Genomic structure of human microtubule-associated protein 2 (MAP-2) and characterization of additional MAP-2 isoforms.

We have determined that the gene for human microtubule-associated protein 2 (MAP-2) spans 19 exons, including 6 exons identified in this study, 1-4, 8, and 13; all six of these exons are transcribed. The alternative splicing of coding exons generates a greater diversity of MAP-2 transcripts and isoforms. The first three exons encode alternate 5' untranslated regions that can be spliced to additional untranslated sequences contained in exons 4 and 5. Exons 8 and 13 are transcribed in human fetal spinal cord, adult brain, MSN cells, and rat brain, and each exon maintains an open reading frame with both high and low molecular weight MAP-2 isoforms. Antibodies generated to synthetic peptides of exons 8 and 13 demonstrate that these exons are translated and MAP-2 isoforms containing these exons are generated.

The versican C-type lectin domain recognizes the adhesion protein tenascin-R.

The core proteins of large chondroitin sulfate proteoglycans contain a C-type lectin domain. The lectin domain of one of these proteoglycans, versican, was expressed as a recombinant 15-kDa protein and shown to bind to insolubilized fucose and GlcNAc. The lectin domain showed strong binding in a gel blotting assay to a glycoprotein doublet in rat brain extracts. The binding was calcium dependent and abolished by chemical deglycosylation treatment of the ligand glycoprotein. The versican-binding glycoprotein was identified as the cell adhesion protein tenascin-R, and versican and tenascin-R were both found to be localized in the granular layer of rat cerebellum. These results show that the versican lectin domain is a binding domain with a highly targeted specificity. It may allow versican to assemble complexes containing proteoglycan, an adhesion protein, and hyaluronan.

Gene therapy for long-term expression of erythropoietin in rats.

The injection of recombinant erythropoietin (Epo) is now widely used for long-term treatment of anemia associated with chronic renal failure, cancer, and human immunodeficiency virus infections. The ability to deliver this hormone by gene therapy rather than by repeated injections could provide substantial clinical and economic benefits. As a preliminary approach, we investigated in rats the expression and biological effects of transplanting autologous vascular smooth muscle cells transduced with a retroviral vector encoding rat Epo cDNA. Vector-derived Epo secretion caused increases in reticulocytes, with peak levels of 7.8-9.6% around day 10 after implantation. The initial elevation in reticulocytes was followed by clinically significant increases in hematocrit and hemoglobin for up to 11 weeks. Ten control and treated animals showed mean hematocrits of 44.9 +/- 0.4% and 58.7 +/- 3.1%, respectively (P < 0.001), and hemoglobin values of 15.6 +/- 0.1 g/dl and 19.8 +/- 0.9 g/dl, respectively (P < 0.001). There were no significant differences between control and treated animals in the number of white blood cells and platelets. Kidney and to a lesser extent liver are specific organs that synthesize Epo in response to tissue oxygenation. In the treated animals, endogenous Epo mRNA was largely down regulated in kidney and absent from liver. These results indicate that vascular smooth muscle cells can be genetically modified to provide treatment of anemias due to Epo deficiency and suggest that this cell type may be targeted in the treatment of other diseases requiring systemic therapeutic protein delivery.

Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss.

We have generated herpes simplex virus (HSV) vectors vIE1GT and v alpha 4GT bearing the GLUT-1 isoform of the rat brain glucose transporter (GT) under the control of the human cytomegalovirus ie1 and HSV alpha 4 promoters, respectively. We previously reported that such vectors enhance glucose uptake in hippocampal cultures and the hippocampus. In this study we demonstrate that such vectors can maintain neuronal metabolism and reduce the extent of neuron loss in cultures after a period of hypoglycemia. Microinfusion of GT vectors into the rat hippocampus also reduces kainic acid-induced seizure damage in the CA3 cell field. Furthermore, delivery of the vector even after onset of the seizure is protective, suggesting that HSV-mediated gene transfer for neuroprotection need not be carried out in anticipation of neurologic crises. Using the bicistronic vector v alpha 22 beta gal alpha 4GT, which coexpresses both GT and the Escherichia coli lacZ marker gene, we further demonstrate an inverse correlation between the extent of vector expression in the dentate and the amount of CA3 damage resulting from the simultaneous delivery of kainic acid.

