Modulation of Pro-Oxidant and Pro-Inflammatory Activities of M1 Macrophages by the Natural Dipeptide Carnosine.

Carnosine is a natural endogenous dipeptide widely distributed in mammalian tissues, existing at particularly high concentrations in the muscles and brain and possesses well-characterized antioxidant and anti-inflammatory activities. In an in vitro model of macrophage activation, induced by lipopolysaccharide + interferon-gamma (LPS + IFN-γ), we here report the ability of carnosine to modulate pro-oxidant and pro-inflammatory activities of macrophages, representing the primary cell type that is activated as a part of the immune response. An ample set of parameters aimed to evaluate cytotoxicity (MTT assay), energy metabolism (HPLC), gene expressions (high-throughput real-time PCR (qRT-PCR)), protein expressions (western blot) and nitric oxide production (qRT-PCR and HPLC), was used to assess the effects of carnosine on activated macrophages challenged with a non cytotoxic LPS (100 ng/mL) + IFN-γ (600 U/mL) concentration. In our experimental model, main carnosine beneficial effects were: (1) the modulation of nitric oxide production and metabolism; (2) the amelioration of the macrophage energy state; (3) the decrease of the expressions of pro-oxidant enzymes (Nox-2, Cox-2) and of the lipid peroxidation product malondialdehyde; (4) the restoration and/or increase of the expressions of antioxidant enzymes (Gpx1, SOD-2 and Cat); (5) the increase of the transforming growth factor-ß1 (TGF-ß1) and the down-regulation of the expressions of interleukins 1ß and 6 (IL-1ß and IL-6) and 6) the increase of the expressions of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). According to these results carnosine is worth being tested in the treatment of diseases characterized by elevated levels of oxidative stress and inflammation (atherosclerosis, cancer, depression, metabolic syndrome, and neurodegenerative diseases).

Astrocytes contribute to Aß-induced blood-brain barrier damage through activation of endothelial MMP9.

The blood-brain barrier (BBB) plays an important role in the maintenance of the brain homeostasis, and its proper functions are warranted by the interplay between different cellular components (endothelial cells, astrocytes and pericytes). BBB dysfunctions in pathological conditions, and particularly in Alzheimer's disease, have been documented. Here, using an in vitroBBB model, the interaction between endothelial cells and astrocytes exposed to Aß1-42 was investigated. Human endothelial cells, cultured in monolayer or co-cultured with astrocytes, were exposed to Aß1-42 (2 µM for 18 h). Aß induced dysfunction of endothelial barrier, as assessed by enhanced permeability to FITC-conjugated dextran and reduced expression of claudin-5; these modifications were observed in the co-culture model, but not in endothelial cells cultured in monolayer. Similarly, Aß-induced damage at the barrier was observed when endothelial cells were challenged in the presence of conditioned medium generated by astrocytes previously exposed to Aß (ACM Aß). Endothelial barrier damages were associated with enhanced matrix metalloprotease 9 (MMP9) activity, known to mediate claudin-5 disruption. These events were not related to the direct effects played by Aß on endothelial cells, but they were rather the consequence of Aß-induced vascular endothelial growth factor (VEGF) expression in astrocytes. Indeed, when vascular endothelial growth factor expression was down-regulated in astrocytes, neither barrier properties or MMP9 expression in endothelial cells were affected after Aß exposure both in the co-culture model or in the presence of ACM Aß. These data point out the importance of astrocytes' mediation in inducing endothelial sensitivity to Aß1-42.

Identification of 5-Methoxyflavone as a Novel DNA Polymerase-Beta Inhibitor and Neuroprotective Agent against Beta-Amyloid Toxicity.

Cell-cycle reactivation is a core feature of degenerating neurons in Alzheimer's disease (AD) and Parkinson's disease (PD). A variety of stressors, including ß-amyloid (Aß) in the case of AD, can force neurons to leave quiescence and to initiate an ectopic DNA replication process, leading to neuronal death rather than division. As the primary polymerase (pol) involved in neuronal DNA replication, DNA pol-ß contributes to neuronal death, and DNA pol-ß inhibitors may prove to be effective neuroprotective agents. Currently, specific and highly active DNA pol-ß inhibitors are lacking. Nine putative DNA pol-ß inhibitors were identified in silico by querying the ZINC database, containing more than 35 million purchasable compounds. Following pharmacological evaluation, only 5-methoxyflavone (1) was validated as an inhibitor of DNA pol-ß activity. Cultured primary neurons are a useful model to investigate the neuroprotective effects of potential DNA pol-ß inhibitors, since these neurons undergo DNA replication and death when treated with Aß. Consistent with the inhibition of DNA pol-ß, 5-methoxyflavone (1) reduced the number of S-phase neurons and the ensuing apoptotic death triggered by Aß. 5-Methoxyflavone (1) is the first flavonoid compound able to halt neurodegeneration via a definite molecular mechanism rather than through general antioxidant and anti-inflammatory properties.

