Four-kilobase sequence of the mouse CNP gene directs spatial and temporal expression of lacZ in transgenic mice

The gene encoding 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) is one of the earliest myelin genes to be expressed in the brain. It is expressed at basal levels in some non-neural tissues but at much higher levels in the nervous system, and its relevance and mechanism are unknown. Using transgenic mice, we examined the expression pattern conferred by a 4-kilobase (-kb) 5′-flanking sequence of the mouse CNP gene coupled to the bacterial lacZ reporter gene. Here we report that this 4-kb fragment contains sufficient information to direct expression of the transgene to the tissue and/or cell type, in which CNP is normally expressed. In the central nervous system (CNS), CNP-lacZ expression was regulated in a temporal manner, consistent with endogenous CNP expression. Transgene expression was detected in embryonic brain and spinal cord in immature oligodendrocytes, and it significantly increased with age. In adult mice, β-galactosidase activity (which appeared to be oligodendrocyte specific) was found essentially in white matter areas of the CNS. Moreover, the transgene was expressed in peripheral nervous system, testis, and thymus—tissues that normally express CNP. Taken together, our results provide strong evidence that cis-acting regulatory elements, necessary to direct spatial and temporal expression of the transgene in oligodendrocytes, are located within the 4-kb 5′-flanking sequence of the mouse CNP gene. This promoter could be a valuable tool to target specific expression of other transgenes to oligodendrocytes, and may provide important new insights into myelination or dysmyelination. J. Neurosci. Res. 53:393–404, 1998. © 1998 Wiley-Liss, Inc.     

Inhibitors of free radical formation fail to attenuate direct β-amyloid25–35 peptide-mediated neurotoxicity in rat hippocampal cultures

The direct neurotoxic action of the β-amyloid protein, the major constituent of senile plaques, may represent the underlying cause of neuronal degeneration observed in Alzheimer's disease. The apoptotic-mediated neuronal death induced by β-amyloid appears to reside in its ability to form Ca^>2+-permeable pores in neuronal membranes resulting in an excessive influx of Ca²⁺ and the induction of neurotoxic cascades. It is possible that during β-amyloid exposure a Ca²⁺-mediated increase in free radical generation may exceed the defensive capacity of cells and thus lead to cell death. Consequently, in the present study we have investigated the effect of a panoply of antioxidants and inhibitors of free radical formation on the development of β-amyloid neurotoxicity. Acute exposure of rat hippocampal neurons to “aged” β-amyloid_25–35 peptide (5–50 m?M) induced a slow, concentration-dependent apoptotic neurotoxicity (25–85%) during a 6 day exposure. Co-incubation of cultures with β-amyloid_25–35 peptide (25 μM) and inhibitors of nitric oxide synthase and/or xanthine oxidase (N^G-monomethyl-L-arginine [1 mM], Nω-nitro-L-arginine [1 mM], oxypurinol [100 m?M], allopurinol [100 m?M]), important mediators of nitric oxide, superoxide, and hydroxyl radical formation, did not attenuate β-amyloid neurotoxicity. Similarly, a reduction in free radical generation by selective inhibition of phospholipase-A₂ cyclooxygenase, and lipoxygenase activities with quinacrine (0.5 m?M), indomethacin (50 m?M), and nor-dihydroguaiaretic acid (0.5 m?M), respectively, did not reduce the proclivity of β-amyloid to induce cell death. Exposure of cultures to catalase (25 U/ml) and/or superoxide dismutase (10 U/ml) as well as the free radical scavengers vitamin E (100 m?M), vitamin C (100 m?M), glutathione (100 m?M), L-cysteine (100 m?M), N-acetyl-cysteine (100 m?M), deferoxamine (5 m?M), or haemoglobin (35 m?g/ml) failed to attenuate the neurotoxic action of β-amyloid. On the other hand, pre-treatment of cultures with subtoxic concentrations of β-amyloid peptide significantly increased the vulnerability of neurons to H₂O₂ exposure and suggest that β-amyloid peptide renders neurons more sensitive to free radical attack. However, a potential β-amyloid-mediated increase in free radical formation is not a proximate cause of the neurotoxic mechanism of β-amyloid in vitro. © 1994 Wiley-Liss, Inc.     

