Changes in Activity of Cysteine Cathepsins B and L in Brain Structures of Mice with Aggressive and Depressive-Like Behavior Formed under Conditions of Social Stress.

We studied activity of lysosomal cysteine proteases, cathepsins B and L, in brain structures (frontal cortex, caudate nucleus, hippocampus, and hypothalamus) of C57Bl/6J mice with aggressive and depressive-like behavior formed under conditions of chronic social stress (repeated experience of victories and defeats within 20 days). Mice with depressive-like behavior showed increased activity of cathepsin В in the hypothalamus and nucleus caudatus and increased activity of cathepsin L in the hippocampus compared to control animals not subjected to agonistic confrontations. In mice with aggressive behavior, protease activity in the studied brain structures was not changed. In 4 h after immune system activation with LPS (250 µg/kg), cathepsin L activity in the hippocampus of control mice increased in comparison with mice receiving saline. In contrast to control animals, LPS caused a decrease in activity of the enzyme in the caudate nucleus and frontal cortex of aggressive mice and in the hippocampus of mice with depressive-like behavior.

Brain acetylcholinesterase activity in Wistar and August rats with low and high motor activity (a cytochemical study).

Acetylcholinesterase activity was quantitatively evaluated by cytochemical method in brain structures (layers III and V of the sensorimotor cortex, caudate nucleus, nucleus accumbens, hippocampus CA3 field) of August and Wistar rats demonstrating high and low motor activity in the open field test. In August rats, acetylcholinesterase activity in the analyzed brain structures prevailed in animals with high motor activity in comparison with rats with low motor activity. In Wistar rats, the differences between the animals demonstrating high and low motor activity were less pronounced, but varied depending on the experimental series of studies. Comparisons of August rats with low motor activity and Wistar rats with high motor activity (maximum difference of motor function in these animals) revealed significant excess of acetylcholinesterase activity in layer III of the sensorimotor cortex in August rats and no differences in other brain structures of the examined animals.

Vascular and brain dopamine beta-hydroxylase activity in young spontaneously hypertensive rats.

Dopamine beta-hydroxylase activity was higher in mesenteric vessels, adrenal glands, and serum of 3-week-old spontaneously hypertensive rats but lower in the locus coeruleus than it was in the control Wistar-Kyoto rats. The results support the concept that the nervous system is an important regulator of blood pressure.

Parkinson's disease: activity of L-dopa decarboxylase in discrete brain regions.

The activity of L-dopa decarboxylase was greatly reduced in the striatum, less so in the hypothalamus, and unchanged in the cortex of brains of patients with Parkinson's disease. However, it appears that even in the striatum enough activity remained to allow for the formation of dopamine from L-dopa in patients treated with large doses of L-dopa.

Short- and long-term lithium administration: effects on the brain's serotonergic biosynthetic systems.

Short-term treatment with lithium chloride stimulates the uptake of tryptophan and its conversion to serotonin by striate synaptosomes. Preincubation of striate synaptosomes with L-tryptophan and in vivo administration of L-tryptophan appear to act in a similar manner. Midbrain tryptophan hydroxylase activity is reduced in temporal continuity with the lithium-induced activation of tryptophan uptake and conversion. By 10 days, conversion of tryptophan to serotonin in nerve endings becomes a joint function of the maintained increased uptake of tryptophan and a decreased level of tryptophan hydroxylase activity in nerve endings. The occurrence of this delayed alteration corresponds in time to the previously described axoplasmic flow rate for tryptophan hydroxylase.

Angiotensin-forming enzyme in brain tissue.

A renin-like enzyme is present in brain tissue and is independent of kidney and plasma renin. In the presence of homologous substrate it forms angiotensin. Administration of aldosterone significantly decreases this angiotensinforming enzyme activity, while administration of progesterone markedly enhances it.

Selective sparing of a class of striatal neurons in Huntington's disease.

A distinct subpopulation of striatal aspiny neurons, containing the enzyme nicotinamide adenine dinucleotide phosphate diaphorase, is preserved in the caudate nucleus in Huntington's disease. Biochemical assays confirmed a significant increase in the activity of this enzyme in both the caudate nucleus and putamen in postmortem brain tissue from patients with this disease. The resistance of these neurons suggests that the gene defect in Huntington's disease may be modifiable by the local biochemical environment. This finding may provide insight into the nature of the genetically programmed cell death that is a characteristic of the disease.

