Molecular Brain Research最新文献

Tetrahydrobiopterin is a necessary cofactor for the synthesis of nitric oxide by the hemeprotein enzyme, NO-synthase (NOS). It is widely thought that inadequate levels of tetrahydrobiopterin lead to tissue injury and organ dysfunction due, in part, to formation of superoxide from pterin-deficient NOS. In the course of studies on the ubiquitylation of neuronal NOS (nNOS), we have found that certain substrate analogs, such as N^G-nitro-l-arginine, stabilize the dimeric form of nNOS and protect the enzyme from ubiquitylation. Since tetrahydrobiopterin is known to bind near heme and confers stability to the active dimeric structure of nNOS, we wondered if the loss of tetrahydrobiopterin could be an endogenous signal for nNOS ubiquitylation and degradation. We show here in HEK293 cells stably transfected with nNOS that depletion of tetrahydrobiopterin by treatment with 2,4-diamino-6-hydroxypyrimidine leads to destabilization of the dimeric form and enhances ubiquitylation of nNOS. Sepiapterin, a precursor to tetrahydrobiopterin in the salvage pathway, completely reverses the effect of 2,4-diamino-6-hydroxypyrimidine on nNOS ubiquitylation. Consistent with that found in cells, the in vitro ubiquitylation of nNOS by reticulocyte proteins decreases when tetrahydrobiopterin is present. Thus, inadequate amounts of tetrahydrobiopterin may lead to a sustained decrease in the steady state level of nNOS that is not readily reversed.

Neurons in the hilus of the dentate gyrus are lost following a lateral fluid percussion injury. Environmental enrichment is known to increase neurogenesis in the dentate in intact rats, suggesting that it might also do so following fluid percussion injury, and potentially provide replacements for lost neurons. We report that 1 h of daily environmental enrichment for 3 weeks increased the number of progenitor cells in the dentate following fluid percussion injury, but only on the ipsilesional side. In the dentate granule cell layer, but not the hilus, most progenitors had a neuronal phenotype. The rate of on going cell proliferation was similar across groups. Collectively, these results suggest that the beneficial effects of environmental enrichment on behavioral recovery following FP injury are not attributable to neuronal replacement in the hilus but may be related to increased neurogenesis in the granule cell layer.

Behavioral studies have demonstrated that chronic food restriction augments the rewarding and motor-activating effects of centrally injected psychostimulants and direct dopamine (DA) receptor agonists. Recently, it has been shown that intracerebroventricular (i.c.v.) injection of the D-1 DA receptor agonist, SKF-82958, produces an enhanced locomotor-activating effect as well as increased activation of striatal ERK 1/2 MAP kinase, CaM kinase II, CREB, and c-fos in food-restricted (FR) relative to ad libitum fed (AL) rats. Striatal neurons that express the D-1 DA receptor coexpress dynorphin and substance P, and CREB is known to couple D-1 DA receptor stimulation to preprodynorphin (ppD) gene expression. The purpose of the present study was to examine possible genomic consequences of FR using real-time quantitative RT-PCR to measure striatal neuropeptide gene expression 3 h after i.c.v. injection of SKF-82958 (20 μg). Results indicate that, in nucleus accumbens (NAc), basal levels of ppD and preprotachykinin (ppT) mRNA are lower in FR than AL rats. This may reflect a decrease in tonic DA transmission during FR which precedes the compensatory upregulation of postsynaptic D-1 DA receptor-mediated cell signaling. In response to SKF-82958 challenge, however, FR subjects displayed greater levels of ppD and ppT mRNA in NAc than did AL subjects. A similar trend was seen in caudate-putamen (CPu). SKF-82958 also increased preproenkephalin (ppE) mRNA in Nac, but not CPu, with no difference between feeding groups. The present findings regarding ppD and ppT are consistent with prior findings of increased behavioral and cellular responses to acute D-1 DA agonist challenge in FR rats. The functional consequences of increased neuropeptide gene expression in response to acute drug challenge remain to be investigated but may include modulation of behavioral effects that emerge with repeated drug exposure, including sensitization, tolerance, and addiction.