Estrogen activates matrix metalloproteinases-2 and -9 to increase beta amyloid degradation.

Estrogen is known to affect different aspects of ß-amyloid (Aß) synthesis and degradation. The present work was undertaken to evaluate specifically whether matrix metalloproteinases (MMP) -2 and -9 are involved in Aß degradation induced by estrogen and whether this is relevant to estrogen-induced neuroprotection. In SH-SY5Y human neuroblastoma cells, 10 nM 17ß-estradiol (17ß-E2) increases mRNA and intracellular protein expression of MMP-2 and -9, as well as the levels of the active forms of both enzymes released in the medium. Specificity of the effect is proved by prevention with the estrogen receptor (ER) antagonist ICI 182,780 (1 µM) and involvement of the ERα subtype is confirmed by the use of selective ERα or ERß agonists (PPT, DPN) and antagonists (MPP, PHTPP). 17ß-E2 significantly increases the degradation of Aß, either transferred with the conditioned medium of H4-APPSw human neuroglioma cells, engineered to overproduce Aß(1-40) and Aß(1-42), or exogenously added as 2 µM Aß(1-42). Both these effects are completely prevented by preexposure to the broad spectrum MMP inhibitor GM6001 (5 µM). Importantly, the 17ß-E2-induced rescue of neuroblastoma cells challenged with 2 µM Aß(1-42), an effect prevented by ICI 182,780 (1 µM), is mediated by MMPs, as it appears significantly reduced by GM6001 (5 µM) as well as by both MMP-2 (200 nM) and MMP-9 (200nM) selective inhibitors. In conclusion, the present study shows for the first time that MMP-2 and -9 give a main contribution to estrogen's neuroprotective effect.

Alzheimer's disease and neuroinflammation: will new drugs in clinical trials pave the way to a multi-target therapy?

Despite extensive research, no disease-modifying therapeutic option, able to prevent, cure or halt the progression of Alzheimer's disease [AD], is currently available. AD, a devastating neurodegenerative pathology leading to dementia and death, is characterized by two pathological hallmarks, the extracellular deposits of amyloid beta (Aß) and the intraneuronal deposits of neurofibrillary tangles (NFTs) consisting of altered hyperphosphorylated tau protein. Both have been widely studied and pharmacologically targeted for many years, without significant therapeutic results. In 2022, positive data on two monoclonal antibodies targeting Aß, donanemab and lecanemab, followed by the 2023 FDA accelerated approval of lecanemab and the publication of the final results of the phase III Clarity AD study, have strengthened the hypothesis of a causal role of Aß in the pathogenesis of AD. However, the magnitude of the clinical effect elicited by the two drugs is limited, suggesting that additional pathological mechanisms may contribute to the disease. Cumulative studies have shown inflammation as one of the main contributors to the pathogenesis of AD, leading to the recognition of a specific role of neuroinflammation synergic with the Aß and NFTs cascades. The present review provides an overview of the investigational drugs targeting neuroinflammation that are currently in clinical trials. Moreover, their mechanisms of action, their positioning in the pathological cascade of events that occur in the brain throughout AD disease and their potential benefit/limitation in the therapeutic strategy in AD are discussed and highlighted as well. In addition, the latest patent requests for inflammation-targeting therapeutics to be developed in AD will also be discussed.

Melatonin Activates Anti-Inflammatory Features in Microglia in a Multicellular Context: Evidence from Organotypic Brain Slices and HMC3 Cells.

