Ana Patricia Fernández

Chronic Stress Induces the Expression of Inducible Nitric Oxide Synthase in Rat Brain Cortex

Abstract: Long‐term exposure to stress has detrimental effects on several brain functions in many species, including humans, and leads to neurodegenerative changes. However, the underlying neural mechanisms by which stress causes neurodegeneration are still unknown. We have investigated the role of endogenously released nitric oxide (NO) in this phenomenon and the possible induction of the inducible NO synthase (iNOS) isoform. In adult male rats, stress (immobilization for 6 h during 21 days) increases the activity of a calcium‐independent NO synthase and induces the expression of iNOS in cortical neurons as seen by immunohistochemical and western blot analysis. Three weeks of repeated immobilization increases immunoreactivity for nitrotyrosine, a nitration product of peroxynitrite. Repeated stress causes accumulation of the NO metabolites NO2‐ + NO3‐ (NOx‐) accumulation in cortex, and these changes occur in parallel with lactate dehydrogenase (LDH) release and impairment of glutamate uptake in synaptosomes. Administration of the selective iNOS inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) prevents NOx‐ accumulation in cortex, LDH release, and impairment of glutamate uptake in synaptosomes. Taken together, these findings indicate that a sustained overproduction of NO via iNOS expression may be responsible, at least in part, for some of the neurodegenerative changes caused by stress and support a possible neuroprotective role for specific iNOS inhibitors in this situation.

Neuronal and inducible nitric oxide synthase and nitrotyrosine immunoreactivities in the cerebral cortex of the aging rat.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) and nitrotyrosine immunoreactivities were localized and semiquantitatively assessed in the cerebral cortex of aged rats by means of light microscopic immunocytochemistry and Western blotting, using a new series of specific polyclonal antibodies. In the aged rats the strongly nNOS‐immunoreactive multipolar neurons found in layers II–VI of the cortex of young rats were seen in similar numbers, but showed varicose, vacuolated, and fragmented processes, with an irregular outline and loss of spines. A large number of more weakly nNOS‐positive neurons, characterized by a ring of immunoreactive cytoplasm, and not seen in young rats, were observed in layers II–VI of aged rat cortex. While no iNOS‐immunopositive neurons were found in the cortex of young rats, a large number of such neurons appeared throughout the aged rat cortex. Nitrotyrosine‐positive cells outnumbered total NOS‐positive neurons in the cortex of young rats, but this relation was inverted in the aged rats, although these showed a slight increase in the number and staining intensity of nitrotyrosine‐positive cells. Western blots of brain extracts showed a several‐fold increase in both nNOS‐ and iNOS‐immunoreactive bands in the aged rat, but a less marked increase in nitrotyrosine‐containing proteins. The results suggest that while nNOS and iNOS expression is substantially increased in the aged rat cortex, this is not necessarily accompanied by a proportionate increase in nitric oxide synthesis. The mechanisms underlying the increased expression of nNOS and iNOS, and the functional implications of this increase, require elucidation. Microsc. Res. Tech. 43:75–88, 1998.

Neuronal expression of inducible nitric oxide synthase after oxygen and glucose deprivation in rat forebrain slices

Nitric oxide (NO) overproduction has been postulated to contribute significantly to ischaemia‐reperfusion neurotoxicity. Inducible or type II NO synthase (iNOS) synthesizes NO in large quantities for long periods of time. Therefore we investigated the expression and localization of iNOS after oxygen and glucose deprivation in rat forebrain slices. In this experimental model, calcium‐independent NOS activity reached a maximum 180 min after the end of a 20 min oxygen–glucose deprivation period. During the same period of time, the calcium‐independent activity was absent in control forebrain slices. To test whether this calcium‐independent NOS activity was due to the expression of iNOS, the effects of the addition of dexamethasone, cycloheximide and pyrrolidine dithiocarbamate were determined. All of them inhibited the induction of the calcium‐independent NOS activity measured in the rat forebrain slices after oxygen and glucose deprivation. Furthermore, oxygen and glucose deprivation caused the expression of the gene encoding iNOS in rat forebrain slices, as assessed by the detection of iNOS message and protein in these samples. A sixfold increase in the iNOS mRNA levels was observed at 180 min and the time‐course of the expression of iNOS mRNA was in agreement with the temporal profile of iNOS enzymatic activity. Immunohistochemistry analysis revealed that iNOS was highly expressed in neurones, astrocytes and microglial cells. These results demonstrate for the first time that iNOS is expressed in neurones after oxygen and glucose deprivation, and that this expression occurs in short periods of time. These findings suggest that NO can play an important pathogenic role in the tissue damage that occurs after cerebral ischaemia.

