Tolga Uz

Antidepressants alter cell proliferation in the adult brain in vivo and in neural cultures in vitro.

The action of antidepressants on cell proliferation (bromodeoxyuridine (BrdU) or [3H]thymidine incorporation) was studied in the adult rat hippocampus in vivo and in neural precursors (immature rat cerebellar granule cells) in vitro. In vivo, prolonged (21 days) but not acute (single) intraperitoneal treatment with fluoxetine (5 mg/kg) resulted in a 3.4-fold increase of bromodeoxyuridine-positive cells in the subgranular zone of the dentate gyrus. In cell cultures, at 1 and 10 days in vitro, 48-h fluoxetine exposure (1 microM, which is comparable to therapeutic plasma concentrations) reduced thymidine incorporation when initiated at 1 day in vitro, but increased cell proliferation when initiated at 10 days in vitro. Clomipramine and imipramine produced similar action in vitro; desipramine was ineffective.

Increased brain damage after stroke or excitotoxic seizures in melatonin-deficient rats.

The pineal hormone melatonin is neuroprotective in vitro, and in vivo it is neuropro‐ tective when given in pharmacological doses. Conse‐quently, it has been hypothesized that with aging, as circulating levels of melatonin in mammals normally decrease, the brain might be at increased risk of neurodegeneration. However, direct evidence that melatonin deficiency leads to increased brain vulnerability is still lacking. We created melatonin deficiency in rats by pinealectomy and induced neurodegeneration by two models of focal brain ischemia/stroke and by glutamate receptor‐medi‐ ated, epilepsy‐like seizures. We observed greater neurodegeneration in melatonin‐deficient animals than in controls. Our results suggest that endogenous melatonin may play a neuroprotective role, and that melatonin deficiency might be a pathophysiological mechanism in neurodegenerative diseases.—Manev, H., Uz, T., Kharlamov, A., Joo, J.‐Y. Increased brain damage after stroke or excitotoxic seizures in mela‐ tonin‐deficient rats. FASEB J. 10, 1546‐1551

Characterization of zinc-induced neuronal death in primary cultures of rat cerebellar granule cells.

Although zinc is essential for the activity of numerous biological systems, and zinc deficiency has been associated with various pathologies, this metal can also exert direct neurotoxic action. In primary cultures of rat cerebellar granule neurons, a brief, 15- to 30-min exposure to zinc (100-500 microM) resulted in concentration-dependent delayed neuronal death. The toxicity of zinc depended on the maturity of the neuronal cultures-it was not apparent prior to Day 5 and it reached a plateau at about 9-10 days in vitro. We assayed cell injury by measuring mitochondrial functioning (MTT assay) and cell death with the trypan blue exclusion assay. Apoptosis was assayed by the morphological appearance of cells following fluorescence staining with propidium iodide and by the in situ TUNEL technique. Mitochondrial injury was an early result of zinc treatment. Actinomycin D, an inhibitor of macromolecular synthesis, attenuated delayed cell death. The calcium channel blockers nimodipine and amlodipine reduced both mitochondrial injury and cell death; the blockade of ionotropic glutamate receptors with MK-801 or CNQX was ineffective. These results suggest that calcium channel-blocker-sensitive mitochondrial injury and DNA damage are operative in the protein-synthesis-dependent neurotoxicity of zinc. An imbalance of zinc homeostasis might play a role in the pathophysiology of apoptosis-associated neurodegenerative disorders.

Protective effect of melatonin against hippocampal dna damage induced by intraperitoneal administration of kainate to rats

The pineal hormone melatonin protects neurons in vitro from excitotoxicity mediated by kainate-sensitive glutamate receptors and from oxidative stress-induced DNA damage and apoptosis. Intraperitoneal injection on kainate into experimental animals triggers DNA damage in several brain areas, including the hippocampus. It is not clear whether melatonin is neuroprotective in vivo. In this study, we tested the in vivo efficacy of melatonin in preventing kainate-induced DNA damage in the hippocampus of adult male Wistar rats. Melatonin and kainate were injected i.p. Rats were killed six to 72 h later and their hippocampi were examined for evidence of DNA damage (in situ dUTP-end-labeling, i.e. TUNEL staining) and for cell viability (Nissl staining). Quantitative assay was performed using computerized image analysis. At 48 and 72 h after kainate we found TUNEL-positive cells in the CA1 region of the hippocampus; in the adjacent sections that were Nissl-stained, we found evidence of cell loss. Both the number of TUNEL-positive cells and the loss of Nissl staining were reduced by i.p. administration of melatonin (4 x 2.5 mg/kg; i.e. 20 min before kainate, immediately after, and 1 and 2 h after the kainate). Our results suggest that melatonin might reduce the extent of cell damage associated with pathologies such as epilepsy that involve the activation of kainate-sensitive glutamate receptors.

