Early role for a Na+,K+-ATPase (ATP1A3) in brain development.

Osmotic equilibrium and membrane potential in animal cells depend on concentration gradients of sodium (Na+) and potassium (K+) ions across the plasma membrane, a function catalyzed by the Na+,K+-ATPase α-subunit. Here, we describe ATP1A3 variants encoding dysfunctional α3-subunits in children affected by polymicrogyria, a developmental malformation of the cerebral cortex characterized by abnormal folding and laminar organization. To gain cell-biological insights into the spatiotemporal dynamics of prenatal ATP1A3 expression, we built an ATP1A3 transcriptional atlas of fetal cortical development using mRNA in situ hybridization and transcriptomic profiling of ∼125,000 individual cells with single-cell RNA sequencing (Drop-seq) from 11 areas of the midgestational human neocortex. We found that fetal expression of ATP1A3 is most abundant to a subset of excitatory neurons carrying transcriptional signatures of the developing subplate, yet also maintains expression in nonneuronal cell populations. Moving forward a year in human development, we profiled ∼52,000 nuclei from four areas of an infant neocortex and show that ATP1A3 expression persists throughout early postnatal development, most predominantly in inhibitory neurons, including parvalbumin interneurons in the frontal cortex. Finally, we discovered the heteromeric Na+,K+-ATPase pump complex may form nonredundant cell-type-specific α-ß isoform combinations, including α3-ß1 in excitatory neurons and α3-ß2 in inhibitory neurons. Together, the developmental malformation phenotype of affected individuals and single-cell ATP1A3 expression patterns point to a key role for α3 in human cortex development, as well as a cell-type basis for pre- and postnatal ATP1A3-associated diseases.

Major amyloid-ß-degrading enzymes, endothelin-converting enzyme-2 and neprilysin, are expressed by distinct populations of GABAergic interneurons in hippocampus and neocortex.

Impaired clearance of amyloid-ß peptide (Aß) has been postulated to significantly contribute to the amyloid accumulation typical of Alzheimer's disease. Among the enzymes known to degrade Aß in vivo are endothelin-converting enzyme (ECE)-1, ECE-2, and neprilysin (NEP), and evidence suggests that they regulate independent pools of Aß that may be functionally significant. To better understand the differential regulation of Aß concentration by its physiological degrading enzymes, we characterized the cell and region-specific expression pattern of ECE-1, ECE-2, and NEP by in situ hybridization and immunohistochemistry in brain areas relevant to Alzheimer's disease. In contrast to the broader distribution of ECE-1, ECE-2 and NEP were found enriched in GABAergic neurons. ECE-2 was majorly expressed by somatostatin-expressing interneurons and was active in isolated synaptosomes. NEP messenger RNA was found mainly in parvalbumin-expressing interneurons, with NEP protein localized to perisomatic parvalbuminergic synapses. The identification of somatostatinergic and parvalbuminergic synapses as hubs for Aß degradation is consistent with the possibility that Aß may have a physiological function related to the regulation of inhibitory signaling.

Matrix metalloproteinase-9 (MMP-9) in human intractable epilepsy caused by focal cortical dysplasia.

Focal cortical dysplasia (FCD) is a developmental brain disorder characterized by localized abnormalities of cortical layering and neuronal morphology. It is associated with pharmacologically intractable forms of epilepsy in both children and adults. The mechanisms that underlie FCD-associated seizures and lead to the progression of the disease are unclear. Matrix metalloproteinases (MMPs) are enzymes that are able to influence neuronal function through extracellular proteolysis in various normal and pathological conditions. The results of experiments that have used rodent models showed that extracellular MMP-9 can play an important role in epileptogenesis. However, no studies have shown that MMP-9 is involved in the pathogenesis of human epilepsy. The aim of the present study was to determine whether MMP-9 plays a role in intractable epilepsy. Using an unbiased antibody microarray approach, we found that up regulation of MMP-9 is prominent and consistent in FCD tissue derived from epilepsy surgery, regardless of the patient's age. Additionally, an up regulation of MMP-1, -2, -8, -10, and -13 was found but was either less pronounced or limited only to adult cases. In the dysplastic cortex, immunohistochemistry revealed that the highest MMP-9 immuno reactivity occurred in the cytoplasm of abnormal neurons and balloon cells. The neuronal over expression of MMP-9 also occurred in sclerotic hippocampi that were excised together with the dysplastic cortex, but sclerotic hippocampi were free of dysplastic features. In both locations, MMP-9 was also found in reactive astrocytes, albeit to a lesser extent. At the subcellular level, increased MMP-9 immunoreactivity was prominently upregulated at synapses. Thus, although upregulation of the enzyme in FCD is not causally linked to the developmental malformation, it may be a result of ongoing abnormal synaptic plasticity. The present findings support the hypothesis of the pathogenic role of MMP-9 in human epilepsy and may stimulate discussions about whether MMPs could be novel therapeutic targets for intractable epilepsy.

