Inhibition of phospholipase A(2) increases Tau phosphorylation at Ser214 in embryonic rat hippocampal neurons

Background: Arachidonic acid is released from cellular membranes by the action of phospholipase A(2) (PLA(2)) and is implicated in microtubule-associated protein Tau phosphorylation. Tau hyperphosphorylation affects its ability to stabilize microtubules. Objective: To determine the effect of PLA(2) inhibition on the phosphorylation state of Tau phosphoepitopes in primary cultures of hippocampal neurons. Methods: 4 DIC neurons were incubated at different concentrations of methyl-arachidonylfluorophosphonate (MAFP), an irreversible inhibitor of cPLA(2) and iPLA(2). Changes on Tau phosphorylation were determined by Western blotting with a panel of anti-Tau antibodies (C-terminal, Ser199/202, Ser202/205, Ser396 and Ser214). Results: The Ser214 site was hyperphosphorylated upon MAFP treatment. Significant differences were observed with 10 mu M (p = 0.01), 50 mu M (p = 0.01) and 100 mu M (p = 0.05) of MAFP. Less-intense changes were found in other phosphoepitopes. Conclusion: The present findings indicate that the phosphorylation of Ser214 is regulated by c- and/or iPLA(2), whereas other phosphoepitopes primarily regulated by GKS3b were not affected. (C) 2009 Elsevier Ltd. All rights reserved.

Expression Profile of Rat Hippocampal Neurons Treated with the Neuroprotective Compound 2,4-Dinitrophenol: Up-Regulation of cAMP Signaling Genes

2,4-Dinitrophenol (DNP) is classically known as a mitochondrial uncoupler and, at high concentrations, is toxic to a variety of cells. However, it has recently been shown that, at subtoxic concentrations, DNP protects neurons against a variety of insults and promotes neuronal differentiation and neuritogenesis. The molecular and cellular mechanisms underlying the beneficial neuroactive properties of DNP are still largely unknown. We have now used DNA microarray analysis to investigate changes in gene expression in rat hippocampal neurons in culture treated with low micromolar concentrations of DNP. Under conditions that did not affect neuronal viability, high-energy phosphate levels or mitochondrial oxygen consumption, DNP induced up-regulation of 275 genes and down-regulation of 231 genes. Significantly, several up-regulated genes were linked to intracellular cAMP signaling, known to be involved in neurite outgrowth, synaptic plasticity, and neuronal survival. Differential expression of specific genes was validated by quantitative RT-PCR using independent samples. Results shed light on molecular mechanisms underlying neuroprotection by DNP and point to possible targets for development of novel therapeutics for neurodegenerative disorders.

Circulating levels of brain-derived neurotrophic factor: correlation with mood, cognition and motor function

Brain-derived neurotrophic factor (BDNF) is the most widely distributed neurotrophin in the CNS, where it plays several pivotal roles in synaptic plasticity and neuronal survival. As a consequence, BDNF has become a key target in the physiopathology of several neurological and psychiatric diseases. Recent studies have consistently reported altered levels of BDNF in the circulation (i.e., serum or plasma) of patients with major depression, bipolar disorder, Alzheimer`s disease, Huntington`s disease and Parkinson`s disease. Correlations between serum BDNF levels and affective, cognitive and motor symptoms have also been described. BDNF appears to be an unspecific biomarker of neuropsychiatric disorders characterized by neurodegenerative changes.

The neuroprotective effect of dental pulp cells in models of Alzheimer`s and Parkinson`s disease

Aim of the present study was to investigate the neuroprotective effect of dental pulp cells (DPCs) in in vitro models of Alzheimer and Parkinson disease. Primary cultures of hippocampal and ventral mesencephalic neurons were treated for 24 h with amyloid beta (A beta(1-42)) peptide 1-42 and 6-OHDA, respectively. DPCs isolated from adult rat incisors were previously cultured in tissue culture inserts and added to the neuron cultures 2 days prior to neurotoxin treatment. Cell viability was assessed by the MTT assay. The co-culture with DPCs significantly attenuated 6-OHDA and A beta(1-42)-induced toxicity in primary cultures of mesencephalic and hippocampal neurons, and lead to an increase in neuronal viability in untreated cultures, suggesting a neurotrophic effect in both models. Furthermore, human dental pulp cells expressed a neuronal phenotype and produced the neurotrophic factors NGF, GDNF, BDNF, and BMP2 shown by microarray screening and antibody staining for the representative proteins. DPCs protected primary neurons in in vitro models of Alzheimer`s and Parkinson`s disease and can be viewed as possible candidates for studies on cell-based therapy.