Caveolin-1 deficiency causes cholesterol dependent mitochondrial dysfunction and apoptotic susceptibility

Caveolins (CAVs) are essential components of caveolae, plasma membrane invaginations with reduced fluidity, reflecting cholesterol accumulation. CAV proteins bind cholesterol, and CAVs ability to move between cellular compartments helps control intracellular cholesterol fluxes. In humans, CAV1 mutations result in lipodystrophy, cell transformation, and cancer. CAV1 gene-disrupted mice exhibit cardiovascular diseases, diabetes, cancer, atherosclerosis, and pulmonary fibrosis. The mechanism or mechanisms underlying these disparate effects are unknown, but our past work suggested that CAV1 deficiency might alter metabolism: CAV1(-/-) mice exhibit impaired liver regeneration unless supplemented with glucose, suggesting systemic inefficiencies requiring additional metabolic intermediates. Establishing a functional link between CAV1 and metabolism would provide a unifying theme to explain these myriad pathologies. Here we demonstrate that impaired proliferation and low survival with glucose restriction is a shortcoming of CAV1-deficient cells caused by impaired mitochondrial function. Without CAV1, free cholesterol accumulates in mitochondrial membranes, increasing membrane condensation and reducing efficiency of the respiratory chain and intrinsic antioxidant defense. Upon activation of oxidative phosphorylation, this promotes accumulation of reactive oxygen species, resulting in cell death. We confirm that this mitochondrial dysfunction predisposes CAV1-deficient animals to mitochondrial-related diseases such as steatohepatitis and neurodegeneration.

Intra- and extracellular Aβ and PHF in clinically evaluated cases of Alzheimer's disease

Temporal cortical sections from postmortem brains of individuals without any dementing condition and with different degrees of severity of Alzheimers disease (AD) evaluated by the Clinical Dementia Rating scale (CDR 0-CDR 3) were analyzed using immunohistochemical procedures. To demonstrate the amyloid-beta-peptide (Abeta) deposition and the neurofibrillary pathology, two monoclonal antibodies were used, a human CERAD Abeta (10D5) antibody raised against the N-terminal region of the Abeta-peptide, and an antibody raised against paired helical filaments (PHF-1). The neuron cell bodies and the glial cells were also recognized by two polyclonal antibodies raised, respectively, against the protein gene peptide (PGP 9.5) and glial fibrillary acidic protein (GFAP). Directly related to severity of AD, progressive deposits of Abeta-peptide were found within cortical pyramidal-like neurons and forming senile plaques. Ultrastructurally, Abeta-peptide deposits were related to neuronal intracytoplasmic organelles, such as the ER, the mitochondria, the Nissl bodies and lipofuscin. We have also found that the intracellular deposition of the Abeta peptide is a neuropathological finding prior to the appearance of PHF-immunoreactive structures. We suggest that the intracellular Abeta deposition in cortical pyramidal neurons is a first neurodegenerative event in AD development and that it is involved in cell dysfunction, neuronal death, and plaque formation.