Epistasis between tau phosphorylation regulating genes (CDK5R1 and GSK‐3β) and Alzheimer’s disease risk

Objective – Glycogen synthase kinase‐3β (GSK‐3β) and cyclin‐dependent kinase 5 (CDK5) have been implicated as two major protein kinases involved in the abnormal hyperphosphorylation of tau in Alzheimer’s disease (AD) brain, and the development of neurofibrillary tangles. CDK5 regulatory subunit 1 (CDK5R1) encodes for p35, a protein required for activation of CDK5. As both CDK5R1 and GSK‐3β genes are related to phosphorylation of tau, we examined the combined contribution of these genes to the susceptibility for AD. Methods – In a case–control study in 283 AD patients and 263 healthy controls, we examined the combined effects between CDK5R1 (3′‐UTR, rs735555) and GSK‐3β (?50, rs334558) polymorphisms on susceptibility to AD. Results – Subjects carrying both the CDK5R1 (3′‐UTR, rs735555) AA genotype and the GSK‐3β (?50, rs334558) CC genotype had a 12.5‐fold decrease in AD risk (adjusted by age, sex and APOE status OR = 0.08, 95% CI = 0.01–0.76, P = 0.03), suggesting synergistic effects (epistasis) between both genes. Conclusion – These data support a role for tau phosphorylation regulating genes in risk for AD.     

Differential effects of morphine withdrawal on cerebral β1- and β2-adrenergic receptors

Effect of morphine dependence and its withdrawal on the ³H-dihydroalprenolol (³H-DHA) binding for βadrenergic receptors,β₁ andβ₂, was examined by a computerized analysis of biphasic Hofstee plots. The relative density ofβ₁ andβ₂ receptors in the rat cerebral cortex was found to be approximately 70% and 30%, respectively. In rats rendered dependent on morphine by a subcutaneous implantation of a morphine pellet, the ³H-DHA binding toβ₁ andβ₂ receptors was not altered. During the stage of withdrawal induced by administration of naloxone, however, the ³H-DHA binding to the cerebral particulate fractions was increased, and this increase was due to the increased binding sites inβ₁ but not inβ₂ receptor. On the other hand, the apparent affinities ofβ₁ andβ₂ receptors for atenolol and salbutamol, selective antagonists forβ₁- andβ₂-adrenergic receptors, respectively, were not altered under these experimental conditions. These results suggest that an abrupt increase in cerebralβ₁-receptor binding sites occurs at morphine withdrawal, and the occurrence of such a supersensitivity in cerebralβ₁ receptor may be involved in the exhibition and/or maintenance of the abstinence syndrome in morphine-dependent subjects.     

Regulatory volume increase in astrocytes exposed to hypertonic medium requires β1‐adrenergic Na+/K+‐ATPase stimulation and glycogenolysis

The cotransporter of Na⁺, K⁺, 2Cl^–, and water, NKKC1, is activated under two conditions in the brain, exposure to highly elevated extracellular K⁺ concentrations, causing astrocytic swelling, and regulatory volume increase in cells shrunk in response to exposure to hypertonic medium. NKCC1‐mediated transport occurs as secondary active transport driven by Na⁺/K⁺‐ATPase activity, which establishes a favorable ratio for NKCC1 operation between extracellular and intracellular products of the concentrations of Na⁺, K⁺, and Cl^– × Cl^–. In the adult brain, astrocytes are the main target for NKCC1 stimulation, and their Na⁺/K⁺‐ATPase activity is stimulated by elevated K⁺ or the β‐adrenergic agonist isoproterenol. Extracellular K⁺ concentration is normal during regulatory volume increase, so this study investigated whether the volume increase occurred faster in the presence of isoproterenol. Measurement of cell volume via live cell microscopic imaging fluorescence to record fluorescence intensity of calcein showed that this was the case at isoproterenol concentrations of ≥1 µM in well‐differentiated mouse astrocyte cultures incubated in isotonic medium with 100 mM sucrose added. This stimulation was abolished by the β₁‐adrenergic antagonist betaxolol, but not by ICI118551, a β₂‐adrenergic antagonist. A large part of the β₁‐adrenergic signaling pathway in astrocytes is known. Inhibitors of this pathway as well as the glycogenolysis inhibitor 1,4‐dideoxy‐1,4‐imino‐D‐arabinitol hydrochloride and the NKCC1 inhibitors bumetanide and furosemide abolished stimulation by isoproterenol, and it was weakened by the Na⁺/K⁺‐ATPase inhibitor ouabain. These observations are of physiological relevance because extracellular hypertonicity occurs during intense neuronal activity. This might trigger a regulatory volume increase, associated with the post‐excitatory undershoot. © 2014 Wiley Periodicals, Inc.     