Aging-associated up-regulation of neuronal 5-lipoxygenase expression: putative role in neuronal vulnerability

Aging is associated with neurodegenerative processes. 5‐Lipoxygenase (5‐LO), which is also expressed in neurons, is the key enzyme in the synthesis of leukotrienes, inflammatory eicosanoids that are capable of promoting neurodegeneration. We hypothesized that neuronal 5‐LO expression can be up‐regulated in aging and that this may increase the brains vulnerability to neurodegeneration. We observed differences in the distribution of 5‐LO‐like immunoreactivity in various brain areas of adult young (2‐month‐old) vs. old (24‐month‐old) male rats. Greater 5‐LO‐like immunoreactivity was found in old vs. young rats, in particular in the dendrites of pyramidal neurons in limbic structures, including the hippocampus, and in layer V pyramidal cells of the frontoparietal cortex and their apical dendrites. The aging‐increased expression of neuronal 5‐LO protein appears to be due to increased 5‐LO gene expression. Using a quantitative reverse transcription/polymer‐ase chain reaction assay and 5‐LO‐specific oligonucleotide primers and their mutated internal standards, we observed about a 2.5‐fold greater hippocampal 5‐LO mRNA content in old rats. 5‐LO‐like immunoreactivity was also observed in small, nonpyramidal cells, which were positive for glutamic acid decarboxylase or glial fibrillary acid protein. This type of 5‐LO immunostaining did not increase in the old rats. Hippocampal excitotoxic injury induced by systemic injection of kainate was greater in old rats. Neuroprotection was observed with the 5‐LO inhibitor, caffeic acid. Together, these results suggest that aging increases both neuronal 5‐LO expression and neuronal vulnerability to 5‐LO inhibitor‐sensitive ex‐citotoxicity, and indicate that the 5‐LO system might play a significant role in the pathobiology of aging‐associated neurodegenerative diseases.—Uz, T., Pesold, C., Longone, P., Manev, H. Aging‐associated up‐regulation of neuronal 5‐lipoxygenase expression: putative role in neuronal vulnerability. FASEB J. 12, 439–449 (1998)

Putative role of neuronal 5-lipoxygenase in an aging brain

Aging is associated with increased incidence and/or severity of neurodegenerative pathologies. Oxygen‐mediated events are being considered as possible mechanisms responsible for the increasing neuronal vulnerability. Lipoxygenases are enzymes that, as cyclooxygenases (COX), can insert oxygen into the molecule of arachidonic acid and thereby synthesize inflammatory eicosanoids: leukotrienes [due to 5‐lipoxygenase (5‐LOX) activity] and prostaglandins (via COX activity). It appears that 5‐LOX is expressed in central nervous system neurons and may participate in neurodegeneration. 5‐LOX‐triggered cell death may be initiated by the enzymatic activity of 5‐LOX but could also occur via the nonenzymatic actions of the 5‐LOX protein; new data point to the possibility that 5‐LOX protein exerts actions such as interaction with tyrosine kinase receptors, cytoskeletal proteins, and the nucleus. The expression of neuronal 5‐LOX is susceptible to hormonal regulation, presumably due to the presence of hormone‐responsive elements in the structure of the 5‐LOX gene promoter. The expression of the 5‐LOX gene and the activity of the 5‐LOX pathway are increased in elderly subjects. One possible mechanism of such 5‐LOX up‐regulation implies the contribution of aging‐associated hormonal changes: relative melatonin deficiency and/or hyper‐glucocorticoidemia. Thus, the 5‐LOX pathway could become a promising target of neuroprotective therapies for the aging brain.—Manev, H., Uz, T., Sugaya, K., Qu, T. Putative role of neuronal 5‐lipoxygenase in an aging brain. FASEB J. 14, 1464–1469 (2000)

Impaired hippocampal long-term potentiation in melatonin MT2 receptor-deficient mice

