CHF5074 (CSP-1103) induces microglia alternative activation in plaque-free Tg2576 mice and primary glial cultures exposed to beta-amyloid.

Activation of microglia associated with neuroinflammation and loss of phagocytic activity is considered to play a prominent role in the pathogenesis of Alzheimer's disease (AD). CHF5074 (CSP-1103) has been shown to improve cognition and reduce brain inflammation in patients with mild cognitive impairment (MCI). CHF5074 was also found to reverse impairments in recognition memory and improve hippocampal long-term potentiation when administered to plaque-free Tg2576 mice (5-month-old) for 4 weeks. Though, no investigation has focused on the consequence of CHF5074 treatment on microglia polarization yet. In this study we evaluated the effect of CHF5074 administration (375 ppm in the diet) to 5-month-old Tg2576 mice on the expression of pro-inflammatory (M1) genes, Interleukin 1 beta (IL-1ß), Tumor Necrosis Factor alpha (TNFα) and inducible Nitric Oxide Synthase (iNOS), and anti-inflammatory/phagocytic (M2) markers Mannose Receptor type C 1 (MRC1/CD206), Triggering Receptor Expressed on Myeloid cells 2 (TREM2) and Chitinase 3-like 3 (Ym1). No changes of pro-inflammatory gene transcription but a reduced expression of MRC1/CD206, TREM2 and Ym1 were detected in the hippocampus of young Tg2576 mice receiving normal diet, when compared to wild-type littermates. CHF5074 did not affect the pro-inflammatory transcription but significantly increased the expression of MRC1/CD206 and Ym1. CHF5074 effects appeared to be hippocampus-specific, as the M2 transcripts were only slightly modified in the cerebral cortex. In primary cultures of mouse astrocyte-microglia, CHF5074 totally suppressed the expression of TNF-α, IL-1ß and iNOS induced by 10 µM ß-amyloid1-42 (Aß42). Moreover, CHF5074 significantly increased the expression of anti-inflammatory/phagocytic markers MRC1/CD206 and TREM2, reduced by the Aß42 application alone. The effect of CHF5074 was not reproduced by ibuprofen (3 µM or 500 µM) or R-flurbiprofen (3 µM or 100 µM), as both compounds limited the pro-inflammatory gene expression but did not modify the anti-inflammatory/phagocytic transcription. These data show that CHF5074 specifically drives the expression of microglia M2 markers either in young Tg2576 hippocampus or in primary astrocyte-microglia cultures, suggesting its potential therapeutic efficacy as microglial modulator in the early phase of AD.

The acetylation of RelA in Lys310 dictates the NF-κB-dependent response in post-ischemic injury.

The activation of nuclear factor kappa B (NF-κB) p50/RelA is a key event in ischemic neuronal injury, as well as in brain ischemic tolerance. We tested whether epigenetic mechanisms affecting the acetylation state of RelA might discriminate between neuroprotective and neurotoxic activation of NF-κB during ischemia. NF-κB activation and RelA acetylation were investigated in cortices of mice subjected to preconditioning brain ischemia or lethal middle cerebral artery occlusion (MCAO) and primary cortical neurons exposed to preconditioning or lethal oxygen-glucose deprivation (OGD). In mice subjected to MCAO and in cortical neurons exposed to lethal OGD, activated RelA displayed a high level of Lys310 acetylation in spite of reduced total acetylation. Also, acetylated RelA on Lys310 interacted strongly with the CREB-binding protein (CBP). Conversely, RelA activated during preconditioning ischemia appeared deacetylated on Lys310. Overexpressing RelA increased Bim promoter activity and neuronal cell death both induced by lethal OGD, whereas overexpressing the acetylation-resistant RelA-K310R, carrying a mutation from Lys310 to arginine, prevented both responses. Pharmacological manipulation of Lys310 acetylation by the sirtuin 1 activator resveratrol repressed the activity of the Bim promoter and reduced the neuronal cell loss. We conclude that the acetylation of RelA in Lys310 dictates NF-κB-dependent pro-apoptotic responses and represents a suitable target to dissect pathological from neuroprotective NF-κB activation in brain ischemia.