A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.

Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its gene-delivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.

Evidence that the 50-kDa substrate of brefeldin A-dependent ADP-ribosylation binds GTP and is modulated by the G-protein beta gamma subunit complex.

Brefeldin A, a fungal metabolite that inhibits membrane transport, induces the mono(ADP-ribosyl)ation of two cytosolic proteins of 38 and 50 kDa as judged by SDS/PAGE. The 38-kDa substrate has been previously identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We report that the 50-kDa BFA-induced ADP-ribosylated substrate (BARS-50) has native forms of 170 and 130 kDa, as determined by gel filtration of rat brain cytosol, indicating that BARS-50 might exist as a multimeric complex. BARS-50 can bind GTP, as indicated by blot-overlay studies with [alpha-32P]GTP and by photoaffinity labeling with guanosine 5'-[gamma-32P] [beta,gamma-(4-azidoanilido)]triphosphate. Moreover, ADP-ribosylation of BARS-50 was completely inhibited by the beta gamma subunit complex of G proteins, while the ADP-ribosylation of GAPDH was unmodified, indicating that this effect was due to an interaction of the beta gamma complex with BARS-50, rather than with the ADP-ribosylating enzyme. Two-dimensional gel electrophoresis and immunoblot analysis shows that BARS-50 is a group of closely related proteins that appear to be different from all the known GTP-binding proteins.

Bridging grafts and transient nerve growth factor infusions promote long-term central nervous system neuronal rescue and partial functional recovery.

Grafts of favorable axonal growth substrates were combined with transient nerve growth factor (NGF) infusions to promote morphological and functional recovery in the adult rat brain after lesions of the septohippocampal projection. Long-term septal cholinergic neuronal rescue and partial hippocampal reinnervation were achieved, resulting in partial functional recovery on a simple task assessing habituation but not on a more complex task assessing spatial reference memory. Control animals that received transient NGF infusions without axonal-growth-promoting grafts lacked behavioral recovery but also showed long-term septal neuronal rescue. These findings indicate that (i) partial recovery from central nervous system injury can be induced by both preventing host neuronal loss and promoting host axonal regrowth and (ii) long-term neuronal loss can be prevented with transient NGF infusions.

D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release.

Using an antibody highly specific for D-serine conjugated to glutaraldehyde, we have localized endogenous D-serine in rat brain. Highest levels of D-serine immunoreactivity occur in the gray matter of the cerebral cortex, hippocampus, anterior olfactory nucleus, olfactory tubercle, and amygdala. Localizations of D-serine immunoreactivity correlate closely with those of D-serine binding to the glycine modulatory site of the N-methyl-D-aspartate (NMDA) receptor as visualized by autoradiography and are inversely correlated to the presence of D-amino acid oxidase. D-Serine is enriched in process-bearing glial cells in neuropil with the morphology of protoplasmic astrocytes. In glial cultures of rat cerebral cortex, D-serine is enriched in type 2 astrocytes. The release of D-serine from these cultures is stimulated by agonists of non-NMDA glutamate receptors, suggesting a mechanism by which astrocyte-derived D-serine could modulate neurotransmission. D-Serine appears to be the endogenous ligand for the glycine site of NMDA receptors.

Challenging the dogma: the hidden layer of non-protein-coding RNAs in complex organisms

The central dogma of biology holds that genetic information normally flows from DNA to RNA to protein. As a consequence it has been generally assumed that genes generally code for proteins, and that proteins fulfil not only most structural and catalytic but also most regulatory functions, in all cells, from microbes to mammals. However, the latter may not be the case in complex organisms. A number of startling observations about the extent of non-protein-coding RNA (ncRNA) transcription in the higher eukaryotes and the range of genetic and epigenetic phenomena that are RNA-directed suggests that the traditional view of the structure of genetic regulatory systems in animals and plants may be incorrect. ncRNA dominates the genomic output of the higher organisms and has been shown to control chromosome architecture, mRNA turnover and the developmental timing of protein expression, and may also regulate transcription and alternative splicing. This paper re-examines the available evidence and suggests a new framework for considering and understanding the genomic programming of biological complexity, autopoletic development and phenotypic variation. BioEssays 25:930-939,2003. (C) 2003 Wiley Periodicals, Inc.