Neuron-specific expression of reporter gene in transgenic mice carrying the 5′-upstream region of mouse P/Q-type Ca channel α1A subunit gene fused to E. coli lacZ reporter gene

To dissect the molecular mechanisms underlying the neuron-specific expression of the P/Q type calcium channel α_1A subunit gene, transgenic mice carrying a 0.5-kb, 1.5-kb, 3.0-kb or 6.3-kb 5′-upstream region of the gene fused to Escherichia coli lacZ reporter gene were produced. In transgenic mice carrying the 1.5-kb, 3.0-kb or 6.3-kb 5′-upstream region, the reporter gene was exclusively expressed in the nervous system, although those with the 0.5-kb 5′-upstream region failed to show reporter expression. Histological examinations showed that the three 5′-upstream regions induced distinct expression patterns of the reporter gene in the CNS and adrenal medulla. The 1.5-kb 5′-upstream region drove reporter gene expression in the olfactory bulb, dorsal cortex and hippocampus, while the regulatory element for the expression in the amygdaloid nucleus, septum, habenula medial nucleus, choroid plexus, substantia nigra, inferior colliculus, pontine nucleus and cerebellum was located in the 5′-upstream sequence between 1.5 kb and 6.3 kb. In the cerebellum, the expression of the reporter gene was induced by the 3.0-kb region in granule cells, whereas it was induced by the 6.3-kb region in Purkinje cells. The expression of the reporter gene in chromaffin cells in the adrenal medulla was induced only by the 6.3-kb 5′-upstream region. These results suggest that the expression of the mouse P/Q-type Ca²⁺ channel α_1A subunit gene is regulated in a complex fashion by both positive and negative cis-regulatory elements.     

Role of CNPase in the oligodendrocytic extracellular 2′,3′‐cAMP‐adenosine pathway

Extracellular adenosine 3′,5′‐cyclic monophosphate (3′,5′‐cAMP) is an endogenous source of localized adenosine production in many organs. Recent studies suggest that extracellular 2′,3′‐cAMP (positional isomer of 3′,5′‐cAMP) is also a source of adenosine, particularly in the brain in vivo post‐injury. Moreover, in vitro studies show that both microglia and astrocytes can convert extracellular 2′,3′‐cAMP to adenosine. Here, we examined the ability of primary mouse oligodendrocytes and neurons to metabolize extracellular 2′,3′‐cAMP and their respective adenosine monophosphates (2′‐AMP and 3′‐AMP). Cells were also isolated from mice deficient in 2′,3′‐cyclic nucleotide‐3′‐phosphodiesterase (CNPase). Oligodendrocytes metabolized 2′,3′‐cAMP to 2′‐AMP with 10‐fold greater efficiency than did neurons (and also more than previously examined microglia and astrocytes); whereas, the production of 3′‐AMP was minimal in both oligodendrocytes and neurons. The production of 2′‐AMP from 2′,3′‐cAMP was reduced by 65% in CNPase ?/? versus CNPase +/+ oligodendrocytes. Oligodendrocytes also converted 2′‐AMP to adenosine, and this was also attenuated in CNPase ?/? oligodendrocytes. Inhibition of classic 3′,5′‐cAMP‐3′‐phosphodiesterases with 3‐isobutyl‐1‐methylxanthine did not block metabolism of 2′,3′‐cAMP to 2′‐AMP and inhibition of classic ecto‐5′‐nucleotidase (CD73) with α,β‐methylene‐adenosine‐5′‐diphosphate did not attenuate the conversion of 2′‐AMP to adenosine. These studies demonstrate that oligodendrocytes express the extracellular 2′,3′‐cAMP‐adenosine pathway (2′,3′‐cAMP → 2′‐AMP → adenosine). This pathway is more robustly expressed in oligodendrocytes than in all other CNS cell types because CNPase is the predominant enzyme that metabolizes 2′,3′‐cAMP to 2‐AMP in CNS cells. By reducing levels of 2′,3′‐cAMP (a mitochondrial toxin) and increasing levels of adenosine (a neuroprotectant), oligodendrocytes may protect axons from injury. GLIA 2013;61:1595–1606     