Neuronal and inducible nitric oxide synthase expression and protein nitration in rat cerebellum after oxygen and glucose deprivation

A perfusion model of global cerebral ischemia was used for the immunohistochemical study of changes in the glutamate-nitric oxide (NO) system in the rat cerebellum and cerebellar nuclei during a 0-14 h reperfusion period after 30 min of oxygen and glucose deprivation, with and without administration of 1.5 mM N(omega)-nitro-L-arginine methyl ester (L-NAME). While immunostaining for N-methyl-D-aspartate receptor subunit 1 (NMDAR1) showed no marked changes during the reperfusion period, neuronal NO synthase (nNOS) immunostaining increased in stellate and basket cells, granule cells and neurons of the cerebellar nuclei. However, global cerebellar nNOS concentrations determined by Western blotting remained largely unchanged in comparison with actin expression. Inducible NOS (iNOS) immunostaining appeared in Purkinje cells and neurons of the cerebellar nuclei after 2-4 h of reperfusion and intensified during the 6-14 h period. This was reflected by an increase in global cerebellar iNOS expression determined by Western blotting. Immunostaining for protein nitrotyrosine was seen in Purkinje cells, stellate and basket cells, neurons of the cerebellar nuclei and glial cells in controls, and showed a progressive translocation in Purkinje cells and neurons of the cerebellar nuclei from an initial perinuclear or nuclear location towards the periphery. At the end of the reperfusion period the Purkinje cell apical dendrites were notably retracted and tortuous. Prior and concurrent L-NAME administration eliminated nitrotyrosine immunostaining in controls and blocked or reduced most of the postischemic changes observed. The results suggest that while nNOS expression may be modified in certain cells, iNOS is induced after a 2-4 h period, and that changes in protein nitration may be associated with changes in cell morphology.

Nitric oxide in the cerebral cortex of amyloid-precursor protein (SW) Tg2576 transgenic mice.

Changes in the amyloid-peptide (Abeta), neuronal and inducible nitric oxide (NO)synthase (nNOS, iNOS), nitrotyrosine, glial fibrillary acidic protein, and lectin from Lycopersicon esculentum (tomato) were investigated in the cerebral cortex of transgenic mice (Tg2576) to amyloid precursor protein (APP), by immunohistochemistry (bright light, confocal, and electron microscopy). The expression of nitrergic proteins and synthesis of nitric oxide were analyzed by immunoblotting and NOS activity assays, respectively. The cerebral cortex of these transgenic mice showed an age-dependent progressive increase in intraneuronal aggregates of Abeta-peptide and extracellular formation of senile plaques surrounded by numerous microglial and reactive astrocytes. Basically, no changes to nNOS reactivity or expression were found in the cortical mantle of either wild or transgenic mice. This reactivity in wild mice corresponded to numerous large type I and small type II neurons. The transgenic mice showed swollen, twisted, and hypertrophic preterminal and terminal processes of type I neurons, and an increase of the type II neurons. The calcium-dependent NOS enzymatic activity was higher in wild than in the transgenic mice. The iNOS reactivity, expression and calcium-independent enzymatic activity increased in transgenic mice with respect to wild mice, and were related to cortical neurons and microglial cells. The progressive elevation of NO production resulted in a specific pattern of protein nitration in reactive astrocytes. The ultrastructural study carried out in the cortical mantle showed that the neurons contained intracellular aggregates of Abeta-peptide associated with the endoplasmic reticulum, mitochondria, and Golgi apparatus. The endothelial vascular cells also contained Abeta-peptide deposits. This transgenic model might contribute to understand the role of the nitrergic system in the biological changes related to neuropathological progression of Alzheimers disease.

Up-regulation of neuronal NO synthase immunoreactivity in opiate dependence and withdrawal.

