Molecular Brain Research最新文献

Abnormal iron accumulations are frequently observed in the brains of patients with Parkinson's disease and in normal aging. Iron metabolism is regulated in the CNS by iron regulatory proteins (IRP-1 and IRP-2). Mice engineered to lack IRP-2 develop abnormal motoric behaviors including tremors at rest, abnormal gait, and bradykinesia at middle to late age (18 to 24 months). To further characterize the dopamine (DA) systems of IRP-2 −/− mice, we harvested CNS tissue from age-matched wild type and IRP-2 −/− (16–19 months) and analyzed the protein levels of tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter (VMAT2), and DA levels in dorsal striatum, ventral striatum (including the core and shell of nucleus accumbens), and midbrain. We further analyzed the phosphorylation of TH in striatum at serine 40, serine 31, and serine 19. In both dorsal and ventral striatum of IRP-2 knockout mice, there was a 20–25% loss of TH protein and accompanied by a ∼50% increase in serine 40 phosphorylation above wild-type levels. No change in serine 31 phosphorylation was observed. In the ventral striatum, there was also a significant loss (∼40%) of DAT and VMAT2. Levels of DA were decreased (∼20%) in dorsal striatum, but turnover of DA was also elevated (∼30%) in dorsal striatum of IRP-2 −/− mice. We conclude that iron misregulation associated with the loss of IRP-2 protein affects DA regulation in the striatum. However, the modest loss of DA and DA-regulating proteins does not reflect the pathology of PD or animal models of PD. Instead, these observations support that the IRP-2 −/− genotype may enable neurobiological events associated with aging.

Selective serotonin reuptake inhibitors (SSRIs) are widely used for the treatment of depressive mood disorders and well known to inhibit the reuptake of neurotransmitter serotonin into nerve terminals. Thus, it seems conceivable that these drugs may induce the outflow of serotonin from the synapse as a consequence of inhibiting the reuptake, resulting in the stimulation of glial cells surrounding nerve terminals. On this hypothesis, the effect of serotonin on steroid 5α-reductase type 1 (5α-R) gene expression in rat C6 glioma cells was examined as one of the in vitro model experiments for investigating the indirect influence of SSRIs on glial cells. Serotonin elevated 5α-R mRNA and protein levels through the stimulation of serotonin 5-HT_2A receptors, and also elevated Egr-1 mRNA and protein levels prior to 5α-R gene expression in the glioma cells. Furthermore, serotonin failed to significantly increase 5α-R mRNA levels in the cells preloaded with the antisense oligodeoxynucleotide targeted on Egr-1 gene. These results indicate that serotonin may stimulate 5α-R gene expression via transcription factor Egr-1 in glial cells, thus suggesting that serotonin flowing out of the serotonergic synapse may be implicated in SSRI-induced changes in neurosteroid metabolism in brain.

Anorexia (anx) is a recessive mutation that causes lethal starvation in homozygous mice. Studies of anx/anx mice hypothalamus have shown abnormalities in the orexigenic (NPY/AGRP neurons) and the anorexigenic (POMC/CART neurons) pathways. By gene expression profiling using cDNA and oligonucleotide microarrays, we have shown that a surexpression of genes involved in inflammatory process occurred in anx mice hypothalamus. This inflammatory process could be the cause of the anorexia phenotype observed in these mice.

Neocarzinostatin (NCS), an enediyne antimitotic agent, induces cell death in both p75NTR neurotrophin receptor (NTR)-positive and p75NTR-negative PC12 cells in a concentration-dependent fashion. However, p75NTR-positive cells demonstrate a higher susceptibility to NCS-induced cell damage. Furthermore, treatment of p75NTR-positive cells with the p75NTR-specific ligand, MC192, resulted in apoptosis, while treatment of these cells with the TrkA-specific ligand, NGF–mAbNGF30, protected them from NCS-induced death, implying that both the naked and liganded p75NTR receptors have a pro-apoptotic effect on PC12 cells. Microarray studies aimed at examining differential gene expression between p75NTR-positive and p75NTR-negative cells suggested that enzymes of the cholesterol biosynthetic pathway are differentially expressed. We therefore tested the hypothesis that altered cholesterol biosynthesis contributes directly to the pro-apoptotic effects of p75NTR in this PC12 cell-NCS model. Subsequent Northern blotting studies confirmed that the expression of p75NTR is associated with the upregulation of cholesterol biosynthetic enzymes including 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase), farnesyl-diphosphate synthase, and 7-dehydro-cholesterol reductase. Mevastatin, an HMG CoA reductase inhibitor, converts the apoptosis susceptibility of p75NTR-positive cells to that of p75NTR-negative cells. It does so at concentrations that do not themselves alter cell survival. These studies provide evidence that the pro-apoptotic effects of p75NTR in PC12 cells are related to the upregulation of cholesterol biosynthetic enzymes and consequent increased cholesterol biosynthesis.

Apolipoprotein A-I (apo A-I), a major component of high density lipoproteins, has been shown to be involved in lipid metabolism, cholesterol homeostasis and degeneration/regeneration of brain tissues and was proposed as a useful marker for the extent and severity of CNS injury. We searched for aberrant protein expression in hippocampus from patients with mesial temporal lobe epilepsy (MTLE) by an analytical method based on two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF) and unambiguously identified 2 spots as apolipoprotein A-I forms in brain of MTLE patients with 7.5-fold increased levels (controls: 0.046 ± 0.046; MTLE patients: 0.343 ± 0.154, mean ± SD, P = 0.003). Western blot analysis confirmed increased apo A-I levels in MTLE. Immunohistochemistry detected staining for apo A-I extracellularly in perivasal brain parenchyma, neuropil and areas with increased glial fibrillary acidic protein (GFAP) expression as well as some pyramidal neurons and subpial astrocytes. These findings indicate that the increase of apo A-I in MTLE was possibly not reflecting a pathogenetic role but was rather due to extravasates, bleedings or increase of microvascular endothelial cells known to synthesize apo A-I. Care has to be taken when protein expressional findings are to be interpreted in the presence of plasma proteins, including apo A-I, thus clearly representing a confounding factor.