Role of alpha 7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP(C)) is a conserved glycosylphosphatidyl-inositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP(C) extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP(C) engagement induces an increase in intracellular Ca(2+) levels. This effect was not detected in PrP(C)-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP(C). Using a best candidate approach to test for potential channels involved in Ca(2+) influx evoked by STI1-PrP(C), we found that alpha-bungarotoxin, a specific inhibitor for alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when alpha 7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP(C) and allowed reconstitution of signaling by PrP(C)-STI1 interaction. These results indicate that STI1 can interact with the PrP(C).alpha 7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

EXPRESSION OF COCAINE- AND AMPHETAMINE-REGULATED TRANSCRIPT IN THE RAT FOREBRAIN DURING POSTNATAL DEVELOPMENT

Cocaine- and amphetamine-regulated transcript (CART) is widespread in the rodent brain. CART has been implicated in many different functions including reward, feeding, stress responses, sensory processing, learning and memory formation. Recent studies have suggested that CART may also play a role in neural development. Therefore, in the present study we compared the distribution pattern and levels of CART mRNA expression in the forebrain of male and female rats at different stages of postnatal development: P06, P26 and P66. At 6 days of age (P06), male and female rats showed increased CART expression in the somatosensory and piriform cortices, indusium griseum, dentate gyrus, nucleus accumbens, and ventral premammillary nucleus. Interestingly, we found a striking expression of CART mRNA in the ventral posteromedial and ventral posterolateral thalamic nuclei. This thalamic expression was absent at P26 and P66. Contrastingly, at P06 CART mRNA expression was decreased in the arcuate nucleus. Comparing sexes, we found increased CART mRNA expression in the anteroventral periventricular nucleus of adult females. In other regions including the CA1, the lateral hypothalamic area and the dorsomedial nucleus of the hypothalamus, CART expression was not different comparing postnatal ages and sexes. Our findings indicate that CART gene expression is induced in a distinct temporal and spatial manner in forebrain sites of male and female rats. They also suggest that CART peptide participate in the development of neural pathways related to selective functions including sensory processing, reward and memory formation. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Amyloid beta-peptide activates nuclear factor-kappa B through an N-methyl-D-aspartate signaling pathway in cultured cerebellar cells

Amyloid P-peptide (A beta) likely causes functional alterations in neurons well prior to their death. Nuclear factor-kappa B (NF-kappa B), a transcription factor that is known to play important roles in cell survival and apoptosis, has been shown to be modulated by A beta in neurons and glia, but the mechanism is unknown. Because A beta has also been shown to enhance activation of N-methyl-D-aspartate (NMDA) receptors, we investigated the role of NMDA receptor-mediated intracellular signaling pathways in A beta-induced NF-kappa B activation in primary cultured rat cerebellar cells. Cells were treated with different concentrations of A beta 1-40 (1 or 2 mu M) for different periods (6, 12, or 24 hr). MK-801 (NMDA antagonist), manumycin A and FTase inhibitor 1 (farnesyltransferase inhibitors), PP1 (Src-family tyrosine kinase inhibitor), PD98059 [mitogen-activated protein kinase (MAPK) inhibitor], and LY294002 [phosphatidylinositol 3-kinase (PI3-k) inhibitor] were added 20 min before A beta treatment of the cells. A beta induced a time- and concentration-dependent activation of NF-kappa B (1 mu M, 12 hr); both p50/p65 and p50/p50 NF-kappa B dimers were involved. This activation was abolished by MK-801 and attenuated by manumycin A, FTase inhibitor 1, PP1, PD98059, and LY294002. AP at 1 mu M increased the expression of inhibitory protein I kappa B, brain-derived neurotrophic factor, inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1 beta as shown by RTPCR assays. Collectively, these findings suggest that AP activates NF-kappa B by an NMDA-Src-Ras-like protein through MAPK and PI3-k pathways in cultured cerebellar cells. This pathway may mediate an adaptive, neuroprotective response to A beta. (c) 2007 Wiley-Liss, Inc.