Systemic apomorphine alters HPA axis responses to interleukin-1 beta adminstration but not sound stress

Apomorphine is a dopamine receptor agonist that was recently licensed for the treatment of erectile dysfunction. However, although sexual activity can be stressful, there has been little investigation into whether treatments for erectile dysfunction affect stress responses. We have examined whether a single dose of apomorphine, sufficient to produce penile erections (50 mug/kg, i.a.), can alter basal or stress-induced plasma ACTH levels, or activity of central pathways thought to control the hypothalamic-pituitary-adrenal axis in rats. An immune challenge (interleukin-1beta, 1 mug/kg, i.a.) was used as a physical stressor while sound stress (100 dB white noise, 30 min) was used as a psychological stressor. Intravascular administration of apomorphine had no effect on basal ACTH levels but did substantially increase the number of Fos-positive amygdala and nucleus tractus solitarius catecholamine cells. Administration of apomorphine prior to immune challenge augmented the normal ACTH response to this stressor at 90 min and there was a corresponding increase in the number of Fos-positive paraventricular nucleus corticotropin-releasing factor cells, paraventricular nucleus oxytocin cells and nucleus tractus solitarius catecholamine cells. However, apomorphine treatment did not alter ACTH or Fos responses to sound stress. These data suggest that erection-inducing levels of apomorphine interfere with hypothalamic-pituitary-adrenal axis inhibitory feedback mechanisms in response to a physical stressor, but have no effect on the response to a psychological stressor. Consequently, it is likely that apomorphine acts on a hypothalamic-pituitary-adrenal axis control pathway that is unique to physical stressors. A candidate for this site of action is the nucleus tractus solitarius catecholamine cell population and, in particular, A2 noradrenergic neurons. (C) 2003 Elsevier Science Ltd. All rights reserved.

Medial prefrontal cortex suppression of the hypothalamic–pituitary–adrenal axis response to a physical stressor, systemic delivery of interleukin-1β

Previous studies have shown that the medial prefrontal cortex can suppress the hypothalamic-pituitary-adrenal axis response to stress. However, this effect appears to vary with the type of stressor. Furthermore, the absence of direct projections between the medial prefrontal cortex and corticotropin-releasing factor cells at the apex of the hypothalamic-pituitary-adrenal axis suggest that other brain regions must act as a relay when this inhibitory mechanism is activated. In the present study, we first established that electrolytic lesions involving the prelimbic and infralimbic medial prefrontal cortex increased plasma adrenocorticotropic hormone levels seen in response to a physical stressor, the systemic delivery of interleukin-1beta. However, medial prefrontal cortex lesions did not alter plasma adrenocorticotropic hormone levels seen in response to a psychological stressor, noise. To identify brain regions that might mediate the effect of medial prefrontal cortex lesions on hypothalamic-pituitary-adrenal axis responses to systemic interleukin-1beta, we next mapped the effects of similar lesions on interleukin-1beta-induced Fos expression in regions previously shown to regulate the hypothalamic-pituitary-adrenal axis response to this stressor. It was found that medial prefrontal cortex lesions reduced the number of Fos-positive cells in the ventral aspect of the bed nucleus of the stria terminalis. However, the final experiment, which involved combining retrograde tracing with Fos immunolabelling, revealed that bed nucleus of the stria terminalis-projecting medial prefrontal cortex neurons were largely separate from medial prefrontal cortex neurons recruited by systemic interleukin-1beta, an outcome that is difficult to reconcile with a simple medial prefrontal cortex-bed nucleus of the stria terminalis-corticotropin-releasing factor cell control circuit.