Melatonin (MEL) is a neurohormone endowed with neuroprotective activity, exerted both directly on neuronal cells and indirectly through modulation of responsive glial cells. In particular, MEL's effects on microglia are receptor-mediated and in part dependent on SIRT1 activation. In the present study, we exploited the highly preserved cytoarchitecture of organotypic brain cultures (OC) to explore the effects of MEL on hippocampal microglia in a 3D context as compared to a single cell type context represented by the human HMC3 cell line. We first evaluated the expression of MEL receptor MT1 and SIRT1 and then investigated MEL action against an inflammatory stimulation with LPS: OCs were cultured for a total of 2 weeks and during this time exposed to 0.1 µg/mL of LPS for 24 h either on day 1 (LPS 1°) or on day 11 (LPS 11°). MEL was added immediately after plating and kept for the entire experiment. Under these conditions, both MEL and LPS induced amoeboid microglia. However, the same round phenotype matched different polarization features. LPS increased the number of nuclear-NF-kB+ round cells and MEL alone or in combination with LPS increased BDNF+ round microglia. In addition, MEL contrasted LPS effects on NF-kB expression. Data from HMC3 microglia confirmed MEL's anti-inflammatory effects against LPS in terms of CASP1 induction and BDNF release, identifying SIRT1 as a mediator. However, no effects were evident for MEL alone on HMC3 microglia. Overall, our results point to the importance of the multicellular context for full MEL activity, especially in a preventive view, and support the use of OCs as a favorable model to explore inflammatory responses.

Increased Heat Pain Tolerance but Hyperalgesia to Tonic Inflammatory Pain in the CRND8 Mouse Model of Alzheimer's Disease.

BACKGROUND: The effects of Alzheimer's disease (AD) pathology on the experience of pain are poorly understood. OBJECTIVE: To understand the pathophysiological mechanisms underlying pain sensory transmission in the transgenic mouse model of AD, CRND8. METHODS: We explored AD-related pathology in the spinal cord and dorsal root ganglia of 18-week-old female CRND8 mice. We assessed nociceptive responses to both acute heat stimuli and persistent inflammatory pain in CRND8 mice and non-transgenic (non-Tg) littermates. In addition, we searched for differences in biochemical correlates of inflammatory pain between CRND8 and non-Tg mice. Finally, we investigated the excitability of dorsal horn noc iceptive neurons in spinal cord slices from CRND8 and non-Tg mice. RESULTS: We demonstrated the presence of intracellular AD-like pathology in the spinal cord and in the dorsal root ganglia nociceptive sensory neurons of CRND8 mice. We found that CRND8 mice had a reduced susceptibility to acute noxious heat stimuli and an increased sensitivity to tonic inflammatory pain. Tonic inflammatory pain correlated with a lack of induction of pro-opiomelanocortin in the spinal cord of CRND8 mice as compared to non-Tg mice. Electrophysiological recording in acute spinal cord slice preparations indicated an increased probability of glutamate release at the membrane of dorsal horn nociceptive neurons in CRND8 mice. CONCLUSION: This study suggests that an increased thermal tolerance and a facilitation of nociception by peripheral inflammation can coexist in AD.

Estrogen receptors and type 1 metabotropic glutamate receptors are interdependent in protecting cortical neurons against ß-amyloid toxicity.

We examined the interaction between estrogen receptors (ERs) and type 1 metabotropic glutamate receptors (mGlu1 receptors) in mechanisms of neurodegeneration/neuroprotection using mixed cultures of cortical cells challenged with ß-amyloid peptide. Both receptors were present in neurons, whereas only ERα but not mGlu1 receptors were found in astrocytes. Addition of 17ß-estradiol (17ßE2) protected cultured neurons against amyloid toxicity, and its action was mimicked by the selective ERα agonist, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) as well as by a cell-impermeable bovine serum albumin conjugate of 17ßE2. The selective ERß agonist, diarylpropionitrile (DPN), was only slightly neuroprotective. The mGlu1/5 receptor agonist, 3,5-dihydroxyphenylglycine (DHPG), was also neuroprotective against amyloid toxicity, and its action was abolished by the mGlu1 receptor antagonist, (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone (JNJ 16259685). Neuroprotection by 17ßΕ2 or PPT (but not DPN) and DHPG was less than additive, suggesting that ERα and mGlu1 receptors activate the same pathway of cell survival. More important, neuroprotection by 17ßΕ2 was abolished not only by the ER antagonist fulvestrant (ICI 182,780) but also by JNJ 16259685, and neuroprotection by DHPG was abolished by ICI 182,780. ERα and mGlu1 receptors were also interdependent in activating the phosphatidylinositol-3-kinase pathway, and pharmacological blockade of this pathway abolished neuroprotection by 17ßE2, DHPG, or their combination. These data provide the first evidence that ERα and mGlu1 receptors critically interact in promoting neuroprotection, information that should be taken into account when the impact of estrogen on neurodegeneration associated with central nervous system disorders is examined.