Extracellular matrix modulator lysyl oxidase colocalizes with amyloid-beta pathology in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis--Dutch type.

Accumulation of amyloid-beta (Aß) in brain vessel walls and parenchyma, known as cerebral amyloid angiopathy (CAA) and senile plaques (SPs), respectively, plays a key role in Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) pathogenesis. Although the mechanisms underlying CAA and SP formation remain largely unknown, evidence is mounting that local alterations of the extracellular matrix (ECM) in the brain vessel wall and/or parenchyma play an important role. Lysyl oxidase (LOX, E.C. 1.4.3.13) is an inducible amine oxidase that modulates the ECM by catalyzing the formation of molecular covalent cross-links in ECM proteins. The aim of this study is to investigate the association of LOX with CAA and with classic and diffuse SPs in both AD and HCHWA-D cases. We observed an association of LOX with Aß in CAA and with Aß in both classic and diffuse SPs in AD and HCHWA-D cases. In addition, LOX staining was observed in reactive astrocytes associated with these lesions. We conclude that the ECM modulating enzyme LOX is associated with the Aß-related pathological hallmarks of both AD and HCHWA-D, and that our findings provide additional insights into the mechanisms underlying the formation of these lesions.

Aromatase distribution in the monkey temporal neocortex and hippocampus.

Numerous studies have shown that neuronal plasticity in the hippocampus and neocortex is regulated by estrogen and that aromatase, the key enzyme for estrogen biosynthesis, is present in cerebral cortex. Although the expression pattern of aromatase mRNA has been described in the monkey brain, its precise cellular distribution has not been determined. In addition, the degree to which neuronal aromatase is affected by gonadal estrogen has not been investigated. In this study, we examined the immunohistochemical distribution of aromatase in young ovariectomized female rhesus monkeys with or without long-term cyclic estradiol treatment. Both experimental groups showed that aromatase is localized in a large population of CA1-3 pyramidal cells, in granule cells of the dentate gyrus and in some interneurons in which it was co-expressed with the calcium-binding proteins calbindin, calretinin, and parvalbumin. Moreover, numerous pyramidal cells were immunoreactive for aromatase in the neocortex, whereas only small subpopulations of neocortical interneurons were immunoreactive for aromatase. The widespread expression of the protein in a large neuronal population suggests that local intraneuroral estrogen synthesis may contribute to estrogen-induced synaptic plasticity in monkey hippocampus and neocortex of female rhesus monkeys. In addition, the apparent absence of obvious differences in aromatase distribution between the two experimental groups suggests that these localization patterns are not dependent on plasma estradiol levels.

Upregulation of the glutamine transporter through transactivation mediated by cAMP/protein kinase A signals toward exacerbation of vulnerability to oxidative stress in rat neocortical astrocytes.