Catecholamine enzymes in the degenerative neurological disease idiopathic orthostatic hypotension.

Discrete brain areas and sympathetic ganglia obtained at autopsy from patients with idiopathic orthostatic hypotension were assayed for tyrosine hydroxylase and dopamine beta-hydroxylase. Dopamine beta-hydroxylase activity was decreased 7.5-fold in sympathetic ganglia, while tyrosine hydroxylase activity was reduced more than 50-fold in the pontine nucleus locus coeruleus. These observations indicate that noradrenergic neurons of both brain and ganglion are affected in idiopathic orthostatic hypotension, but suggest that the central and peripheral biochemical deficits differ.

Choline administration: activation of tyrosine hydroxylase in dopaminergic neurons of rat brain.

The administration of choline in doses previously shown to elevate brain acetylcholine concentrations also increases the activity of tyrosine hydroxylase in rat caudate nuclei. This response can be blocked by atropine, a muscarinic antagonist. These findings indicate that choline-induced increases in acetylcholine concentrations may be associated with parallel changes in the amount of the neurotransmitter released into synapses.

Changes in hyporesponsiveness to acute amphetamine and age differences in tyrosine hydroxylase immunoreactivity in the brain over adolescence in male and female rats.

We investigated hyposensitivity after amphetamine in early (postnatal Day 30; P30) and late (P45) adolescent rats compared to adults (P70) in experiment 1. Locomotor activity was measured for 1 hr after the first (acute) and second (24 hr later) injection of amphetamine (0.5 or 1.5 mg/kg). P30 and P45 rats were transiently hypoactive compared to adults, as indicated by reduced locomotor activity after acute amphetamine and enhanced activity after the second injection in adolescents only. In experiment 2, ovariectomy did not alter locomotor activity during habituation at any age compared to intact rats, and, as for intact adolescents, ovariectomized adolescents continued to be less active after amphetamine than adults, suggesting gonadal immaturity alone cannot account for age differences in experiment 1. However, ovariectomy attenuated the increase in activity after the second treatment. In experiment 3 involving untreated rats, tyrosine hydroxylase immunoreactivity was reduced in P30, P40, and P50 compared to P90 rats in the nucleus accumbens core and the medial prefrontal cortex. Thus, adolescents may have an increased threshold of behavioral activation that can be overcome with either a higher dose or with repeated amphetamine treatment, and may be related to changes in the dopamine system over development.

Repeated Administration of 3,4-Methylenedioxymethamphetamine (MDMA) Elevates the Levels of Neuronal Nitric Oxide Synthase in the Nigrostriatal System: Possible Relevance to Neurotoxicity.

Previous studies have consistently demonstrated that the amphetamine-related drug 3,4-methylenedioxymethamphetamine (MDMA) induces dopaminergic damage in the mouse brain, and that this effect is most marked in the nigrostriatal system. Moreover, it has been suggested that the overproduction of nitric oxide (NO) may participate in the dopaminergic damage induced by MDMA. To further elucidate this issue, we evaluated the levels of the enzyme nitric oxide synthase (nNOS), which catalyzes the production of NO, in mice treated with regimens of MDMA that induce progressive and persistent neurotoxicity in the dopaminergic nigrostriatal system. Mice received 14, 28, or 36 administrations of MDMA (10 mg/kg i.p.), twice a day/twice a week, and were sacrificed at different time-points after treatment discontinuation. Thereafter, the number of nNOS-positive neurons was quantified by immunohistochemistry in the caudate-putamen (CPu) and substantia nigra pars compacta (SNc). MDMA elevated the numbers of nNOS-positive neurons in the CPu of mice that received 28 or 36 drug administrations. This effect was still detectable at 3 months after treatment discontinuation. Moreover, MDMA elevated the numbers of nNOS-positive neurons in the SNc. However, this effect occurred only in mice that received 28 drug administrations and were sacrificed 3 days after treatment discontinuation. These results are in line with the hypothesis that activation of the NO cascade participates in the toxic effects induced by MDMA in the dopaminergic nigrostriatal system. Moreover, they suggest that activation of the NO cascade induces toxic effects that are more marked in striatal terminals, compared with nigral neurons.