Lithium reduces Gsk3b mRNA levels: implications for Alzheimer Disease

There is evidence of increased systemic expression of active GSK3B in Alzheimer`s disease patients, which apparently is associated with the formation of senile plaques and neurofibrillary tangles. Due to its central role in the pathogenesis of AD, GSK3B is currently a promising target of the pharmaceutical industry. Whilst trials with specific GSK inhibitors in AD are under way, major attention has been focused on the neuroprotective effects of lithium. Whereas the direct and indirect inhibitory effects of lithium over GSK3 activity have been documented by several groups, its effects over Gsk3 transcription have not yet been addressed. We used quantitative PCR to evaluate the transcriptional regulation of Gsk3a and Gsk3b in lithium-treated primary cultures of rat cortical and hippocampal neurons. We found a significant and dose-dependent reduction in the expression of Gsk3b, which was specific to hippocampal cells. This same effect was further confirmed in vivo by measuring Gsk3 expression in different brain regions and in peripheral leukocytes of adult rats treated with lithium. Our studies show that LiCl can modulate Gsk3b transcription in vitro and in vivo. This observation suggest new regulatory effects of lithium over Gsk3b, contributing to the better understanding of its mechanisms of action, offering a new and complementary explanation for Gsk3b modulation and reinforcing its potential for the inhibition of key pathological pathways in Alzheimer`s disease.

Plasma hormonal profiles and dendritic spine density and morphology in the hippocampal CA1 stratum radiatum, evidenced by light microscopy, of virgin and postpartum female rats

Successful reproduction requires that changes in plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), oxytocin (OT), estrogen (E-2) and progesterone (P-4) occur together with the display of maternal behaviors. Ovarian steroids and environmental stimuli can affect the dendritic spines in the rat hippocampus. Here, studying Wistar rats, it is described: (a) the sequential and concomitant changes in the hormonal profile of females at postpartum days (PP) 4, 8, 12, 16, 20 and 24, comparing to estrous cycle referential values; (b) the dendritic spine density in the stratum radiatum of CA1 (CA1-SR) Golgi-impregnated neurons in virgin females across the estrous cycle and in multiparous age-matched ones; and (c) the proportion of different types of spines in the CAI-SR of virgin and postpartum females, both in diestrus. Plasma levels of gonadotrophins and ovarian hormones remained low along PP while LH increased and PRL decreased near the end of the lactating period. The lowest dendritic spine density was found in virgin females in estrus when compared to diestrus and proestrus phases or to postpartum females in diestrus (p < 0.03). Other comparisons among groups were not statistically significant (p > 0.4). There were no differences in the proportions of the different spine types in nulliparous and postpartum females (p > 0.2). Results suggest that medium layer CA1-SR spines undergo rapid modifications in Wistar females across the estrous cycle (not quite comparable to Sprague-Dawley data or to hormonal substitutive therapy following ovariectomy), but persistent effects of motherhood on dendritic spine density and morphology were not found in this area. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Muscarinic acetylcholine neurotransmission enhances the late-phase of long-term potentiation in the hippocampal-prefrontal cortex pathway of rats in vivo: A possible involvement of monoaminergic systems