Decreased Astrocytic CCL2 Accounts for BAF-312 Effect on PBMCs Transendothelial Migration Through a Blood Brain Barrier in Vitro Model.

Disruption of the blood brain barrier (BBB) is a common event in several neurological diseases and in particular, in multiple sclerosis (MS), it contributes to the infiltration of the central nervous system by peripheral inflammatory cells. Sphingosine-1-phosphate (S1P) is a bioactive molecule with pleiotropic effects. Agonists of S1P receptors such as fingolimod and siponimod (BAF-312) are in clinical practice for MS and have been shown to preserve BBB function in inflammatory conditions. Using an in vitro BBB model of endothelial-astrocytes co-culture exposed to an inflammatory insult (tumor necrosis factor-α and interferon-γ; T&I), we show that BAF-312 reduced the migration of peripheral blood mononuclear cells (PBMCs) through the endothelial layer, only in the presence of astrocytes. This effect was accompanied by decreased expression of the adhesion molecule ICAM-1. BAF-312 also reduced the activation of astrocytes, by controlling NF-kB and NLRP3 induction and preventing the increase of proinflammatory cytokine and chemokines. Reduction of CCL2 by BAF-312 may be responsible for the observed effects and, accordingly, addition of exogenous CCL2 was able to counteract BAF-312 effects and rescued T&I responses on PBMC migration, ICAM-1 expression and astrocyte activation. The present results further point out BAF-312 effects on BBB properties, suggesting also the key role of astrocytes in mediating drug effects on endothelial function.

Microglia Contributes to BAF-312 Effects on Blood-Brain Barrier Stability.

Microglia, together with astrocytes and pericytes, cooperate to ensure blood-brain barrier (BBB) stability, modulating endothelial responses to inflammatory insults. Agonists of the sphingosine 1 phosphate (S1P) receptors, such as siponimod (BAF-312), are important pharmacological tools in multiple sclerosis and other inflammatory diseases. Modulation of S1P receptors may result in a reduced inflammatory response and increased BBB stability. An in vitro BBB model was reproduced using human-derived endothelial cells, astrocytes and microglia. Co-cultures were exposed to inflammatory cytokines (TNFα, 10 UI and IFNγ, 5 UI) in the presence of BAF-312 (100 nM), and the BBB properties and microglia role were evaluated. The drug facilitated microglial migration towards endothelial/astrocyte co-cultures, involving the activity of the metalloprotease 2 (MMP2). Microglia actively cooperated with astrocytes in the maintenance of endothelial barrier stability: in the triple co-culture, selective treatment of microglial cells with BAF-312 significantly prevented cytokines' effects on the endothelial barrier. In conclusion, BAF-312, modulating S1P receptors in microglia, may contribute to the reinforcement of the endothelial barrier at the BBB, suggesting an additional effect of the drug in the treatment of multiple sclerosis.

Microglial polarization differentially affects neuronal vulnerability to the ß-amyloid protein: Modulation by melatonin.

Microglial cells play a central but yet debated role in neuroinflammatory events occurring in Alzheimer's disease (AD). We here explored how microglial features are modulated by melatonin following ß-amyloid (Aß42)-induced activation and examined the cross-talk with Aß-challenged neuronal cells. Human microglial HMC3 cells were exposed to Aß42 (200 nM) in the presence of melatonin (MEL; 1 µM) added since the beginning (MELco) or after a 72 h-exposure to Aß42 (MELpost). In both conditions, MEL favored an anti-inflammatory activation and rescued SIRT1 and BDNF expression/release. Caspase-1 up-regulation and phospho-ERK induction following a prolonged exposure to Aß42 were prevented by MEL. In addition, MEL partially restored proteasome functionality that was altered by long-term Aß42 treatment, re-establishing both 20S and 26S chymotrypsin-like activity. Differentiated neuronal-like SH-SY5Y cells were exposed to Aß42 (200 nM for 24 h) in basal medium or in the presence of conditioned medium (CM) collected from microglia exposed for different times to Aß42 alone or in combination with MELco or MELpost. Aß42 significantly reduced pre-synaptic proteins synaptophysin and VAMP2 and mean neuritic length. These effects were prevented by CM from anti-inflammatory microglia (Aß42 for 6 h), or from MELco and MELpost microglia, but the reduction of neuritic length was not rescued when the SIRT1 inhibitor EX527 was added. In conclusion, our data add to the concept that melatonin shows a promising anti-inflammatory action on microglia that is retained even after pro-inflammatory activation, involving modulation of proteasome function and translating into neuroprotective microglial effects.