Localization of Parkinson's disease-associated LRRK2 in normal and pathological human brain.

Mutations in the LRRK2 gene cause autosomal dominant, late-onset parkinsonism, which presents with pleomorphic pathology including alpha-synucleopathy. To promote our understanding of the biological role of LRRK2 in the brain we examined the distribution of LRRK2 mRNA and protein in postmortem human brain tissue from normal and neuropathological subjects. In situ hybridization and immunohistochemical analysis demonstrate the expression and localization of LRRK2 to various neuronal populations in brain regions implicated in Parkinson's disease (PD) including the cerebral cortex, caudate-putamen and substantia nigra pars compacta. Immunofluorescent double labeling studies additionally reveal the prominent localization of LRRK2 to cholinergic-, calretinin- and GABA(B) receptor 1-positive, dopamine-innervated, neuronal subtypes in the caudate-putamen. The distribution of LRRK2 in brain tissue from sporadic PD and dementia with Lewy bodies (DLB) subjects was also examined. In PD brains, LRRK2 immunoreactivity localized to nigral neuronal processes is dramatically reduced which reflects the disease-associated loss of dopaminergic neurons in this region. However, surviving nigral neurons occasionally exhibit LRRK2 immunostaining of the halo structure of Lewy bodies. Moreover, LRRK2 immunoreactivity is not associated with Lewy neurites or with cortical Lewy bodies in sporadic PD and DLB brains. These observations indicate that LRRK2 is not a primary component of Lewy bodies and does not co-localize with mature fibrillar alpha-synuclein to a significant extent. The localization of LRRK2 to key neuronal populations throughout the nigrostriatal dopaminergic pathway is consistent with the involvement of LRRK2 in the molecular pathogenesis of familial and sporadic parkinsonism.

In vitro reactivation potency of acetylcholinesterase reactivators--K074 and K075--to reactivate tabun-inhibited human brain cholinesterases.

In this work, two oximes for the treatment of tabun-inhibited acetylcholinesterase (AChE; EC 3.1.1.7), K074 (1,4-bis(4-hydroxyiminomethylpyridinium)butane dibromide) and K075 ((E)-1,4-bis(4-hydroxyiminomethylpyridinium)but-2-en dibromide), were tested in vitro as reactivators of AChE. Comparison was made with currently used AChE reactivators (pralidoxime, HI-6, methoxime and obidoxime). Human brain homogenate was taken as an appropriate source of the cholinesterases. As resulted, oxime K074 appears to be the most potent reactivator of tabun-inhibited AChE, with reactivation potency comparable to that of obidoxime. A second AChE reactivator, K075, does not attain as great a reactivation potency as K074, although its maximal reactivation (17%) was achieved at relevant concentrations for humans.

The COMT Val158Met polymorphism and temporal lobe morphometry in healthy adults.

We examined the relationship between COMT Val158Met genotype and temporal lobe volumes, including the caudate as a control region. Thirty-one healthy subjects completed 1.5T brain MRI and genotyping. After controlling for demographics, Val158 allele homozygotes exhibited significantly smaller temporal lobe and hippocampal volumes, with a trend for smaller amygdala volumes.

Cyclic adenosine 3':5'-monophosphate phosphodiesterase activity in malignant and cyclic adenosine 3':5'-monophosphate-induced "differentiated" neuroblastoma cells.

The regulation of cyclic adenosine 3:5-monophosphate (cyclic AMP) phosphodiesterase activity in homogenates of malignant and cyclic AMP-induced "differentiated" neuroblastoma cells was studied. Neuroblastoma cells of at least three mouse and one human clone had both the low (2 to 4 muM) and the high (66 to 106 muM) Km phosphodiesterase. In cyclic AMP-induced differentiated cells the values of Km were decreased, whereas the values of Vmax appeared to be slightly increased. Magnesium and manganese stimulated phosphodiesterase activity. Calcium, zinc, copper, mercury, ethylenediaminetetraacetic acid, and imidazole completely inhibited phosphodiesterase activity in malignant cells, whereas the above agents, except ethylenediaminetetraacetic acid, only partially inhibited enzyme activity in differentiated cells. Ethylenediaminetetraacetic acid completely reduced phosphodiesterase activity in differentiated cells. The pH optimum for phosphodiesterase activity was about 8 in both malignant and differentiated cells. The present studies show that the values of Km and Vmax and the sensitivity of phosphodiesterase activity to divalent ions change in cyclic AMP-induced differentiated neuroblastoma cells, and therefore we propose that the reverse may be true during malignant transformation of nerve cells.