In the present study, we have evaluated the possible functionality in astrocytes of the glutamine (Gln) transporter (GlnT) known to predominate in neurons for the neurotransmitter pool of glutamate. Sustained exposure to the adenylyl cyclase activator forskolin for 24 h led to a significant increase in mRNA expression of GlnT among different membrane transporters capable of transporting Gln, with an increase in [(3)H]Gln accumulation sensitive to a system A transporter inhibitor, in cultured rat neocortical astrocytes, but not neurons. Forskolin drastically stimulated GlnT promoter activity in a manner sensitive to a protein kinase A (PKA) inhibitor in rat astrocytic C6 glioma cells, while deletion mutation analysis revealed that the stimulation was mediated by a cAMP responsive element (CRE)/activator protein-1 (AP-1) like site located on GlnT gene promoter. Forskolin drastically stimulated the promoter activity in a fashion sensitive to a PKA inhibitor in C6 glioma cells transfected with a CRE or AP-1 reporter plasmid, in association with the phosphorylation of CRE binding protein on serine133. Transient overexpression of GlnT significantly exacerbated the cytotoxicity of hydrogen peroxide in cultured astrocytes. These results suggest that GlnT expression is upregulated by cAMP/PKA signals for subsequent exacerbation of the vulnerability to oxidative stress in astrocytes.

Dopachrome tautomerase (Dct) regulates neural progenitor cell proliferation.

DOPAchrome tautomerase (Dct) functions downstream of tyrosinase in the biosynthetic pathway of eumelanin by catalyzing the conversion of dopachrome to 5,5-dihydroxyindole-2-carboxylic acid (DHICA) in pigment cells. Dct transcription is regulated directly or synergistically by Pax3, Sox10 and microphthalmia transcription factor (MITF). Using Dct-lacZ transgenic mice, we measured the spatial and temporal pattern of Dct expression in vivo during neocortical neurogenesis in the brain. Dct was expressed in all layers of the dorsal telencephalon in E10.5. At E15.5 and E17.5 when cortical neurogenesis occurs, expression of Dct was primarily localized to the ventricular zone (VZ) where neuronal stem cells reside. Blocking endogenous Dct by RNAi decreased proliferation of embryonic cortical neural progenitor cells (by 48%, P < 0.05), as determined by BrdU incorporation. In adult brain, Dct/Dct expression decreased in the subventricular zone (SVZ), dentate gyrus and olfactory bulb (OB). However, strong expression of Dct was observed in rostral migratory stream (RMS) and septum. Overexpression of Dct in SVZ cells derived from the adult mice significantly increased the number of cells by 260%, whereas silencing Dct by RNAi decreased cell numbers by 25.8% at 48 h post-nucleofection (P < 0.05). The results of RT-PCR analysis revealed that Dct in the brain lacks exon 7 and is identical to the form of Dct found in neural-crest-derived melanocytes. Our data indicate that Dct, previously known as a melanoblast marker, regulates neural progenitor cell proliferation.

Identification of endophilins 1 and 3 as selective binding partners for VGLUT1 and their co-localization in neocortical glutamatergic synapses: implications for vesicular glutamate transporter trafficking and excitatory vesicle formation.

1. Selective protein-protein interactions between neurotransmitter transporters and their synaptic targets play important roles in regulating chemical neurotransmission. We screened a yeast two-hybrid library with bait containing the C-terminal amino acids of VGLUT1 and obtained clones that encode endophilin 1 and endophilin 3, proteins considered to play an integral role in glutamatergic vesicle formation. 2. Using a modified yeast plasmid vector to enable more cost-effective screens, we analyzed the selectivity and specificity of this interaction. Endophilins 1 and 3 selectively recognize only VGLUT1 as the C-terminus of VGLUT2 and VGLUT3 do not interact with either endophilin isoform. We mutagenized four conserved stretches of primary sequence in VGLUT1 that includes two polyproline motifs (Pro1, PPAPPP, and Pro2, PPRPPPP), found only in VGLUT1, and two conserved stretches (SEEK, SYGAT), found also in VGLUT2 and VGLUT3. The absence of the VGLUT conserved regions does not affect VGLUT1-endophilin association. Of the two polyproline stretches, only one (Pro2) is required for binding specificity to both endophilin 1 and endophilin 3. 3. We also show that endophilin 1 and endophilin 3 co-localize with VGLUT1 in synaptic terminals of differentiated rat neocortical neurons in primary culture. These results indicate that VGLUT1 and both endophilins are enriched in a class of excitatory synaptic terminals in cortical neurons and there, may interact to play an important role affecting the vesicular sequestration and synaptic release of glutamate.