Inverse correlation between the expression of AMPK/SIRT1 and NAMPT in psoriatic skin: A pilot study.

PURPOSE: Epidermal hyperplasia and the involvement of immune cells characterize the clinical picture of psoriasis. Among the several factors involved, attention has been focused on sirtuin 1 (SIRT1) - a deacetylase endowed with a variety of functions including the control of metabolic and inflammatory processes-, and on nicotinamide phosphoribosyltransferase (NAMPT), important for SIRT1 activation and involved in inflammatory events. The aim of the study was to analyze changes of SIRT1 and NAMPT expression in psoriatic skin. PATIENTS AND METHODS: Samples from healthy controls and psoriatic patients were subjected to immunohistochemical analysis. RESULTS: A strong downregulation of SIRT1 expression was observed in skin samples from psoriatic patients compared to healthy controls. This was accompanied by a parallel reduction of adenosine monophosphate-activated kinase (AMPK) expression and, more strikingly, by the disappearance of cells immunolabeled for its active, phosphorylated form (pAMPK). In both cases, analysis of the distribution of immunopositive cells revealed a shift towards reduced intensity of staining. In contrast, NAMPT expression was upregulated in psoriatic samples in line with its pro-inflammatory role. This was again more visible with an intensity-based distribution analysis that evidenced a shift towards more intensely immunostained cell populations. CONCLUSIONS: The present data correlate in the same samples the expression of SIRT1, pAMPK/AMPK and NAMPT in psoriasis and open the way for novel pharmacological targets in the treatment of the disease.

Targeting group II metabotropic glutamate (mGlu) receptors for the treatment of psychosis associated with Alzheimer's disease: selective activation of mGlu2 receptors amplifies beta-amyloid toxicity in cultured neurons, whereas dual activation of mGlu2 and mGlu3 receptors is neuroprotective.

Dual orthosteric agonists of metabotropic glutamate 2 (mGlu2) and mGlu3 receptors are being developed as novel antipsychotic agents devoid of the adverse effects of conventional antipsychotics. Therefore, these drugs could be helpful for the treatment of psychotic symptoms associated with Alzheimer's disease (AD). In experimental animals, the antipsychotic activity of mGlu2/3 receptor agonists is largely mediated by the activation of mGlu2 receptors and is mimicked by selective positive allosteric modulators (PAMs) of mGlu2 receptors. We investigated the distinct influence of mGlu2 and mGlu3 receptors in mixed and pure neuronal cultures exposed to synthetic ß-amyloid protein (Aß) to model neurodegeneration occurring in AD. The mGlu2 receptor PAM, N-4'-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride (LY566332), devoid of toxicity per se, amplified Aß-induced neurodegeneration, and this effect was prevented by the mGlu2/3 receptor antagonist (2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl)glycine (LY341495). LY566332 potentiated Aß toxicity regardless of the presence of glial mGlu3 receptors, but it was inactive when neurons lacked mGlu2 receptors. The dual mGlu2/3 receptor agonist, (-)-2-oxa-4-aminobicyclo[3.1.0]exhane-4,6-dicarboxylic acid (LY379268), was neuroprotective in mixed cultures via a paracrine mechanism mediated by transforming growth factor-ß1. LY379268 lost its protective activity in neurons grown with astrocytes lacking mGlu3 receptors, indicating that protection against Aß neurotoxicity was mediated entirely by glial mGlu3 receptors. The selective noncompetitive mGlu3 receptor antagonist, (3S)-1-(5-bromopyrimidin-2-yl)-N-(2,4-dichlorobenzyl)pyrrolidin-3-amine methanesulfonate hydrate (LY2389575), amplified Aß toxicity on its own, and, interestingly, unmasked a neurotoxic activity of LY379268, which probably was mediated by the activation of mGlu2 receptors. These data indicate that selective potentiation of mGlu2 receptors enhances neuronal vulnerability to Aß, whereas dual activation of mGlu2 and mGlu3 receptors is protective against Aß-induced toxicity.