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase.

Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues.

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I50 = 2 microgram/ml) when compared to other naturally occurring glycosamin oglycans. This inhibition was also apparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E2. Heparin was also found to inhibit glucagon-sensitive rat hepatic adenylate cyclase, and the prostaglandin E1-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfated polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary and was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.

Stimulation of guanylate cyclase by EDTA and other chelating agents.

The partially purified soluble guanylate cyclase (GTP pyrophosphatelyase(cyclizing), EC 4.6.1.2) from human caudate nucleus is stimulated from 2 to 4-fold by metal chelating agents. EDTA (K 1/2 - 4.8 microM) is more potent than CDTA (K 1/2 = 13.2 microM) or EGTA (K 1/2 = 21.8 microM) at stimulating activity. Stimulation by chelating agents is apparently not due to removal of inhibitory divalent cations which contaminate the enzyme or reaction mixture. EDTA increases guanylate cyclase activity in part by increasing the affinity of the enzyme for the substrate (MgGTP) 10-fold. Dopamine inhibits partially purified guanylate cyclase in the presence or absence of EDTA. Dopamine increases the Ka of guanylate cyclase for the activator, free Mn2+, more than 50-fold, from 3 to 150 microM.

Activation of tyrosine hydroxylase by polyanions and salts. An electrostatic effect.

The activity of a partially purified preparation of tyrosine hydroxylase (EC 1.14.16.2) from the bovine caudate nucleus was increased by heparin, chondroitin sulfate, phosphatidylserine, polyacrylic acid, polyvinyl sulfuric acid and both poly-D-, and poly-L-glutamic acids, all polyanions. A variety of salts both activated the enzyme and prevented the activation by the polyanions. The observations that activity is increased when the enzyme interacts with salts and with macromolecules of high negative charge density are used to infer a model for these interactions and for the structural change in the enzyme that accompanies activation.

Evolution of GADD34 expression after focal cerebral ischaemia.

GADD34, a stress response protein associated with cell rescue, DNA repair and apoptosis, is expressed in the ischaemic brain. The C-terminal region of GADD34 has homology with the Herpes Simplex Virus protein, ICP34.5, which overcomes the protein synthesis block after viral infection by actively dephosphorylating eukaryotic translation initiation factor 2alpha (eIF2alpha). The carboxy terminus of GADD34 is also capable of dephosphorylating eIF2alpha and therefore has the capacity to restore the protein synthesis shutoff associated with ischaemia. This study examines the distribution and time course of GADD34 expression after focal cerebral ischaemia. Focal ischaemia or sham procedure was carried out on Sprague-Dawley rats with survival times of 4, 12, 24 h, 7 and 30 days. Brains were processed for histology and immunohistochemistry. Ischaemic damage was mapped onto line diagrams and GADD34 positive cells counted in selected regions of cortex and caudate. GADD34 immunopositive cells (mainly neurones), expressed as cells/mm2, were present in ischaemic brains at 4 h (e.g., peri-infarct cortex 20 +/- 5; contralateral cortex 3 +/- 1, P < 0.05). Of the time points examined, numbers of GADD34 positive cells were highest 24 h after ischaemia (peri-infarct cortex 31 +/- 7.3, contralateral cortex 0.1 +/- 0.1, P < 0.05). Immunopositive cells, following a similar time course, were identified within the peri-infarct zone in the caudate nucleus and in ipsilateral cingulate cortex (possibly as a consequence of cortical spreading depression). GADD34 positive cells did not co-localise with a marker of irreversible cell death (TUNEL). Taken together, GADD34 positive cells in key neuroanatomical locations pertinent to the evolving ischaemic lesion, the lack of co-localisation with TUNEL and the protein's known effects on restoring protein synthesis, repairing DNA and involvement in ischaemic pre-conditioning suggests that it has the potential to influence cell survival in ischaemically compromised tissue.