β-Adrenoceptors regulate myoelectric activity in the small intestine of rats: stimulation by β2 and inhibition by β3 subtypes

Abstract Using β-adrenergic agonists and antagonists this study investigated the importance of three different adrenoceptor subtypes for the regulation of migrating myoelectric complexes (MMCs) of the upper small intestine in conscious, naive rats. After a control period of 60 min with four activity fronts, agonists were given as an intravenous infusion for another 60 min. The non-selective β-adrenoceptor agonist isoprenaline (1 μg kg^?1 min^?1) inhibited MMCs and induced irregular spiking during the infusion period. This effect was blocked by intravenous administration of a bolus dose of either the non-selective β-adrenoceptor antagonist propranolol (1 mg kg^?1), or the β₂-antagonist ICI 118 551 (1 mg kg^?1), both given prior to isoprenaline. However, acebutolol (1 mg kg^?1), a selective β₁-antagonist, failed to antagonize the effect of isoprenaline. Furthermore, prenalterol, a selective β₁-agonist (12.5–800.0 μg kg^?1 min^?1), had no effect on the MMC pattern, whereas the β₂-selective agonist ritodrine (25–100 μg kg^?1 min^?1) induced a myoelectric pattern similar to one induced by isoprenaline. The partial β₃-adrenoceptor agonist D7114 (50–100 μg kg^?1 min^?1), disrupted the MMCs and induced quiescence. Neither of the antagonists, i.e. propranolol (1 mg kg^?1), acebutolol (1 mg kg^?1) nor ICI 118 551 (1 mg kg^?1), given alone induced changes in the MMC pattern. In conclusion, β₂-adrenoceptors in particular but also β₃-adrenoceptors seem to be of importance in the regulation of small intestinal motility by disrupting the regular MMC pattern in rats.     

α2‐Adrenergic drugs modulate the binding of [18F]fallypride to dopamine D2/3 receptors in striatum of living mouse

Aim. To test for α₂ adrenergic modulation of dopamine D_2/3 receptor availability in striatum of living mice using the high‐affinity ligand [¹⁸F]fallypride and microPET. Methods. Groups of anesthetized mice were pretreated with saline, the α₂‐agonist clonidine (1 mg/kg), and the α₂‐antagonists RX821002 (1 mg/kg) and yohimbine (1 mg/kg). Dynamic microPET recordings lasting 120 min were then initiated upon i.v. tracer injection of [¹⁸F]fallypride. Parametric maps of [¹⁸F]fallypride binding potential (BP_ND) were calculated using the Logan method, with cerebellum serving as the reference region. Results. Mean striatal [¹⁸F]fallypride BP_ND was 10.6 ± 1.7 in the saline control animals, 8.9 ± 1.7 (?16%; P < 0.05) in the RX821002 group, 8.3 ± 2.6 (?22%; P < 0.05) in the yohimbine group and 10.3 ± 2.2 (n.s.) in the clonidine group. Conclusions. These findings are consistent with a tonic inhibition of dopamine release by α₂ adrenergic receptors, such that α₂ blockade increased the competition from endogenous dopamine at D_2/3 receptors, thus reducing the [¹⁸F]fallypride BP_ND by about 20%. Absent effects of clonidine suggest a ceiling effect in the tonic inhibition of dopamine release. This in vivo PET evidence for α₂/dopaminergic interaction may be relevant to putative actions of atypical antipsychotic medications via adrenergic receptors. Synapse, 2010. © 2010 Wiley‐Liss, Inc.