Molecular Aspects of Cellular Dysfunction in Alzheimer's Disease: The Need for a Holistic View of the Early Pathogenesis.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, a socio-economic burden destined to worsen with increased population aging [...].

Protective effect of the sphingosine-1 phosphate receptor agonist siponimod on disrupted blood brain barrier function.

Sphingosine 1 phosphate (S1P) is a bioactive sphingolipid that exerts several functions in physiological and pathological conditions. The modulation of one of its receptors, S1P1, plays an important role in the egress of lymphocytes from lymph nodes and is a useful target in multiple sclerosis (MS) treatment. A new drug, siponimod (BAF-312) has been recently approved for the treatment of secondary progressive MS and has affinity for two S1P receptors, S1P1 and S1P5. The two receptors are expressed by endothelial cells that, as components of the blood-brain barrier (BBB), prevent the access of solutes and lymphocytes into the central nervous system, function often compromised in MS. Using an in vitro BBB model exposed to inflammatory cytokines (TNFα and IFNγ, 5 UI and 10 UI respectively), we evaluated the effects of BAF-312 (100 nM) on expression and function of endothelial tight junctional proteins (Zo-1 and claudin-5), regulation of transendothelial electrical resistance (TEER) and permeability to FITC-conjugated dextran. Zo-1 expression, as well as TEER values, were promptly recovered (24 h) when both S1P1 and S1P5 were activated by BAF-312. In contrast, at this time point, activation of S1P5 with the selective agonist UC-42-WP04 (300 nM) or with BAF-312, under blockade of S1P1 with the selective antagonist NIBR-0213 (1 µM), resulted in recovery of expression and localization of claudin-5 and reduction of TNFα/INFγ-induced expression of metalloproteinase 9. Only after a prolonged BAF-312 exposure (48 h), S1P1 was involved through activation of the PI3K/Akt pathway. The PI3K inhibitor LY294002 (10 µM) prevented in fact the effects of BAF-312 on all the parameters examined. In conclusion, BAF-312, by modulating both S1P1 and S1P5, may strengthen BBB properties, thus providing additional effects in the treatment of MS.

SIRT1-Dependent Upregulation of BDNF in Human Microglia Challenged with Aß: An Early but Transient Response Rescued by Melatonin.

Microglia represent a first-line defense in the brain. However, in pathological conditions such as Alzheimer's disease (AD), a pro-inflammatory switch may occur, leading to loss of protective functions. Using the human microglial cell line HMC3, we showed that exposure to low concentrations of ß-amyloid peptide 1-42 (Aß42; 0.2 µM) initially (6 h) upregulated anti-inflammatory markers interleukin (IL)-4, IL-13, and brain-derived neurotrophic factor (BDNF). BDNF increase was prevented by selective inhibition of SIRT1 with EX527 (2 µM). Accordingly, these early effects were accompanied by a significant Aß42-induced increase of SIRT1 expression, nuclear localization, and activity. SIRT1 modulation involved adenosine monophosphate-regulated kinase (AMPK), which was promptly (30 min) phosphorylated by Aß42, while the AMPK inhibitor BML-275 (2 µM) attenuated Aß42-induced SIRT1 increase. Initially observed microglial responses appeared transient, as microglial features changed when exposure to Aß42 was prolonged (0.2 µM for 72 h). While SIRT1 and BDNF levels were reduced, the expression of inflammatory markers IL-1ß and tumor necrosis factor (TNF)-α increased. This coincided with a rise in NF-kB nuclear localization. The effects of melatonin (1 µM) on prolonged microglial exposure to Aß42 were analyzed for their protective potential. Melatonin was able to prolong SIRT1 and BDNF upregulation, as well as to prevent NF-kB nuclear translocation and acetylation. These effects were sensitive to the melatonin receptor antagonist, luzindole (25 µM). In conclusion, our data define an early microglial defensive response to Aß42, featuring SIRT1-mediated BDNF upregulation that can be exogenously modulated by melatonin, thus identifying an important target for neuroprotection.

Long-term incubation with beta-amyloid peptides impairs endothelium-dependent vasodilatation in isolated rat basilar artery.