Effect of NMDA on staurosporine-induced activation of caspase-3 and LDH release in mouse neocortical and hippocampal cells.

To achieve a better understanding of developmentally regulated NMDA- and staurosporine-induced apoptotic processes, we investigated the concerted action of these agents on caspase-3 activity and LDH release in neocortical and hippocampal cell cultures at different stages in vitro (DIV). Hoechst 33342 and MAP-2 stainings were additionally employed to visualize apoptotic changes and cell damage. The vulnerability of neocortical cells to NMDA was more prominent at later culture stages, whereas hippocampal neurons were more susceptible to NMDA treatment at earlier stages. A persistent activation of caspase-3 by staurosporine was found at all experimental stages. Despite of certain differences in susceptibility to NMDA and staurosporine, both tissues responded to regulatory action of NMDA towards staurosporine-activated caspase-3 in a similar way. Combined treatment with NMDA and staurosporine resulted in a substantial increase in caspase-3 activity in neocortical and hippocampal neurons on 2 DIV. Additive effects were also observed in neocortical cultures on 12 DIV. In contrast, NMDA substantially inhibited staurosporine-induced caspase-3 activity on 7 DIV in neocortical and hippocampal cultures. Additionally, pro-apoptotic effects of 17beta-estradiol were attenuated by NMDA on 7 DIV. Changes in vulnerability to NMDA- and staurosporine-mediated activation of caspase-3 were not strictly related to LDH release. Our data revealed that NMDA can both enhance and inhibit the staurosporine-induced neuronal cell apoptosis. The pro-apoptotic effect of NMDA was exhibited at early and late culture stages, whereas the anti-apoptotic effect was transient occurring on 7 DIV only.

Abnormal neocortical development in mice lacking cGMP-dependent protein kinase I.

Cyclic GMP-dependent protein kinase type I (cGKI) is a key signaling intermediate important for synaptic potentiation in the hippocampus and cerebellum, but its expression and function in cortical development have not been elucidated. The expression of cGKI in the developing mouse neocortex was evaluated by immunofluorescence labeling, and effect of cGKI deletion on cortical development was studied in adult cGKI knockout mice. cGKI was expressed at highest levels at embryonic stages in young neurons and radial glial fibers, corresponding to the major period of radial migration and laminar development of pyramidal neurons (embryonic day E13.5-E14.5), declining upon maturation (E17.5-postnatal day P28). The cerebral cortex of homozygous null mutant mice lacking cGKI exhibited heterotopic collections of neurons in the upper cortical layers and abnormal invaginations of layer I, in accord with a neuronal migration or positioning defect. Some cGKI mutant mice displayed defects in midline development resulting in partial fusion of cerebral hemispheres with adjacent neuronal heterotopias. Apical dendrites of cortical pyramidal neurons were misoriented in the cerebral cortex of cGKI null mutants, as shown in reporter mice expressing yellow fluorescent protein in layer V pyramidal neurons and by Golgi impregnation. These results demonstrate a role for cGKI signaling in cortical development related to neuronal migration/positioning that is important for dendritic orientation and connectivity.

Cyto- and chemoarchitecture of the cortex of the tammar wallaby (Macropus eugenii): areal organization.