Thiamine Deficiency Results in Downregulation of the GLAST Glutamate Transporter in Cultured Astrocytes

Pyrithiamine-induced thiamine deficiency (TD) is a well-established model of Wernicke's encephalopathy in which a glutamate-mediated excitotoxic mechanism may play an important role in determining selective vulnerability. In order to examine this possibility, cultured astrocytes were exposed to TD and effects on glutamate transport and metabolic function were studied. TD led to decreases in cellular levels of thiamine and thiamine diphosphate (TDP) after 24 h of treatment and decreased activities of the TDP-dependent enzymes alpha-ketoglutarate dehydrogenase and transketolase after 4 and 7 days, respectively. TD treatment for 10 days led to a reversible decrease in the uptake of [H-3]-D-aspartate, a nonmetabolizable analogue of glutamate. Kinetic analysis revealed that the uptake inhibition was caused by a 47% decrease in the V-max for uptake of [H-3]-D-aspartate, with no change in the K-m value. Immunoblotting showed that this decrease in uptake was due to an 81% downregulation of the astrocyte-specific GLAST glutamate transporter. Loss of uptake activity and GLAST protein were blocked by treatment with the protein kinase C inhibitor H7, while exposure to DCG IV, a group II metabotropic glutamate receptor (mGluR) agonist, resulted in improvement of [H-3]-D-aspartate uptake and a partial reversal of transporter downregulation. These results are consistent with our recent in vivo findings of a loss of astrocytic glutamate transporters in TD and provide evidence that TD conditions may increase phosphorylation. of GLAST, contributing to its downregulation. In addition, manipulation of group II mGluR activity may provide an important strategy in the treatment of this disorder. (C) 2003 Wiley-Liss, Inc.

Synthesis and biological evaluation of nonpeptide mimetics of co-conotoxin GVIA

A benzothiazole-derived compound (4a) designed to mimic the C-alpha-C-beta bond vectors and terminal functionalities of Lys2, TyrI3 and Arg17 in omega-conotoxin GVIA was synthesised, together with analogues (4b-d), which had each side-chain mimic systematically truncated or eliminated. The affinity of these compounds for rat brain N-type and P/Q-type voltage gated calcium channels (VGCCs) was determined. In terms of N-type channel affinity and selectivity, two of these compounds (4a and 4d) were found to be highly promising, first generation mimetics of omega-conotoxin. The fully functionalised mimetic (4a) showed low PM binding affinity to N-type VGCCs (IC50 = 1.9 muM) and greater than 20-fold selectivity for this channel sub-type over P/Q-type VGCCs, whereas the mimetic in which the guanidine-type side chain was truncated back to an amine (4d, IC50 = 4.1 muM) showed a greater than 25-fold selectivity for the N-type channel. (C) 2004 Elsevier Ltd. All rights reserved.

The alpha(2)delta auxiliary subunit reduces affinity of omega-conotoxins for recombinant N-type (Ca(v)2.2) calcium channels

The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on- rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential ( - 120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K] MVIIA and [O10K] GVIA had greater recovery from the block, whereas [K10R] CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states.

Opioids Inhibit Lateral Amygdala Pyramidal Neurons by Enhancing A Dendritic Potassium Current

Pyramidal neurons in the lateral amygdala discharge trains of action potentials that show marked spike frequency adaptation, which is primarily mediated by activation of a slow calcium-activated potassium current. We show here that these neurons also express an alpha-dendrotoxin- and tityustoxin-Kalpha-sensitive voltage-dependent potassium current that plays a key role in the control of spike discharge frequency. This current is selectively targeted to the primary apical dendrite of these neurons. Activation of mu-opioid receptors by application of morphine or D-Ala(2)-N-Me-Phe(4)-Glycol(5)-enkephalin (DAMGO) potentiates spike frequency adaptation by enhancing the alpha-dendrotoxin-sensitive potassium current. The effects of mu-opioid agonists on spike frequency adaptation were blocked by inhibiting G-proteins with N-ethylmaleimide (NEM) and by blocking phospholipase A(2). Application of arachidonic acid mimicked the actions of DAMGO or morphine. These results show that mu-opioid receptor activation enhances spike frequency adaptation in lateral amygdala neurons by modulating a voltage-dependent potassium channel containing Kv1.2 subunits, through activation of the phospholipase A(2)-arachidonic acid-lipoxygenases cascade.