Abstract Rationale: Nitric oxide (NO) has been postulated to contribute significantly to analgesic effects of opiates as well as to the development of tolerance and physical dependence to morphine. Objective: The present study was undertaken to determine the effect of chronic morphine treatment and abstinence on the expression of neuronal NO synthase (neuronal NOS, nNOS) in several brain regions of mice. Methods: Seven days after the implantation of a 75 mg morphine pellet, adult male CD1 mice received a SC dose of 1 mg/kg naloxone. Fifteen minutes after the naloxone injection, brains were removed and nNOS expression was studied by using immunohistochemical methods. Results: Morphine-dependence produced an increase in the number of nNOS-positive cells in the main and accessory olfactory bulb, olfactory nuclei, cerebellum, locus coeruleus, medulla oblongata (nucleus of the solitary tract and prepositus hypoglossal nucleus), and a decrease in nNOS immunoreactivity in hypothalamus. The administration of naloxone to morphine-dependent mice to induce abstinence increased nNOS immunoreactivity in the hypothalamus and locus coeruleus. Conclusions: These results indicate that the chronic treatment with morphine leads to alterations in nNOS expression in important regions implicated in the physical tolerance and dependence to opiates and suggest the use of specific inhibitors of this isoform in these conditions.

Oxidative stress and altered mitochondrial function in neurodegenerative diseases: lessons from mouse models

Oxidative stress has been consistently linked to ageing-related neurodegenerative diseases leading to the generation of lipid peroxides, carbonyl proteins and oxidative DNA damage in tissue samples from affected brains. Studies from mouse models that express disease-specific mutant proteins associated to the major neurodegenerative processes have underscored a critical role of mitochondria in the pathogenesis of these diseases. There is strong evidence that mitochondrial dysfunction is an early event in neurodegeneration. Mitochondria are the main cellular source of reactive oxygen species and key regulators of cell death. Moreover, mitochondria are highly dynamic organelles that divide, fuse and move along axons and dendrites to supply cellular energetic demands; therefore, impairment of any of these processes would directly impact on neuronal viability. Most of the disease-specific pathogenic mutant proteins have been shown to target mitochondria, promoting oxidative stress and the mitochondrial apoptotic pathway. In addition, disease-specific mutant proteins may also impair mitochondrial dynamics and recycling of damaged mitochondria via autophagy. Collectively, these data suggest that ROS-mediated defective mitochondria may accumulate during and contribute to disease progression. Strategies aimed to improve mitochondrial function or ROS scavenging may thus be of potential clinical relevance.

Effects of oxygen and glucose deprivation on the expression and distribution of neuronal and inducible nitric oxide synthases and on protein nitration in rat cerebral cortex.

Changes in the nitric oxide (NO) system of the rat cerebral cortex were investigated by immunohistochemistry, immunoblotting, NO synthase (NOS) activity assay, and magnetic resonance imaging (MRI) in an experimental model of global cerebral ischemia and reperfusion. Brains were perfused transcardially with an oxygenated plasma substitute and subjected to 30 minutes of oxygen and glucose deprivation, followed by reperfusion for up to 12 hours with oxygenated medium containing glucose. A sham group was perfused without oxygen or glucose deprivation, and a further group was treated with the NOS inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME) before and during perfusion. Global ischemia led to cerebrocortical injury as shown by diffusion MRI. This was accompanied by increasing morphologic changes in the large type I interneurons expressing neuronal NOS (nNOS) and the appearance of nNOS immunoreactivity in small type II neurons. The nNOS‐immunoreactive band and calcium‐dependent NOS activity showed an initial increase, followed by a fall after 6 hours of reperfusion. Inducible NOS immunoreactivity appeared in neurons, especially pyramidal cells of layers IV–V, after 4 hours of reperfusion, with corresponding changes on immunoblotting and in calcium‐independent NOS activity. Immunoreactive protein nitrotyrosine, present in the nuclear area of neurons in nonperfused controls and sham‐perfused animals, showed changes in intensity and distribution, appearing in the neuronal processes during the reperfusion period. Prior and concurrent L‐NAME administration blocked the changes on diffusion MRI and attenuated the morphologic changes, suggesting that NO and consequent peroxynitrite formation during ischemia–reperfusion contributes to cerebral injury. J. Comp. Neurol. 443:183–200, 2002.