We have examined the cyto- and chemoarchitecture of the isocortex of a diprotodontid marsupial, the tammar wallaby (Macropus eugenii), using Nissl staining in combination with enzyme histochemical (acetylcholinesterase - AChE, NADPH-diaphorase - NADPHd, cytochrome oxidase) and immunohistochemical (non-phosphorylated neurofilament - SMI-32) markers. The primary sensory cortex showed distinctive patterns of reactivity in cytochrome oxidase, acetylcholinesterase and NADPH diaphorase. For example, in AChE material, S1 showed a heterogeneous appearance, with regions exhibiting a double layer of AChE activity (layers II and IV) adjacent to poorly reactive regions. In NADPHd preparations, activity in S1 was strongest in layers I to IV although, as in AChE material, there were consistent patches of reduced NADPHd activity which corresponded to poorly reactive regions in the AChE sections. Each of the primary sensory areas of the isocortex showed a different pattern of distribution of SMI-32+ neurons. In V1, SMI-32+ neurons were distributed in two layers (III and V) throughout the tangential extent of that region. In S1, SMI-32+ neurons were concentrated in layer V, but large and discrete patches within S1 had additional SMI-32+ neurons in layer III. In primary auditory cortex there was a dense band of SMI-32+ neurons in layer V, with only occasional labeled pyramidal neurons in layer III. In the secondary sensory areas (V2 and S2) SMI-32+ neurons were either distributed in layers III and V (V2) or solely within layer V (S2). The tangential and laminar distribution of Type I reactive NADPH diaphorase neurons in the tammar wallaby cortex was more like that seen in eutheria than in polyprotodontid metatheria.

Receptor-independent depression of DA and 5-HT uptake by cannabinoids in rat neocortex--involvement of Na(+)/K(+)-ATPase.

There is evidence that cannabinoids modulate the reuptake of some neurotransmitters in the central nervous system. In this study, we investigated the effects of the synthetic cannabinoid receptor agonist WIN55212-2, the endocannabinoid anandamide and the chemically related arachidonic acid on serotonin (5-HT) and dopamine (DA) uptake into rat neocortical synaptosomes. At micromolar concentrations, anandamide and arachidonic acid produced steep inhibition curves with Hill coefficients above unity. WIN55212-2 inhibited both DA and 5-HT uptake with Hill coefficients near unity, also within the micromolar range. The effect of WIN55212-2 was not mediated by cannabinoid receptors, since the CB1 receptor antagonist AM251 failed to diminish uptake inhibition by WIN55212-2 and since the Ki estimates of WIN55212-2 were outside the range of the dissociation constants of WIN55212-2 at both CB1 and CB2 receptors. A 100-fold higher concentration of DA, respectively 5-HT, did not induce a shift to the right of the WIN55212-2 concentration-inhibition curves, suggesting a carrier-independent mechanism. The Na(+)/K(+)-ATPase inhibitor ouabain concentration dependently inhibited 5-HT uptake. Possible drug effects on commercial Na(+)/K(+)-ATPase and synaptosomal ATP consumption were investigated using an ATP bioluminescence assay. Ouabain inhibited both commercial and synaptosomal Na(+)/K(+)-ATPase. WIN55212-2 had no effect on commercial Na(+)/K(+)-ATPase, but inhibited synaptosomal ATP consumption. Anandamide produced a sharp decrease in the activity of commercial Na(+)/K(+)-ATPase and on synaptosomal ATP consumption. Presence of ouabain significantly reduced the inhibitory effect of anandamide on synaptosomal ATP consumption, whereas the effect of WIN55212-2 remained unchanged. Our results show that cannabinoids and arachidonic acid inhibit DA and 5-HT uptake into rat neocortical synaptosomes. This effect is neither cannabinoid receptor-mediated nor due to competitive inhibition of membrane transporters, but is partly effected by a decreased Na(+)/K(+)-ATPase activity.

Slight impairment of Na+,K+-ATPase synergistically aggravates ceramide- and beta-amyloid-induced apoptosis in cortical neurons.

Dysfunction of the Na(+),K(+)-ATPase (Na(+),K(+)-pump), due to reduced energy supply or increased endogenous ouabain-like inhibitors, likely occurs under pathological conditions in the central nervous system. In cultured mouse cortical neurons, we examined the hypothesis that a mild non-toxic inhibition of the Na(+),K(+)-ATPase could synergistically sensitize the vulnerability of neurons to normally non-lethal apoptotic signals. Ouabain at a low concentration of 0.1 microM slightly lessened the Na(+),K(+)-pump activity measured as an ouabain-sensitive current, yet did not affect K(+) homeostasis and viability of cortical neurons. Co-exposure to 0.1 microM ouabain plus non-lethal C(2)-ceramide (5 microM) or beta-amyloid 1-42 (5 microM), however, induced marked intracellular K(+) loss, caspase-3 cleavage, DNA laddering, and synergistically triggered neuronal death. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) predominantly blocked the caspase activation and neuronal death. These results suggest that slight impairment of Na(+),K(+)-pump activity may amplify the disruption of K(+) homeostasis in the presence of a non-lethal apoptotic insult, leading to activation of apoptotic cascade and substantial neuronal injury.