Activation of NF-kappaB p65/c-Rel dimer is associated with neuroprotection elicited by mGlu5 receptor agonists against MPP(+) toxicity in SK-N-SH cells.

Nuclear factor-kappaB (NF-kappaB) is a transcriptional regulator of neuron survival eliciting diverse effects according to the specific composition of its active dimer. While p50/p65 mediates neurodegenerative events, c-Rel-containing dimers promote cell survival. Stimulation of metabotropic glutamate receptors type 5 (mGlu5) reduces neuron vulnerability to amyloid-beta through activation of anti-apoptotic, c-Rel-dependent transcription of Bcl-X(L) pathway. We here evaluated the protective activity of mGlu5 agonists in dopaminergic SK-N-SH cells exposed to 1-methyl-4-phenylpyridinium (MPP(+)), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causing parkinsonism in experimental animals. MPP(+) produced a concentration-dependent cell loss. Activation of mGlu5 receptors by CHPG (1 mM) and 3HPG (50 microM) abolished the toxic effect produced by 3 microM MPP(+). The neuroprotection was associated with activation of NF-kappaB p65/c-Rel dimer and reduction of p50/p65. These effects were prevented by the mGlu5 receptor antagonist MPEP (5 microM). It is suggested that mGlu5 receptor agonists through activation of a c-Rel-dependent anti-apoptotic pathway can rescue dopaminergic cell from mitochondrial toxicity.

NF-kappaB factor c-Rel mediates neuroprotection elicited by mGlu5 receptor agonists against amyloid beta-peptide toxicity.

Opposite effects of nuclear factor-kappaB (NF-kappaB) on neuron survival rely on activation of diverse NF-kappaB factors. While p65 is necessary for glutamate-induced cell death, c-Rel mediates prosurvival effects of interleukin-1beta. However, it is unknown whether activation of c-Rel-dependent pathways reduces neuron vulnerability to amyloid-beta (Abeta), a peptide implicated in Alzheimer's disease pathogenesis. We show that neuroprotection elicited by activation of metabotropic glutamate receptors type 5 (mGlu5) against Abeta toxicity depends on c-Rel activation. Abeta peptide induced NF-kappaB factors p50 and p65. The mGlu5 agonists activated c-Rel, besides p50 and p65, and the expression of manganese superoxide dismutase (MnSOD) and Bcl-X(L). Targeting c-Rel expression by RNA interference suppressed the induction of both antiapoptotic genes. Targeting c-Rel or Bcl-X(L) prevented the prosurvival effect of mGlu5 agonists. Conversely, c-Rel overexpression or TAT-Bcl-X(L) addition rescued neurons from Abeta toxicity. These data demonstrate that mGlu5 receptor activation promotes a c-Rel-dependent antiapoptotic pathway responsible for neuroprotection against Abeta peptide.

Expression of nerve growth factor in brown adipose tissue: implications for thermogenesis and obesity.

The presence of nerve growth factor (NGF) and the ability of adrenergic stimulation to affect the rate of its synthesis in mouse, rat, and human brown adipose tissue (BAT) were investigated. Addition of conditioned medium, obtained from preconfluent and confluent brown adipocytes, to PC12 cells induced typical morphological changes similar to those due to NGF itself. Anti-NGF antibodies blocked this action. Moreover, NGF mRNA was detected by RT-PCR both in BAT and in brown adipocyte preparations. That NGF is synthesized in and released from brown fat cells was confirmed by immunoblotting. When the animals were exposed to low temperatures, NGF production declined. The effect of cold exposure could be mimicked by the addition of norepinephrine (NE) at day 4 or 8 (preconfluent and confluent cells, respectively). NE depletion obtained by reserpine injection induced a drastic increase of BAT NGF production. In both rat and human BAT, immunohistochemistry identified distinct anatomical structures that express the low affinity neurotropin receptor, termed p75NGFR. BAT production of NGF was higher in genetically obese rats and mice than in their lean counterparts, a difference that becomes more evident with age. Prolonged exposure to low temperature significantly decreased the BAT NGF synthesis also in obese animals. We conclude that NGF is synthesized in and released from brown fat cells, its production being inversely dependent on sympathetic activity, in both physiological and pathophysiological conditions, and increased in genetic animal models of obesity.