Alzheimer's disease is associated to a cerebral amyloid angiopathy with dysregulation of cerebral blood flow (CBF). In vitro studies have shown that short-term application of beta-amyloid (Abeta) peptides to isolated vessels affects vascular tone within 1h, but no studies have examined the effect of long-term incubation with Abeta. Here we evaluate the effect of Abeta((1-40)) and Abeta((25-35)) in rat basilar artery for up to 24h. Basilar artery segments were incubated with 25microeta((1-40)) or Abeta((25-35)), for 6 or 24h. After treatment, arteries were mounted in a wire myograph, in physiological salt solution gassed with O(2)/CO(2), in the absence of Abeta, and challenged with vasoconstrictors and vasodilators. Vasomotor responses were not significantly changed by 6h treatment with Abeta peptides whereas 24h treatment with either Abeta((25-35)) or Abeta((1-40)) increased vasoconstriction to 5-hydroxytryptamine (5-HT) and reduced endothelium-dependent vasodilatation to acetylcholine (ACh). Analysis of endothelial cells did not show apoptotic changes associated to endothelial dysfunction, as assessed by TUNEL immunostaining and examination of nuclear morphology, but basal phosphorylation of endothelial nitric oxide synthase (at serine 1177) appeared reduced. These data suggest that long incubation with Abeta peptides induces an alteration of endothelial function in isolated basilar artery, involving eNOS activity without changing cell morphology. This endothelial dysfunction may play a role in the pathogenesis of CBF dysregulation occurring in cerebral amyloid angiopathy and Alzheimer's disease.

Modulation of C6 Glioma Cell Proliferation by Ureido-Calix[8]arenes.

Calixarenes are synthetic macrocyclic compounds that may serve as scaffolds for biologically active molecules and have been proposed as potential anticancer agents. We synthesized a ureido-calix[8]arene carrying N-acetyl-D-glucosamine residue (compound 1) and had previously demonstrated that it inhibits C6 glioma cell migration and proliferation, with divergent mechanisms. In the present work we explored in more detail the antiproliferative effect of compound 1, comparing it to related compounds lacking either the sugar moieties (compound 2), the multiple ureido groups (compound 3) or both (compound 4). The results show that the action of compound 1 is independent of the N-acetyl-D-glucosamine residues, requires the presence of multiple ureido groups and does not seem to involve focal adhesion kinase signaling. Inhibition of proliferation is reduced by preincubation with epidermal growth factor (EGF) and vascular endothelial growth factor (20 ng/ml) with compound 1, and extracellular-related kinase phosphorylation is reduced by treatment with compound 1 in both basal and EGF-stimulated conditions, suggesting that the observed effect depends on a direct interference with growth factor signaling.

The Treatment of Impaired Wound Healing in Diabetes: Looking among Old Drugs.

Chronic wounds often occur in patients with diabetes mellitus due to the impairment of wound healing. This has negative consequences for both the patient and the medical system and considering the growing prevalence of diabetes, it will be a significant medical, social, and economic burden in the near future. Hence, the need for therapeutic alternatives to the current available treatments that, although various, do not guarantee a rapid and definite reparative process, appears necessary. We here analyzed current treatments for wound healing, but mainly focused the attention on few classes of drugs that are already in the market with different indications, but that have shown in preclinical and few clinical trials the potentiality to be used in the treatment of impaired wound healing. In particular, repurposing of the antiglycemic agents dipeptidylpeptidase 4 (DPP4) inhibitors and metformin, but also, statins and phenyotin have been analyzed. All show encouraging results in the treatment of chronic wounds, but additional, well designed studies are needed to allow these drugs access to the clinics in the therapy of impaired wound healing.

The Ambiguous Role of Microglia in Aß Toxicity: Chances for Therapeutic Intervention.

Amyloid-ß (Aß) has long been shown to be critical in Alzheimer's disease pathophysiology. Microglia contributes to the earliest responses to Aß buildup, by direct interaction through multiple receptors. Microglial cells operate Aß clearance and trigger inflammatory/regenerative processes that take place in the long years of silent disease progression that precede symptomatic appearance. But in time and with aging, the fine balance between pro- and anti-inflammatory activity of microglia deranges, negatively impacting its Aß-clearing ability. Furthermore, in recent years, microglial activation has proven to be much more complex than the mere dichotomic pro/antiinflammatory polarization previously accepted. Microglia can display a wide spectrum of phenotypes, which can even be mixed. On these bases, it is evident that while pharmacological intervention aiding microglia to prolong its ability to cope with Aß buildup could be extremely relevant, its feasibility is hampered by such high complexity, which still needs to be completely understood.