Progressive brain dysfunction following intracerebroventricular infusion of beta(1-42)-amyloid peptide.

The behavioral, neurochemical and histological changes of rats subjected to 3 days treatment with intracerebroventricular infusion of beta-amyloid peptides(Abeta)(1-42) were investigated 20 days and 80 days after the surgery. Abeta(1-42) produced a dose-dependent and a time-dependent impairment in the spontaneous alternation performance in the Y-maze (spatial working memory), place navigation task in a water maze (spatial reference memory) and passive avoidance retention (non-spatial long-term memory) at doses of 10 and 20 microg/rat. The learning impairments were more severe at 80 days than 20 days after infusion of Abeta(1-42). At 25 days after the infusion, a significant decrease in hemicholinium-3 (HC-3) binding was observed only in the hippocampus, although choline acetyltransferase (ChAT) activity was unchanged in the brain regions tested as compared with the vehicle (Abeta(40-1)) treatment. In contrast, the reduction in ChAT activity 85 days after Abeta(1-42) infusion was significant in hippocampus and striatum. HC-3 binding was also significantly decreased in the posterior cortex, hippocampus and striatum. In the histological analysis, brain atrophy was observed inasmuch as ventricular enlargement and neuronal damage in the CA1 area of the hippocampus were seen 85 days after Abeta(1-42) infusion. These results suggest that the rats subjected to intracerebroventricular infusion of Abeta(1-42) suffered from progressive brain dysfunction, and could be useful as an animal model for evaluating the developmental processes at the early and/or middle stage of Alzheimer's-type dementia.

Neuronal polo-like kinase in Alzheimer disease indicates cell cycle changes.

Neurons of adults apparently lack the components necessary to complete the cell division process. Therefore, in Alzheimer disease, the increased expression of cell cycle-related proteins in degenerating neurons likely leads to an interrupted mitotic process associated with cytoskeletal abnormalities and, ultimately, neuronal degeneration. In this study, to further delineate the role of mitotic processes in the pathogenesis of Alzheimer disease, we undertook a study of polo-like kinase (Plk), a protein that plays a crucial role in the cell cycle. Our results show disease-related increases in Plk in susceptible hippocampal and cortical neurons in comparison to young or age-matched controls. An increase in neuronal Plk further implicates aberrations in cell cycle control in the pathogenesis of Alzheimer disease and provides a novel mechanistic basis for therapeutic intervention.

Constitutive overactivation of protein kinase C in guinea pig brain increases alpha-secretory APP processing without decreasing beta-amyloid generation.

Whilst it is generally accepted that the activation of protein kinase C (PKC) increases amyloid precursor protein (APP) secretion in vitro, the role of PKC in the regulation of APP processing and beta-amyloid generation in vivo is still not well understood. In order to address this question, we established the animal model of neocortical microencephalopathy in guinea pigs caused by in utero treatment with methylazoxymethanol acetate, a DNA-methylating substance that eliminates proliferating cells of neuroepithelial origin. The induction of this neocortical malformation is accompanied by constitutive overactivation of PKC in the neocortex of the offspring. In the cortical and hippocampal tissues of juvenile microencephalic guinea pigs (postnatal day 30), we observed significant increases in basal (by 58% and 74%, respectively,) and phorbol ester-stimulated PKC enzyme activity (by 47% and 71%) as compared to age-matched control animals. In the same cortical/hippocampal preparations of methylazoxymethanol-treated animals, there was increased alpha-secretion of APP by 35% and 30% as measured by Western blot analysis using the antibody 6E10, whilst total APP secretion as well as APP mRNA expression remained unaltered. This upregulation of APP alpha-secretion was limited to brain areas that displayed elevated PKC activity. However, constitutive overactivation of neocortical PKC did not affect the generation of beta-amyloid peptides 1-40 or 1-42 as measured by ELISA, suggesting that only the alpha-secretase pathway of APP processing is affected by chronic PKC overactivation in vivo.

