Expression, but not activity, of neuronal nitric oxide synthase is regionally increased in the alcoholic brain.

The regional distribution of nitric oxide synthase (NOS) was investigated in alcoholic post-mortem brains compared with brains of non-alcoholic control individuals. Total enzyme activity in 28 brain regions was determined using the [(3)H]l-citrulline formation assay, whereas Western blot analyses were used for semi-quantitative measurement of the neuronal isoform of NOS (nNOS). In the alcoholic brain, nNOS protein expression was increased in the following regions: frontal cortex (85%), the cingulate gyrus (294%), the nucleus accumbens (54%), the entorhinal cortex (85%) and the thalamus (51%). These increases were, however, not associated with higher total NOS activity. Interestingly, nNOS protein content was increased in the frontal cortex and the nucleus accumbens, brain regions which are suggested to be involved in the dopaminergic mesolimbic reward system. It is concluded that upregulation of signal transduction pathways, such as the adenosine 3',5'-monophosphate and the protein kinase C-dependent pathway, due to stimulation of G-protein-coupled neurotransmitter receptor regulation, as a form of functional tolerance, could be responsible for increased nNOS protein expression, and downregulation of NOS enzyme activity in these brain regions.

Characterization and regional distribution of nitric oxide synthase in the human brain during normal ageing.

Nitric oxide (NO) is a highly diffusible cellular mediator generated from L-arginine by the enzyme nitric oxide synthase (NOS). As little is known about the regional distribution of NOS in the human brain, we examined the distribution pattern of nitric oxide synthase activity in 28 regions of the human brain using the [(3)H]L-citrulline formation assay. To elucidate which isoforms contribute to the total NOS activity we performed Western blot analysis of neuronal, inducible and endothelial NOS. We further determined brain levels of arginine and citrulline as a potential index of NOS activity pre mortem. NOS activity appears to remain unaltered during ageing and is independent of post mortem delay, gender or sample storage time. We identified a regional pattern of NOS distribution with highest levels of NOS activity in the substantia innominata, cerebellar cortex, nucleus accumbens and subthalamicus, whereas lowest levels were measured in the corpus callosum, thalamus, occipital cortex, and dentate nucleus. nNOS was measured throughout the brain, in contrast iNOS and eNOS were not detectable. We therefore conclude that primarily nNOS is responsible for NOS activity in the human brain. Levels of citrulline were higher than those of arginine, but did not correlate with the enzyme activity, suggesting that these parameters are unsuitable for testing NOS activity premortem. The characterization and topographical pattern of NOS in the human brain during normal ageing may assist our understanding of the physiological role of NO and its relevance in Parkinson's and Alzheimer's disease, alcoholism, schizophrenia and AIDS.

Radical scavenging compound J 811 inhibits hydrogen peroxide-induced death of cerebellar granule cells.

Oxidative stress is considered to be an important pathophysiological condition to promote cell death in a broad variety of disorders, such as cardiovascular and neurodegenerative diseases. Scavestrogens, structurally derived from estradiol, are potent radical scavengers and inhibitors of iron-induced cell damage in vitro. In this study the potential cytoprotective effects of the so-called scavestrogen estra-1,3,5(10),8-tetraene-3,17alpha-diol, J 811, was tested using rat cerebellar granule cells (CGCs) exposed to 25 or 50 microM hydrogen peroxide (H2O2). H2O2-induced apoptotic cell death was detected by the appearance of high molecular weight DNA fragments and nuclear condensation. The addition of J 811 before or shortly after the exposure to H2O2 prevented CGC apoptosis in a dose-dependent manner. The estrogen receptor antagonist ICI 182.780 failed to prevent the protective effect of J 811, suggesting that the latter is not dependent on estrogen receptor activation. The lack of protection against apoptosis caused by colchicine suggests that J 811 is neither interfering with the activation of caspase-3, nor acting downstream of caspase-3. Therefore, the protective effect observed against H2O2 seems to be upstream caspases activation, pointing to a scavenging action of J 811. Thus the scavestrogen J 811 is a powerful antioxidant able to interfere with radical-mediated cell death and is potentially useful in diseases where reactive oxygen species are involved.

A disturbance in the neuronal insulin receptor signal transduction in sporadic Alzheimer's disease.