The prefrontal cortex is continuously required for working memory processing during wakefulness, but is particularly hypoactivated during sleep and in psychiatric disorders such as schizophrenia. Ammon`s horn CA1 hippocampus subfield (CA1) afferents provide a functional modulatory path that is subjected to synaptic plasticity and a prominent monoaminergic influence. However, little is known about the muscarinic cholinergic effects on prefrontal synapses. Here, we investigated the effects of the muscarinic agonist, pilocarpine (PILO), on the induction and maintenance of CA1-medial prefrontal cortex (mPFC) long-term potentiation (LTP) as well as on brain monoamine levels. Field evoked responses were recorded in urethane-anesthetized rats during baseline (50 min) and after LTP (130 min), and compared with controls. LTP was induced 20 min after PILO administration (15 mg/kg, i.p.) or vehicle (NaCl 0.15 M, i.p.). In a separate group of animals, the hippocampus and mPFC were microdissected 20 min after PILO injection and used to quantify monoamine levels. Our results show that PILO potentiates the late-phase of mPFC UP without affecting either post-tetanic potentiation or early LTP (20 min). This effect was correlated with a significant decrease in relative delta (1-4 Hz) power and an increase in sigma (10-15 Hz) and gamma (2540 Hz) powers in CA1. Monoamine levels were specifically altered in the mPFC. We observed a decrease in dopamine, 5-HT, 5-hydroxyindolacetic acid and noradrenaline levels, with no changes in 3,4-hydroxyphenylacetic acid levels. Our data, therefore, suggest that muscarinic activation exerts a boosting effect on mPFC synaptic plasticity and possibly on mPFC-dependent memories, associated to monoaminergic changes. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Postnatal fluoxetine treatment affects the development of serotonergic neurons in rats

The goal of the present study was to investigate morphological changes in the serotonergic neurons/terminals in the dorsal (DR) and median (MnR) raphe nuclei and on the hippocampal dentate gyrus (DG) in neonatal rats treated from the 1st to the 21st postnatal day with fluoxetine (10 mg/kg sc, daily) or drug vehicle (0 9% saline 1 ml/kg). The results show that postnatal chronic treatment with fluoxetine promoted. (1) a smaller body weight increase during the pre-weaning period; (2) smaller number of 5-HT neurons in the DR, (3) smaller 5-HT neuronal cell bodies (area, perimeter and diameter) in the DR and the MnR and (4) diminished serotonergic terminals in the DG. These data suggest that the development of the serotonergic system was impaired and that early exposure to fluoxetine damaged the morphology of 5-HT neurons in young adult rats While these findings are consistent with other work, more studies are needed to better clarify the effects of postnatal chronic treatment with fluoxetine on the serotonergic system and, consequently, on the functions modulated by serotonin (C) 2010 Elsevier Ireland Ltd All rights reserved

Lesion of the subthalamic nucleus reverses motor deficits but not death of nigrostriatal dopaminergic neurons in a rat 6-hydroxydopamine-lesion model of Parkinson's disease

The objective of the present study was to determine whether lesion of the subthalamic nucleus (STN) promoted by N-methyl-D-aspartate (NMDA) would rescue nigrostriatal dopaminergic neurons after unilateral 6-hydroxydopamine (6-OHDA) injection into the medial forebrain bundle (MFB). Initially, 16 mg 6-OHDA (6-OHDA group) or vehicle (artificial cerebrospinal fluid - aCSF; Sham group) was infused into the right MFB of adult male Wistar rats. Fifteen days after surgery, the 6-OHDA and SHAM groups were randomly subdivided and received ipsilateral injection of either 60 mM NMDA or aCSF in the right STN. Additionally, a control group was not submitted to stereotaxic surgery. Five groups of rats were studied: 6-OHDA/NMDA, 6-OHDA/Sham, Sham/NMDA, Sham/Sham, and Control. Fourteen days after injection of 6-OHDA, rats were submitted to the rotational test induced by apomorphine (0.1 mg/kg, ip) and to the open-field test. The same tests were performed again 14 days after NMDA-induced lesion of the STN. The STN lesion reduced the contralateral turns induced by apomorphine and blocked the progression of motor impairment in the open-field test in 6-OHDA-treated rats. However, lesion of the STN did not prevent the reduction of striatal concentrations of dopamine and metabolites or the number of nigrostriatal dopaminergic neurons after 6-OHDA lesion. Therefore, STN lesion is able to reverse motor deficits after severe 6-OHDA-induced lesion of the nigrostriatal pathway, but does not protect or rescue dopaminergic neurons in the substantia nigra pars compacta.