Aquaporin (AQP) 4 is a member of the AQP gene family of water-selective transport proteins. We studied the effect of water deprivation on AQP4 gene expression in chickens. The nucleotide sequence of a chicken aquaporin 4 (AQP4) cDNA that encodes a protein of 335 amino acids showed high homology to mammalian AQP4. Using Northern blotting analysis, AQP4 mRNA in chickens was observed as a band of approximately 5.5 kb in several tissues in addition to the hypothalamus, proventriculus, kidney, and breast muscle. Quantitative analysis by real-time RT-PCR analysis showed that the mRNA expression of AQP4 in the hypothalamus significantly increased after dehydration. On the other hand, the mRNA expression of AQP4 in the kidney significantly decreased after dehydration. This suggests that AQP4 may play a pivotal role in osmoregulation in the chicken brain.

Parkinson's disease is characterized by a severe loss of dopaminergic neurons resulting in a range of motor deficits. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to cause a similar loss of dopaminergic neurons in the human midbrain with corresponding Parkinsonian symptoms. Several animal species have also shown sensitivity to MPTP, including primates, mice, goldfish, and, most recently, zebrafish. This study demonstrates that the effect of MPTP on dopaminergic neurons in zebrafish larvae is mediated by the same pathways that have been demonstrated in mammalian species. MPTP-induced neurodegeneration was prevented by co-incubation with either the monoamine oxidase-B (MAO-B) inhibitor l-deprenyl or the dopamine transporter (DAT) inhibitor nomifensine. Furthermore, targeted inactivation of the DAT gene by antisense morpholinos also protected neurons from MPTP damage. Thus, the mechanism for MPTP-induced dopaminergic neuron toxicity in mammals is conserved in zebrafish larvae. Effects on swimming behavior and touch response that result from MPTP damage are partially ameliorated by both l-deprenyl and DAT knockdown.

Kinesin participates in axonal transport of neurofilaments (NFs), but the mode by which they attach to kinesin is unclear. We compared the association of NFs with kinesin in mice expressing or lacking NF-H or NF-M. In normal and M−/− mice, the leading edge of metabolically labeled NF subunits was selectively co-precipitated with kinesin. By contrast, the entire wave of radiolabeled subunits co-precipitated with kinesin in H−/− mice. Similar bulk levels of NFs co-precipitated with kinesin from normal and H−/− mice, but reduced levels co-precipitated from M−/− mice. These data suggest that both NF-H and NF-M regulate the association of NFs with kinesin. They further indicate that phosphorylation of NF-H dissociates NFs from kinesin and provides a mechanism by which NF-H phosphorylation can contribute to the slowing of NF axonal transport.

Barhl1 and Brn-3c have been identified as transcription factors that are essential for survival and maintenance of hair cells of the inner ear. Little is known about the mechanism of how Brn-3c or Barhl1 may regulate transcription in the inner ear. In this study, the transcriptional function of both Brn-3c and Barhl1 was investigated in the organ of corti derived cell lines, OC-1 and OC-2. We examined regulatory domains in these transcription factors by linking regions of Barhl1 and Brn-3c to the DNA binding domain of the heterologous transcription factor GAL4 and assayed their effect on a heterologous promoter containing GAL4 DNA binding sites by co-transfection into OC-1 and OC-2 cell lines. Brn-3c was found to contain an independent N-terminal activation domain that is sufficient to activate gene transcription in the organ of corti derived cell lines. Barhl1 on the other hand was found to act as a transcriptional repressor with repressive activity not restricted to a particular domain of Barhl1. In addition, we analyzed the effect of Barhl1 on the promoters of the neurotrophin genes NT-3 and BDNF in OC-1 and OC-2 cell lines. However, Barhl1 was not found to directly regulate neurotrophin promoter constructs in these cells.

GABA and glutamate are the major neurotransmitters in the human central nervous system. Disturbances in these transmitter systems have been suggested to influence a variety of neurological and psychiatric diseases. Human platelets have been used as a model for neural amino acid transport, although it has not been known exactly which transporters participate in the transport process. In this study, we identify with reverse transcription-polymerase chain reaction (RT-PCR) BGT-1 and EAAT3 as transporters for GABA and glutamate, respectively. We also show that platelets contain transporters for dopamine, taurine and creatine. The cloning of these transporters confirms that blood platelets can be used as a model for neurotransmitter transport in the CNS.