Disturbances of glucose and energy metabolism are hypothesized as pathogenetic factors in sporadic dementia of Alzheimer type (SDAT). Insulin and is receptors play an important role in the regulation of brain glucose metabolism and neuronal growth. In postmortem brain cortex in SDAT, the densities of brain insulin receptors were decreased compared to adult controls, but were increased in relation to aged controls. Tyrosine kinase activity, a signal transduction mechanism common to insulin and IGF-1 receptors, was reduced in SDAT in comparison to middle-aged and age-matched control groups. The data are consistent with a neurotrophic role of insulin in the human brain and an upregulation of insulin receptors is SDAT brain as a compensatory mechanism, possibly due to impaired signal transduction mechanism.

Scavestrogens protect IMR 32 cells from oxidative stress-induced cell death.

Oxidative stress is considered an important pathophysiological mechanism contributing to promote cell death in a broad variety of diseases including cardiovascular and neurodegenerative disorders. The so-called scavestrogens J811 and J861, structurally derived from 17alpha-estradiol, are potent radical scavengers and inhibitors of iron-induced cell damage in vitro. In this study the potential cytoprotective effects of the scavestrogens J811 and J861 against Fenton reagent-induced cell damage (50 microM FeSO4 plus 200 microM H2O2) were compared with those of 17alpha- and 17beta-estradiol. Cell viability studies using Trypan blue staining showed that estradiols and scavestrogens at concentrations ranging from 0.1 to 10 microM are able to protect IMR 32 neuroblastoma cells from Fenton-mediated death. In addition, these compounds decreased lipid peroxidation measured as thiobarbituric acid reactive substances and renormalize oxidative stress-increased intracellular glutathione levels. When given 6 h after the toxic stimulus, J811 and J861 rescued 60% of cells, whereas 17alpha- and 17beta-estradiol were ineffective. These results suggest that the scavestrogens J811 and J861 are powerful antioxidants capable of interfering with radical-mediated cell death in diseases known to be aggravated by reactive oxygen species. Such compounds may be useful in the development of novel treatments for stroke or neurodegenerative disorders.

Interleukin-6 levels in cerebrospinal fluid inversely correlate to severity of Parkinson's disease.

INTRODUCTION: Several lines of evidence suggest that neuroimmune mechanisms may also be involved in neurodegeneration in Parkinson's disease (PD). The potential role of cytokines such as interleukin 6 (IL-6), in the interaction between neurons and immune system has been emphasized by recent findings. IL-6 induces acute phase protein synthesis, differentiation of neuronal cells and improves catecholaminergic and cholinergic cell survival in the brain. SUBJECTS AND METHODS: We determined levels of IL-6 in cerebrospinal fluid (CSF) of untreated parkinsonian patients and age- and sex-matched controls. Intensity of disease was evaluated by the Unified Parkinson's Disease Rating scale. RESULTS: Significantly elevated levels of IL-6 were found in the CSF of parkinsonian patients. Moreover a significant inverse correlation between severity of PD and IL-6 CSF levels appeared. DISCUSSION: Elevated IL-6 levels in the CSF of untreated parkinsonian patients may reflect the original condition in the course of disease. We speculate that an endogenous upregulation of IL-6 synthesis occurs in order to regenerate lesioned neurons probably at an early phase of the degenerative process in PD.

Brain insulin and insulin receptors in aging and sporadic Alzheimer's disease.

The search for the causes of neurodegenerative disorders is a major theme in brain research. Acquired disturbances of several aspects of cellular metabolism appear pathologically important in sporadic Alzheimer's disease (SDAT). Among these brain glucose utilisation is reduced in the early stages of the disease and the regulatory enzymes important for glucose metabolism are reduced. In the brain, insulin, insulin-like growth factors and their receptors regulate glucose metabolism and promote neuronal growth. To detect changes in the functional activity of the brain insulin neuromodulatory system of SDAT patients, we determined the concentrations of insulin and c-peptide as well as insulin receptor binding and IGF-I receptor binding in several regions of postmortem brain cortex during aging and Alzheimer's disease. Additionally, we performed immunohistochemical staining with antibodies against insulin in neocortical brain areas in SDAT and controls. We show for the first time that insulin and c-peptide concentration in the brain are correlated and decrease with aging, as do brain insulin receptor densities. Weak insulin-immunoreactivity could be demonstrated histochemically in pyramidal neurons of controls, whereas in SDAT a stronger insulin-immunoreactivity was found. On a biochemical level, insulin and c-peptide levels were reduced compared to middle-aged controls, but were unchanged compared to age-matched controls. Brain insulin receptor densities in SDAT were decreased compared to middle-aged controls, but increased in comparison to age-matched controls. IGF-I receptor densities were unchanged in aging and in SDAT. Tyrosine kinase activity, a signal transduction mechanism common to both receptor systems, was reduced in SDAT in comparison to middle-aged and age-matched control groups. These data are consistent with a neurotrophic role of insulin in the human brain and a disturbance of insulin signal transduction in SDAT brain and favor the hypothesis that insulin dependent functions may be of pathogenetic relevance in sporadic SDAT.