[Aldehyde dehydrogenase activity and level of dopamine in certain sections of the brain of rats preferring and refusing ethanol]. / Al'degiddegidrogenaznaia aktivnost' i soderzhanie dofamina v nekotorykh otdelakh mozga krys, predpochitaiushchikh i otvergaiushchikh étanol.

Aldehyde dehydrogenase activity (KF 1.2.1.3) of cytosol fractions of brain structures (hypothalamus, midbrain and new cortex) as well as dophamine content in these structures were studied in comparative aspect in rats preferring and rejection ethanol. It has been shown that there were two isoforms of aldehyde dehydrogenases (aldehyde dehydrogenase 1 and aldehyde dehydrogenase 2) in cytosol fractions of all investigated brain structures of animals preferring ethanol while only aldehyde dehydrogenase 2 has been found in the new cotex of rats rejecting ethanol. Thus, aldehyde-dehydrogenase activity is higher in the animals preferring ethanol than in those ones rejecting ethanol. Content of dophamine in the rats preferring ethanol is higher than in those ones rejecting ethanol both in the hypothalamus and new cortex. Differences between the studied groups of animals can underlie the pathologic attraction to alcohol.

Tissue transglutaminase is increased in Huntington's disease brain.

The polyglutamine-expanded N-terminal region of mutant huntingtin causes neurodegeneration in Huntington's disease (HD). Neuronal intranuclear and cytosolic inclusions composed of mutant huntingtin are found in brains of HD patients. Because tissue transglutaminase cross-links proteins into filamentous aggregates and polypeptide-bound glutamines are primary determining factors for tissue transglutaminase-catalyzed reactions, it has been hypothesized that tissue transglutaminase may contribute to the formation of these aggregates. In this report immunohistochemical and biochemical methods were used to demonstrate that tissue transglutaminase expression and transglutaminase activity are elevated in HD brains in a grade-dependent manner. In the striatum, tissue transglutaminase activity was significantly increased in the grade 3 HD cases compared with controls. When normalized to the neuronal marker calbindin D28k, immunoblot analysis revealed that in the striatum the levels of tissue transglutaminase were significantly increased in all HD cases compared with controls. Immunohistochemical staining of the HD striatum revealed that tissue transglutaminase immunoreactivity was markedly increased in all grades as compared with controls. In the superior frontal cortex, tissue transglutaminase activity was significantly higher in all HD cases as compared with controls. Quantitative analysis of immunoblots demonstrated that tissue transglutaminase levels were elevated in HD grades 2 and 3 cases. Tissue transglutaminase immunoreactivity within the superior frontal neocortex was also greater in all the HD cases compared with controls. These data clearly indicate that tissue transglutaminase is elevated in HD brain and may play a role in the disease process.

Upregulation of COX-2 and CGRP expression in resident cells of the Borna disease virus-infected brain is dependent upon inflammation.

Infection of immunocompetent adult rats with Borna disease virus (BDV) causes severe encephalitis and neural dysfunction. The expression of COX-2 and CGRP, genes previously shown to be implicated in CNS disease and peripheral inflammation, was dramatically upregulated in the cortical neurons of acutely BDV-infected rats. Neuronal COX-2 and CGRP upregulation was predominantly seen in brain areas where ED1-positive macrophages/microglia accumulated. In addition, COX-2 expression was strongly induced in brain endothelial cells and the number of COX-2 immunoreactive microglial cells was increased. In contrast, despite increased expression of viral antigens, neither COX-2 nor CGRP expression was altered in the CNS of BDV-infected rats treated with dexamethasone, or tolerant to BDV. Thus, increased CGRP and COX-2 expression in the BDV-infected brain is the result of the inflammatory response and likely to be involved in the pathogenesis of virus-induced encephalitis.