Sara Merlo

Targeting group II Metabotropic Glutamate (mGlu) receptors for the treatment of psychosis associated with alzheimer's disease: Selective activation of mGlu2 receptors amplifies β-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 Alzheimers 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 β-amyloid protein (Aβ) 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 Aβ-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 Aβ 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-β1. LY379268 lost its protective activity in neurons grown with astrocytes lacking mGlu3 receptors, indicating that protection against Aβ 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 Aβ 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 Aβ, whereas dual activation of mGlu2 and mGlu3 receptors is protective against Aβ-induced toxicity.

Differential involvement of estrogen receptorα and estrogen receptorβ in the healing promoting effect of estrogen in human keratinocytes

Estrogen affects proliferation and migration of different skin components, thus influencing wound healing processes. The human keratinocyte cell line NCTC 2544 has been used to examine the effects of estrogen, dissect its mechanism of action and characterize receptor subtypes involved. Western blot and immunocytochemical analyses confirmed the expression of estrogen receptors (ERs) alpha and beta, with prevalence in the nuclear and extranuclear compartment, for ER alpha and ER beta respectively. Treatment with 10 nM 17beta-estradiol (17beta-E(2)) and the ER alpha and ER beta selective agonists, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT; 100 nM), and diarylpropionitrile (DPN; 1 nM) produced a slight but significant increase in cell proliferation, as by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine incorporation assays, only after a long-term treatment (96 h). Analysis of cell migration by a scratch wound assay showed that 17beta-E(2) (10 nM) accelerated migration between 5 and 24 h after scratching, an effect confirmed by the transwell migration assay. PPT and DPN elicited similar effects. Pre-treatment with the mitogen-activated protein kinase inhibitor, U0126 (1 microM), abolished the ability of 17beta-E(2) and DPN, but not of PPT, to accelerate wound closure. TGF-beta1 (10 ng/ml) produced a similar positive effect on wound closure and the TGF-beta1 receptor antagonist, SB431542 (10 microM), reduced the ability of 17beta-E(2) and PPT to accelerate cell migration, but did not modify DPN effect. It is suggested that estrogen positively affects in vitro wound healing by stimulating cell proliferation after long-term exposure but mainly by accelerating cell migration within a few hours from treatment. Selective activation of ER beta may result in favorable stimulation of wound healing without any increase of transforming growth factor-beta1 production.

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

Estrogen is known to affect different aspects of β-amyloid (Aβ) synthesis and degradation. The present work was undertaken to evaluate specifically whether matrix metalloproteinases (MMP) -2 and -9 are involved in Aβ degradation induced by estrogen and whether this is relevant to estrogen-induced neuroprotection. In SH-SY5Y human neuroblastoma cells, 10 nM 17β-estradiol (17β-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 ERβ agonists (PPT, DPN) and antagonists (MPP, PHTPP). 17β-E2 significantly increases the degradation of Aβ, either transferred with the conditioned medium of H4-APPSw human neuroglioma cells, engineered to overproduce Aβ(1-40) and Aβ(1-42), or exogenously added as 2 μM Aβ(1-42). Both these effects are completely prevented by preexposure to the broad spectrum MMP inhibitor GM6001 (5 μM). Importantly, the 17β-E2-induced rescue of neuroblastoma cells challenged with 2 μM Aβ(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 estrogens neuroprotective effect.

Integrins mediate β-amyloid-induced cell-cycle activation and neuronal death

Early intracellular events responsible for cell‐cycle induction by β‐amyloid (Aβ) in neurons have not been identified yet. Extracellular signal–regulated kinases 1/2 (ERK1/2) have been identified in this pathway, and inhibition of ERK activity prevents cell‐cycle activation and reduces neuronal death induced by Aβ. To identify upstream events responsible for ERK activation, attention has been focused on integrins. Treatment of SH‐SY5Y cells, differentiated by long‐term exposure to 10 μM retinoic acid with a neutralizing anti‐α1‐integrin antibody significantly reduced Aβ‐induced neuronal death. However, cell‐cycle analysis showed that treatment with anti‐α1‐integrin per se produced changes in the distribution of cell populations, thus hampering any effect on Aβ‐induced cell‐cycle activation. 4‐Amino‐5‐(4‐chlorophenyl)‐7(t‐butyl)pyrazol(3,4‐D)pyramide, an inhibitor of src protein kinases that colocalizes with focal adhesion kinase (FAK) and is involved in integrin signaling, was effective in reducing activation of the cell cycle and preventing induction of neuronal death by Aβ while inhibiting ERK1/2 phosphorylation. Similar results were obtained when FAK expression was down‐regulated by siRNA silencing. The present study identifies a sequence of early events in the toxic effect of Aβ in neuronal cultures that involves interaction with integrins, activation of FAK/src, enhanced phosphorylation of ERK1/2, and induction of the cell cycle, all leading to neuronal death.

Alzheimer's disease: brain expression of a metabolic disorder?