Effects of ascorbic acid supplementation in ileum myenteric neurons of streptozotocin-induced diabetic rats

The exacerbation of the oxidative stress and of the polyol pathway which impair damage myenteric plexus are metabolic characteristics of diabetes. The ascorbic acid (AA) is an antioxidant and an aldose reductase inhibitor, which may act as neuroprotector. The effects of AA supplementation on the density and cellular body profile area (CP) of myenteric neurons in STZ-induced diabetes in rats were assessed. Four groups with five animals each were formed: normoglycemic (C); diabetic (D); AA-treated diabetic (DS) and AA-treated normoglycemic (CS). Dosagen of 50mg of AA were given, three times a week, for each animal (group DS and CS). Ninety days later and after euthanasia, the ileum was collected and processed for the NADPH-diaphorase technique. There were no differences (P>0.05) in the neuronal density among the groups. The CP area was lower (P<0.05) in the DS and CS groups, with a higher incidence of neurons with a CP area exceeding 200µm² for groups C and D. The AA had no influence on the neuronal density in the ileum but had a neuroprotective effect, preventing the increase in the CP area and allowing a higher number of neurons with a CP area with less than 200µm².

Active Dendrites Enhance Neuronal Dynamic Range

Since the first experimental evidences of active conductances in dendrites, most neurons have been shown to exhibit dendritic excitability through the expression of a variety of voltage-gated ion channels. However, despite experimental and theoretical efforts undertaken in the past decades, the role of this excitability for some kind of dendritic computation has remained elusive. Here we show that, owing to very general properties of excitable media, the average output of a model of an active dendritic tree is a highly non-linear function of its afferent rate, attaining extremely large dynamic ranges (above 50 dB). Moreover, the model yields double-sigmoid response functions as experimentally observed in retinal ganglion cells. We claim that enhancement of dynamic range is the primary functional role of active dendritic conductances. We predict that neurons with larger dendritic trees should have larger dynamic range and that blocking of active conductances should lead to a decrease in dynamic range.

Survival and Neuronal Differentiation of Mesenchymal Stem Cells Transplanted into the Rodent Brain Are Dependent upon Microenvironment

Introduction: The successful integration of stem cells in adult brain has become a central issue in modern neuroscience. In this study we sought to test the hypothesis that survival and neurodifferentiation of mesenchymal stem cells (MSCs) may be dependent upon microenvironmental conditions according to the site of implant in the brain. Methods: MSCs were isolated from adult rats and labeled with enhanced-green fluorescent protein (eGFP) lentivirus. A cell suspension was implanted stereotactically into the brain of 50 young rats, into one neurogenic area (hippocampus), and into another nonneurogenic area (striatum). Animals were sacrificed 6 or 12 weeks after surgery, and brains were stained for mature neuronal markers. Cells coexpressing NeuN (neuronal specific nuclear protein) and GFP (green fluorescent protein) were counted stereologically at both targets. Results: The isolated cell population was able to generate neurons positive for microtubule-associated protein 2 (MAP2), neuronal-specific nuclear protein (NeuN), and neurofilament 200 (NF200) in vitro. Electrophysiology confirmed expression of voltage-gated ionic channels. Once implanted into the hippocampus, cells survived for up to 12 weeks, migrated away from the graft, and gave rise to mature neurons able to synthesize neurotransmitters. By contrast, massive cell degeneration was seen in the striatum, with no significant migration. Induction of neuronal differentiation with increased cyclic adenosine monophosphate in the culture medium before implantation favored differentiation in vivo. Conclusions: Our data demonstrated that survival and differentiation of MSCs is strongly dependent upon a permissive microenvironment. Identification of the pro-neurogenic factors present in the hippocampus could subsequently allow for the integration of stem cells into nonpermissive areas of the central nervous system.