Rat frontal cortex beta 1-adrenoceptors are activated by the beta 3-adrenoceptor agonists SR 58611A and SR 58878A but not by BRL 37344 or ICI 215,001.

SR 58611A, a selective agonist of gut and brown adipose tissue beta 3-adrenoceptors (beta 3 ARs), has been reported to have antidepressant-like activity in rodents, by indicating brain beta 3ARs as the sites of this property. SR 58611A and its acid metabolite SR 58878A, as opposed to BRL 37344, ICI 215,001, and CGP 12177, increased cyclic AMP levels in rat frontal cortex. ICI 215,001, differently from BRL 37344, at concentrations in the millimolar range antagonized norepinephrine- or (-)-isoproterenol-stimulated adenylyl cyclase partially. The increase of cyclic AMP levels induced by SR 58878A was blocked selectively by beta 1AR antagonist CGP 20712A but not by beta 2AR antagonist ICI 118,551. In addition, PCR analysis did not reveal beta 3AR mRNA, and no specific beta 3AR binding sites were detected by [3H]CGP 12177 in rat frontal cortex. When down-regulation of the beta 1AR ligand binding and mRNA levels had been induced in frontal cortex by chronic administration of imipramine, SR 58878A as well as norepinephrine and (-)-isoproterenol inceased the cyclic AMP production less markedly. Our findings indicate that beta 3ARs are absent in the adult rat frontal cortex, and that various beta 3AR agonists differently affect the frontal cortex beta 1ARs, indicating that SR 58611A may exert its putative antidepressant effect acting on the frontal cortex beta 1ARS.

Activation of dopamine D2 receptors linked to voltage-sensitive potassium channels reduces forskolin-induced cyclic AMP formation in rat pituitary cells.

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptor agonists, including BHT 920 and bromocriptine, and the potassium channel opener minoxidil share the property of hyperpolarizing the plasma membrane by activating voltage-dependent potassium channels. These drugs were tested for their ability to inhibit the cyclic AMP formation induced by forskolin either in intact or in broken pituitary cells. In contrast to bromocriptine, which was active in both experimental systems, BHT 920 and minoxidil inhibited the forskolin-induced cyclic AMP formation in intact-cell but not in broken-cell preparations. The effects of BHT 920 were (a) concentration dependent, with a calculated IC50 of 0.7 microM, (b) dopaminergic in nature, being specifically antagonized by sulpiride, (c) not additive with those induced by minoxidil, and (d) less effective in the presence of potassium channel blockers, such as 4-aminopyridine and tetraethylammonium. These data indicate that the inhibition of forskolin-induced cyclic AMP formation by BHT 920 in intact pituitary cells is not a primary consequence of receptor occupation, but a late event, possibly related to the opening of voltage-dependent potassium channels elicited by this drug through the activation of a subtype of dopamine D2 receptors uncoupled to adenylyl cyclase.

A mechanism additional to cyclic AMP accumulation for vasoactive intestinal peptide-induced prolactin release.

Vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor which has been proposed to exert its secreting property by activating the adenylate cyclase enzyme. The present study shows that the omission of external Ca2+ did not affect the ability of VIP to induce PRL release while it completely abolished the VIP stimulatory effect on adenylate cyclase. We found that VIP (500 nM) stimulated PRL secretion in a time-dependent manner reaching a plateau at 3 min. This pattern was not changed when Ca2+ was omitted from the incubation medium. When tested at different concentrations, VIP stimulated PRL release with EC50 values of 1.3 nM in the presence of Ca2+ and 30 nM in the absence of Ca2+. On the other hand, Ca2+ removal completely suppressed the VIP-induced cAMP formation. VIP (200 nM) was also found to activate Ca2+ influx into pituitary cells. The increase in Ca2+ permeability showed a peak at 5 s and remained significantly higher than control values until 1 min. In conclusion, in an experimental condition where Ca2+ was omitted from the medium, VIP was found to induce PRL release without stimulating cAMP production. This cAMP-independent PRL release was blocked by preincubation of the cells with 1 microgram/ml pertussis toxin. An additional mechanism other than adenylate cyclase activation or Ca2+ entry is proposed to sustain VIP-induced PRL release.