We previously demonstrated that the brain, pituitary, and C6 glioblastoma cells express leptin. To determine the physiological role of brain-derived leptin, we sought to selectively silence its expression using RNA interference (RNAi) in vitro. One of four potential targets, siRNA L7, reduced leptin mRNA by 50% (P < 0.05) and protein by 55% (P < 0.0001) in C6 cells. RNAi also induced a twofold increase in cell death as seen by ethidium homodimer-1 (P < 0.015) and TUNEL (P < 0.005) staining. These data suggest that endogenous leptin may be a critical factor promoting cell survival in the brain.

Cp is an acute phase reactant protein that also acts as a ferroxidase, and thus indirectly decreases the production of the reactive oxygen species hydroxyl radical. Ceruloplasmin (Cp) expression is induced by a variety of central nervous system injuries, but the mechanism by which this occurs is unclear. Based on the fact that peripheral nerve injury induces interleukin-6 (IL-6) expression and that there are three IL-6 response elements in the upstream region of the Cp gene, we studied their role in transcriptional regulation of Cp in astrocytic C6 glioma cells, using transfection of a rat Cp-luciferase construct, followed by sequential and simultaneous mutation of the IL-6 response elements. We found that 0.8 kb of sequence upstream to the rat ceruloplasmin start site was sufficient to drive luciferase expression in C6 glioma cells. Cells transfected with Cp-luc and treated with 100 ng/ml rat IL-6 induced 216.8% ± 4.6% of control activity. Mutagenesis of the IL-6 response elements decreased luciferase activity, with the maximal decline (9.7 ± 0.7% of wild-type) after mutation of the second site. Mutagenesis of multiple sites decreased activity beyond mutagenesis of single sites with mutation of all three sites decreasing activity to 5.3 ± 0.4% of wild-type. Gel shift and supershift assays indicated that activation of Cp in these cells was not via STAT-3. These results are consistent with a signaling process via IL-6 response elements for Cp upregulation.

The effects of HSV-1 amplicon and polyethyleneimine (PEI)-mediated transfection of dominant negative FGF receptor-1 mutant FGFR1(TK−) into the rat brain substantia nigra (SN) were examined in vivo to model the reduced FGF signaling documented to occur in Parkinson's disease. The number of SN neurons that expressed tyrosine hydroxylase (TH) was significantly reduced following HSV-1 FGFR1(TK−) intranigral delivery and similar changes were observed after PEI-mediated FGFR1(TK−) transfections. Further, we also observed a significantly lower striatal dopamine content following the PEI transfection of FGFR1(TK−). Thus, we conclude that reduced FGF signaling in the SN of Parkinsonian patients could play a role in the impaired dopaminergic transmission associated with the degenerative disease.

Reelin-stimulated tyrosine phosphorylation of the Dab1 adaptor protein is required during brain development for Reelin-dependent neuron positioning in the cerebral cortex and various other laminated regions. Dab1 contains an amino-terminal PTB/PI domain through which it can bind to Reelin receptors and membrane phosphoinositides. The relative contributions of these binding activities were unknown. Here, we identify a mutation in the PTB domain of Dab1 that inhibits membrane localization without inhibiting receptor binding. In neurons, this mutation reduces both basal and Reelin-stimulated Dab1 tyrosine phosphorylation. In contrast, a mutation that inhibits receptor binding reduces Reelin-stimulated but not basal tyrosine phosphorylation. These results support a model in which phospholipids recruit Dab1 to membranes but do not play a direct role in relaying the Reelin signal, while direct Dab1–receptor interaction is responsible for relaying the Reelin signal but not for membrane recruitment.

Rho-family GTPases play a central role in the regulation of neuronal morphogenesis. In growth cones, for example, Rho GTPases transduce extracellular stimuli into structural changes such as filopodia and lamellipodia. Although it is generally accepted that Rac1/Cdc42 and RhoA are positive and negative regulators of neurite outgrowth, respectively, the role of each Rho-family member in neuronal morphogenesis may change according to the cell context. At present, the mechanism underlying this complexity is largely unknown. In growth cones, this is partly due to a lack of information on the distribution of active Rho GTPases. Here, we visualized RhoA/Rac1/Cdc42 activities during laminin-induced growth cone advance of DRG neurons and N1E-115 neuroblastoma cells using probes based on fluorescence/Förster resonance energy transfer. The Rac1 and Cdc42 activities were high in the peripheral domain (P-domain) of growth cones. Active Rac1 was uniformly detected throughout the P-domain, whereas Cdc42 activity increased gradually toward the growth cone edge. Against a model involving RhoA down-regulation at the periphery of protruding growth cones, we found that the RhoA activity was higher in the P-domain than in the central domain and axon shaft, and that a high level of RhoA activity was maintained in the extending part of growth cones. In lysophosphatidic acid-treated N1E-115 cells, well-developed neurites with growth cones showed RhoA activation, but sustained their extended morphology until they were drawn toward the contracting somata. On the other hand, suppression of RhoA activity by C3 exoenzyme led to loss or deformation of actin bundles in the growth cones. Thus, RhoA activation in the shaft results in neurite retraction, whereas high RhoA activity in the P-domain is necessary to retain the spread morphology of nerve growth cone.