X11L and APP are expressed, the mutant mice exhibited a significant increase in the level of the C-terminal fragments of APP produced by cleavage with beta-secretase but not alpha-secretase. The levels of Abeta were increased in the hippocampus of aged mutant mice as compared with age-matched controls. These observations clearly indicate that X11L suppresses the amyloidogenic but not amyloidolytic processing of APP in regions of the brain such as the hippocampus, which express significant levels of X11L.     

Correlation of type I insulin-like growth factor receptor (IGF-I-R) and insulin receptor-related receptor (IRR) messenger RNA levels in tumor cell lines from pediatric tumors of neuronal origin.

The insulin receptor-related receptor (IRR) is a member of the insulin receptor family. So far no ligand has yet been discovered for this receptor type (orphan receptor). IRR, insulin receptor (IR), and insulin-like growth factor-I receptor (IGF-I-R) are all tyrosine kinases. The cellular function of the IRR is not known. The expression of IRR mRNA is restricted to a few, e.g. neuronal tissues, and has also been found in neuroblastomas. Since tyrosine kinase receptors, including the IGF-I-R, may be involved in tumor genesis, we examined the expression of IRR mRNA and IGF-I-mRNA in 18 tumor cell lines using RT-PCR and the solution hybridization/RNAse protection assay. In particular, the mRNA levels of IRR and IGF-I-R were compared by semi-quantitative RT-PCR in seven neuroblastomas and 11 soft tissue sarcomas (STS), five of which were of neuronal origin. In all of the seven neuroblastoma cell lines and in five of the 11 STS cell lines, the IRR mRNA was detected. In addition, the IRR mRNA was expressed in rhabdomyosarcoma, in leiomyosarcoma, in one of the Ewing sarcoma and in the neurofibrosarcoma cell line. The last two tumor cell types are of neuronal origin. The levels of expression of IGF-I-R and IRR mRNA of the neuroblastoma cell lines were closely related (r = 0.82, P < 0.002). Furthermore, IRR mRNA was found only in cell lines that also expressed IGF-I-R mRNA. In conclusion, cell lines from pediatric tumors of neuronal origin express IRR mRNA simultaneously with a another tyrosine kinase receptor (IGF-I-R) mRNA. The tight coupling of their mRNA expression suggests a functional association of both receptors in the tumor cells.     

IL-1 gene expression in lymphoid tissues

We examined the expression of IL-1 mRNA in vivo by in situ hybridization. RNA probes for murine IL-1 alpha and IL-1 beta were used to detect IL-1 mRNA in frozen sections of spleen, lymph node, and thymus of mice injected with Salmonella typhi LPS or SRBC. No IL-1 was detected in lymphoid tissues from un-injected mice. This lack of expression correlated with the absence of IL-1 biologic activity. However, after LPS injection, IL-1 alpha and beta mRNA expression was found in macrophages of the red pulp and marginal zone of the spleen. The periarteriolar lymphoid sheath contained cells that only expressed IL-1 beta mRNA. These cells were not lymphocytes and did not stain with the macrophage marker F4/80. A similar cellular response was found after SRBC injection. Scattered macrophages in lymph nodes and thymus were positive, but only after LPS or SRBC injection. The spleens of mice injected with LPS had megakaryocytes containing IL-1 mRNA.     

Targeted disruption of intracellular type I platelet activating factor-acetylhydrolase catalytic subunits causes severe impairment in spermatogenesis

Intracellular type I platelet activating factor-acetylhydrolase is a phospholipase that consists of a dimer of two homologous catalytic subunits alpha1 and alpha2 as well as LIS1, a product of the causative gene for type I lissencephaly. LIS1 plays an important role in neuronal migration during brain development, but the in vivo function of the catalytic subunits remains unclear. In this study, we generated alpha1- and a2-deficient mice by targeted disruption. alpha1(-/-) mice are indistinguishable from wild-type mice, whereas alpha2(-/-) male mice show a significant reduction in testis size. Double-mutant male mice are sterile because of severe impairment of spermatogenesis. Histological examination revealed marked degeneration at the spermatocyte stage and an increase of apoptotic cells in the seminiferous tubules. The catalytic subunits are expressed at high levels in testis as well as brain in mice. In wild-type mice, alpha2 is expressed in all seminiferous tubule cell types, whereas alpha1 is expressed only in the spermatogonia. This expression pattern parallels the finding that deletion of both subunits induces a marked loss of germ cells at an early spermatogenic stage. We also found that the LIS1 protein levels, but not the mRNA levels, were significantly reduced in alpha2(-/-) and double-mutant mice, suggesting that the catalytic subunits, especially alpha2, are a determinant of LIS1 expression level.     

Sex-specific expression of the X-linked histone demethylase gene Jarid1c in brain

Jarid1c, an X-linked gene coding for a histone demethylase, plays an important role in brain development and function. Notably, JARID1C mutations cause mental retardation and increased aggression in humans. These phenotypes are consistent with the expression patterns we have identified in mouse brain where Jarid1c mRNA was detected in hippocampus, hypothalamus, and cerebellum. Jarid1c expression and associated active histone marks at its 5'end are high in P19 neurons, indicating that JARID1C demethylase plays an important role in differentiated neuronal cells. We found that XX mice expressed Jarid1c more highly than XY mice, independent of their gonadal types (testes versus ovaries). This increased expression in XX mice is consistent with Jarid1c escape from X inactivation and is not compensated by expression from the Y-linked paralogue Jarid1d, which is expressed at a very low level compared to the X paralogue in P19 cells. Our observations suggest that sex-specific expression of Jarid1c may contribute to sex differences in brain function.     

Increased Expression of Apolipoprotein D Following Experimental Traumatic Brain Injury

Increasing evidence suggests that apolipoprotein D (apoD) could play a major role in mediating neuronal degeneration and regeneration in the CNS and the PNS. To investigate further the temporal pattern of apoD expression after experimental traumatic brain injury in the rat, male Sprague-Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for apoD mRNA and protein expression and for immunohistological analysis at intervals from 15 min to 14 days after injury. Increased apoD mRNA and protein levels were seen in the cortex and hippocampus ipsilateral to the injury site from 48 h to 14 days after the trauma. Immunohistological investigation demonstrated a differential pattern of apoD expression in the cortex and hippocampus, respectively: Increased apoD immunoreactivity in glial cells was detected from 2 to 3 days after the injury in cortex and hippocampus. In contrast, increased expression of apoD was seen in cortical and hippocampal neurons at later time points following impact injury. Concurrent histopathological examination using hematoxylin and eosin demonstrated dark, shrunken neurons in the cortex ipsilateral to the injury site. In contrast, no evidence of cell death was observed in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. No evidence of increased apoD mRNA or protein expression or neuronal pathology by hematoxylin and eosin staining was detected in the contralateral cortex and hippocampus. Our results reveal induction of apoD expression in the cortex and hippocampus following traumatic brain injury in the rat. Our data also suggest that increased apoD expression may play an important role in cortical neuronal degeneration after brain injury in vivo. However, increased expression of apoD in the hippocampus may not necessarily be indicative of neuronal death.