Advanced glycation endproducts-associated parameters in the peripheral blood of patients with Alzheimer's disease.

Advanced glycation endproducts (AGEs), structural components of beta-amyloid plaques and neurofibrillary tangels, have been implicated in the pathogenesis of Alzheimer's disease. AGE levels, measured by fluorescence, and their precursor molecules such as glucose and its Amadori product, fructosylamine, were measured to examine the question whether the reported increased level of AGEs in the brain is reflected in an increase in AGE-associated parameters in peripheral blood. Lactoferrin, proposed to play an important role in the interaction of AGEs with their receptors, was determined by ELISA. All AGE-associated parameters showed trends to lower values in patients with Alzheimer's disease compared with non-demented controls. Albumin and total iron were not significantly different between the groups. In contrast to diabetes and renal failure, where high levels of AGEs and their precursors are present in tissue as well as in peripheral blood, elevated CNS AGE levels in patients with Alzheimer's disease are manifested without detectable peripheral changes.

Interleukin-1 beta and interleukin-6 are elevated in the cerebrospinal fluid of Alzheimer's and de novo Parkinson's disease patients.

Interleukin-1 beta (IL-1 beta), interleukin-2 (IL-2), and interleukin-6 (IL-6) were measured in the cerebrospinal fluid (CSF) and plasma of 12 control subjects, 11 sporadic Alzheimer's disease (AD) and 22 de novo Parkinson's disease (PD) patients using high sensitivity enzyme-linked immunosorbent assays (ELISA). IL-1 beta and IL-6 contents were significantly elevated in the CSF of de novo PD and AD patients in comparison to the control group. In contrast, the plasma levels were not significantly affected. IL-2 contents in the CSF and plasma samples were unchanged in the three groups compared. Because the two cytokines IL-1 beta and IL-6 are known to play a key role in the interaction between the nervous and immune system, e.g. in the so-called acute phase response, our results support the involvement of immunological events in the complex process of neurodegeneration in AD and PD.

Regional distribution and characterization of nitric oxide synthase activity in the brain of the common marmoset.

The distribution of nitric oxide synthase (NOS) within the brain of the common marmoset, a non-human primate species, was investigated using the [3H]L-citrulline formation assay and Western blot analysis. No hemispheric asymmetry of specific NOS activity was shown. The highest levels of NOS were found in the putamen and caudate nucleus--more than twice those in the cortex and the cerebellum, the brain regions with the lowest activities. The regional distribution pattern was similar to that in the ferret brain and contrasted to that in the mouse and bovine brain. Analysis of NOS catalytic activities in subcellular fractions revealed marked differences in the subcellular localization. Neuronal NOS accounted mainly for the measured catalytic activity in the brain. Differences in the regional distribution pattern of brain NOS activity among species may be indicative of diversities in the functional role of nitric oxide and NOS in mammals.

Altered regulation of brain glucose metabolism as a cause of neurodegenerative disorders?

At present, search for the causes of neurodegenerative diseases represents a major topic in brain research. Acquired disturbances of cell metabolism are supposed to be a cause of the two most important neurodegenerative disorders in ageing, like senile dementia of the Alzheimer type and Parkinson's disease, resulting in measurable decreases of in vivo and post mortem cerebral glucose metabolism. Accumulating evidence indicates that insulin plays an important role in the regulation of brain glucose homeostasis in the central nervous system and has trophic effects on neurons. It has been suggested that the reduction of brain glucose metabolism in neuro-degenerative disorders may be related to a defect of the neuronal insulin-insulin receptor-interaction. It will be the aim of our study to demonstrate whether there exist any changes in the content of insulin, its receptor and/or in the functionality of the insulin receptor and its signal transduction in neurodegenerative disorders as Alzheimer's and Parkinson's disease.