Alzheimers disease (AD) is the most common form of dementia and is of rapidly increasing health, social and economic impact. Recent evidence suggests a strict link between metabolic disorders and AD. In the last decade much attention has focused specifically on the connection between dysfunction of lipid metabolism and AD. Here we discuss aspects of lipid regulation, including changes in cholesterol levels, function of apolipoproteins and leptin, and how these relate to AD pathogenesis. Despite the vast literature available, many aspects still need clarification. Nevertheless, the route is already delineated to directly connect aspects of lipid regulation to AD. This could represent a starting point to identify novel potential targets for a preventive and/or treatment strategy of the disease.

Enhanced expression of ERα in astrocytes modifies the response of cortical neurons to β-amyloid toxicity

Estrogen receptor alpha (ERalpha) is over-expressed in reactive glia under conditions of neuronal damage. To elucidate the functional significance of ERalpha overexpression, an in vitro model of reactive astrocytes with enhanced expression of ERalpha was obtained by growth in G5 culture supplement. Exposure of cortical neurons to beta-amyloid in the presence of either conditioned medium from reactive astrocytes previously treated with 17beta-estradiol (17betaE2) or transferring of 17betaE2-pretreated astrocytes, caused a greater neuroprotective effect compared to the respective control conditions, although reactive glia resulted being per se neuroprotective. Blockade of ERalpha overexpression by the ER antagonist ICI182,780 was not successful as ICI182,780 behaved as an agonist. However, complete prevention of 17betaE2 effect by ICI182,780 produced an increased sensitivity of neurons to beta-amyloid toxicity. A similar effect was observed when ERalpha knock-down was induced by siRNA. It is suggested that increased ERalpha expression in reactive glia may have a role in limiting neuronal damage.

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

Alzheimers 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 Alzheimers disease.

Estrogen Receptors and Type 1 Metabotropic Glutamate Receptors Are Interdependent in Protecting Cortical Neurons against β-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 β-amyloid peptide. Both receptors were present in neurons, whereas only ERα but not mGlu1 receptors were found in astrocytes. Addition of 17β-estradiol (17βE2) 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 17βE2. The selective ERβ 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 17βΕ2 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 17βΕ2 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 17βE2, 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.

Fluoxetine Prevents Aβ1-42-Induced Toxicity via a Paracrine Signaling Mediated by Transforming-Growth-Factor-β1

Selective reuptake inhibitors (SSRIs), such as fluoxetine and sertraline, increase circulating Transforming-Growth-Factor-β1 (TGF-β1) levels in depressed patients, and are currently studied for their neuroprotective properties in Alzheimer’s disease. TGF-β1 is an anti-inflammatory cytokine that exerts neuroprotective effects against β-amyloid (Aβ)-induced neurodegeneration. In the present work, the SSRI, fluoxetine, was tested for the ability to protect cortical neurons against 1 μM oligomeric Aβ1-42-induced toxicity. At therapeutic concentrations (100 nM–1 μM), fluoxetine significantly prevented Aβ-induced toxicity in mixed glia-neuronal cultures, but not in pure neuronal cultures. Though to a lesser extent, also sertraline was neuroprotective in mixed cultures, whereas serotonin (10 nM–10 μM) did not mimick fluoxetine effects. Glia-conditioned medium collected from astrocytes challenged with fluoxetine protected pure cortical neurons against Aβ toxicity. The effect was lost in the presence of a neutralizing antibody against TGF-β1 in the conditioned medium, or when the specific inhibitor of type-1 TGF-β1 receptor, SB431542, was added to pure neuronal cultures. Accordingly, a 24 h treatment of cortical astrocytes with fluoxetine promoted the release of active TGF-β1 in the culture media through the conversion of latent TGF-β1 to mature TGF-β1. Unlike fluoxetine, both serotonin and sertraline did not stimulate the astrocyte release of active TGF-β1. We conclude that fluoxetine is neuroprotective against Aβ toxicity via a paracrine signaling mediated by TGF-β1, which does not result from a simplistic SERT blockade.

Inhibition of rat glioma cell migration and proliferation by a calix[8]arene scaffold exposing multiple GlcNAc and ureido functionalities

β1,4‐Galactosyltransferases (β1,4‐GalTase) exposed on the cell surface are involved in cell migration. Specifically, β1,4‐GalTase V is highly expressed in glioma and promotes invasion, growth, and survival of glioma cells. A glycocalix[8]arene exposing N‐acetylglucosamine (GlcNAc) residues (compound 1) inhibited rat C6 glioma cell migration as assessed in a scratch wound model. This effect was related to inhibition of focal adhesion kinase phosphorylation, measured by western blot analysis, and specifically observed in the area bordering the scratch wound. Compound 1 inhibited also C6 cell proliferation, an effect unrelated to its ability to interact with GalTase as it was mimicked by different calix[8]arene derivatives, all characterized by multivalency and ureido groups. Compound 1 did not induce apoptotic death, but caused a different distribution of C6 cells within the cell cycle. The results here reported identify compound 1 as a molecule able to exert inhibitory effects on C6 cell migration and proliferation, independently, because of distinct components in its structure.