The pineal product melatonin that acts on specific melatonin receptors has been implicated in pathobiological mechanisms of neuropsychiatric disorders including Alzheimers disease. We used mice lacking melatonin MT(2) receptors (MT(2) knockouts) to investigate the role of these receptors in synaptic plasticity and learning-dependent behavior. In field CA1 of hippocampal slices from wild-type mice, theta burst stimulation induced robust and stable long-term potentiation that was smaller and decremental in slices from MT(2) knockouts. Tested in an elevated plus-maze on two consecutive days, wild-type mice showed shorter transfer latencies to enter a closed arm on the second day; this experience-dependent behavior did not occur in MT(2) knockouts. These results suggest that MT(2) receptors participate in hippocampal synaptic plasticity and in memory processes.

The pineal gland is critical for circadian Period1 expression in the striatum and for circadian cocaine sensitization in mice.

Sensitization to psychostimulants can be influenced by circadian rhythms. The pineal gland, the main source of circadian melatonin synthesis, may influence behavioral sensitization to cocaine; mice with normal melatonin rhythms do not get sensitized at night. Clock genes such as Period1 (Per1) show rhythmic region- and strain-dependent expression in the mouse brain, and mice mutant for the Per1 gene lack cocaine sensitization. Here, for the first time we show circadian changes of PER1 protein levels in the mouse striatum, a brain region crucial for the development of locomotor sensitization to cocaine. In male C3H/HeJ mice, we found peak striatal PER1 protein levels during the day; this was preceded by a Per1 mRNA peak 16 h earlier. Pinealectomized mice did not show this circadian pattern. We analyzed circadian cocaine sensitization at times when striatal PER1 protein levels in control mice (naive and sham-pinealectomized) were high and low, respectively. Only mice with circadian changes in striatal Per1 expression showed the night-time absence of cocaine sensitization, whereas pinealectomized mice were without circadian changes in striatal Per1 and were sensitized to cocaine regardless of diurnal rhythm. Our results indicate that both the striatal circadian Per1 expression and diurnal locomotor cocaine sensitization are strongly influenced by pineal products. Since we found evidence for the expression of melatonin receptor mRNA in the striatum, we suggest that further studies on pineal-driven mechanisms will help us better understand the mechanisms of drug abuse and identify novel targets for the prevention and/or treatment of addictions.

Fluoxetine increases the content of neurotrophic protein S100β in the rat hippocampus

Recent studies indicate that a protracted daily administration of the antidepressant fluoxetine to adult rats increases cell proliferation/neurogenesis in the hippocampus. It has been hypothesized that this action of fluoxetine might be mediated by neurotrophic factors. We hypothesized that glial S100beta could be such a factor, and using quantitative Western immunoblotting, we investigated the effect of a 21-day treatment of rats with fluoxetine (5 mg/kg), and found that fluoxetine increases the content of hippocampal S100beta.

Increased 5-Lipoxygenase Immunoreactivity in the Hippocampus of Patients With Alzheimer's Disease

The proinflammatory enzyme 5-lipoxygenase (5-LOX) is upregulated in Alzheimers disease (AD), but its localization and association with the hallmark lesions of the disease, β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs), is unknown. This study examined the distribution and cellular localization of 5-LOX in the medial temporal lobe from AD and control subjects. The spatial relationship between 5-LOX immunoreactive structures and AD lesions was also examined. We report that, in AD subjects, 5-LOX immunoreactivity is elevated relative to controls, and its localization is dependent on the antibody-targeted portion of the 5-LOX amino acid sequence. Carboxy terminus-directed antibodies detected 5-LOX in glial cells and neurons, but less frequently in neurons with dystrophic (NFT) morphology. In contrast, immunoreactivity observed using 5-LOX amino terminus-directed antibodies was virtually absent in neurons and abundant in NFTs, neuritic plaques, and glia. Double-labeling studies showed a close association of 5-LOX-immunoreactive processes and glial cells with Aβ immunoreactive plaques and vasculature and also detected 5-LOX in tau immunoreactive and amyloid containing NFTs. Different immunolabeling patterns with antibodies against carboxy vs amino terminus of 5-LOX may be caused by post-translational modifications of 5-LOX protein in Aβ plaques and NFTs. The relationship between elevated intracellular 5-LOX and hallmark AD pathological lesions provides further evidence that neuroinflammatory pathways contribute to the pathogenesis of AD.