Maria Laura Giuffrida

β-Amyloid Monomers Are Neuroprotective

The 42-aa-long β-amyloid protein—Aβ1-42—is thought to play a central role in the pathogenesis of Alzheimers disease (AD) (Walsh and Selkoe, 2007). Data from AD brain (Shankar et al., 2008), transgenic APP (amyloid precursor protein)-overexpressing mice (Lesné et al., 2006), and neuronal cultures treated with synthetic Aβ peptides (Lambert et al., 1998) indicate that self-association of Aβ1-42 monomers into soluble oligomers is required for neurotoxicity. The function of monomeric Aβ1-42 is unknown. The evidence that Aβ1-42 is present in the brain and CSF of normal individuals suggests that the peptide is physiologically active (Shoji, 2002). Here we show that synthetic Aβ1-42 monomers support the survival of developing neurons under conditions of trophic deprivation and protect mature neurons against excitotoxic death, a process that contributes to the overall neurodegeneration associated with AD. The neuroprotective action of Aβ1-42 monomers was mediated by the activation of the PI-3-K (phosphatidylinositol-3-kinase) pathway, and involved the stimulation of IGF-1 (insulin-like growth factor-1) receptors and/or other receptors of the insulin superfamily. Interestingly, monomers of Aβ1-42 carrying the Arctic mutation (E22G) associated with familiar AD (Nilsberth et al., 2001) were not neuroprotective. We suggest that pathological aggregation of Aβ1-42 may also cause neurodegeneration by depriving neurons of the protective activity of Aβ1-42 monomers. This “loss-of-function” hypothesis of neuronal death should be taken into consideration when designing therapies aimed at reducing Aβ burden.

TGF-β1 pathway as a new target for neuroprotection in alzheimer's disease

Alzheimers disease (AD) is a neurodegenerative disorder that affects more than 37 million people worldwide. Current drugs for AD are only symptomatic, but do not interfere with the underlying pathogenic mechanisms of the disease. AD is characterized by the presence of ß‐amyloid (Aβ) plaques, neurofibrillary tangles, and neuronal loss. The identification of the molecular determinants underlying AD pathogenesis is a fundamental step to design new disease‐modifying drugs. Recently, a specific impairment of transforming‐growth‐factor‐β1 (TGF‐β1) signaling pathway has been demonstrated in AD brain. The deficiency of TGF‐β1 signaling has been shown to increase both Aβ accumulation and Aβ‐induced neurodegeneration in AD models. The loss of function of TGF‐ß1 pathway seems also to contribute to tau pathology and neurofibrillary tangle formation. Growing evidence suggests a neuroprotective role for TGF‐β1 against Aβ toxicity both in vitro and in vivo models of AD. Different drugs, such as lithium or group II mGlu receptor agonists are able to increase TGF‐β1 levels in the central nervous system (CNS), and might be considered as new neuroprotective tools against Aβ‐induced neurodegeneration. In the present review, we examine the evidence for a neuroprotective role of TGF‐β1 in AD, and discuss the TGF‐β1 signaling pathway as a new pharmacological target for the treatment of AD.

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.

Aβ(25–35) and its C‐ and/or N‐blocked derivatives: Copper driven structural features and neurotoxicity

The toxic properties of β‐amyloid protein, Aβ(1–42), the major component of senile plaques in Alzheimers disease, depend on nucleation‐dependent oligomerization and aggregation. In addition, Aβ(1–42) toxicity is favored by the presence of trace metals, which affect the secondary structure of the peptide. A peptide comprising 11 residues within Aβ(1–42) [Aβ(25–35)] aggregates and retains the neurotoxic activity of Aβ(1–42). We have used both Aβ(25–35) and its C‐amidated or N‐acetylated/C‐amidated derivatives to investigate the role of copper(II) in modulating the conformation and aggregation state as well as the neurotoxic properties of amyloid peptides. Electrospray ionization mass spectrometry (ESI‐MS) and electron paramagnetic resonance (EPR) measurements were performed to verify the formation of copper(II)/Aβ(25–35) complexes and to determine the coordination mode, respectively. Aβ(25–35) and its derivatives were analyzed by circular dichroism spectroscopy to assess their secondary structure, subjected to thioflavine‐T (Th‐T) binding assay to reveal β‐sheet structured aggregates formation, and imaged by scanning force microscopy. Toxicity was assessed on mature cultures of rat cortical neurons. We found that β‐sheet‐structured species of Aβ(25–35) were neurotoxic, whereas the random‐coil‐structured derivatives were devoid of effect. Interestingly, copper promoted the random‐coil/β‐sheet transition of Aβ(25–35), with ensuing peptide toxicity, but it induced the toxicity of the N‐acetylated/C‐amidated derivative without affecting peptide folding. Moreover, copper did not influence either the folding or the activity of the C‐amidated Aβ(25–35), suggesting that blockade of the C‐terminus of Aβ peptides might be sufficient to prevent Aβ toxicity.

Carnosine interaction with nitric oxide and astroglial cell protection

The neuropeptide carnosine (β‐amyloid peptide aggregation has been demonstrated. Carnosine protection against peroxynitrite damage is particularly relevant, but until now there has been no evidence of any direct interaction with nitric oxide. In this study we examined the protection that carnosine provides against nitric oxide (NO)–induced cell death in primary rat astroglial cell cultures treated with lipopolysaccharide (LPS) and interferon gamma (INFγ), a well‐known neurotoxic proinflammatory condition. A correlation was found between cell protection and NO free‐radical scavenging activity of carnosine. Moreover, by competitive spectrophotometric measurement and electrospray mass spectrometry analysis in cell‐free experiments, we demonstrated a direct interaction of the dipeptide with NO. A comparison of carnosine with its homologues or derivatives (homocarnosine and carcinine) as well as with its amino acid constituents (L‐histidine and β‐alanine) highlighted that only histidine showed significant scavenging activity. Therefore, carnosine shows direct NO‐trapping ability and may be a valuable multifunctional molecule in the treatment of neurodegenerative disorders.

Neurotoxic properties of the anabolic androgenic steroids nandrolone and methandrostenolone in primary neuronal cultures.

Anabolic‐androgenic steroid (AAS) abuse is associated with multiple neurobehavioral disturbances. The sites of action and the neurobiological sequels of AAS abuse are unclear at present. We investigated whether two different AASs, nandrolone and methandrostenolone, could affect neuronal survival in culture. The endogenous androgenic steroid testosterone was used for comparison. Both testosterone and nandrolone were neurotoxic at micromolar concentrations, and their effects were prevented by blockade of androgen receptors (ARs) with flutamide. Neuronal toxicity developed only over a 48‐hr exposure to the steroids. The cell‐impermeable analogues testosterone‐BSA and nandrolone‐BSA, which preferentially target membrane‐associated ARs, were also neurotoxic in a time‐dependent and flutamide‐sensitive manner. Testosterone‐BSA and nandrolone‐BSA were more potent than their parent compounds, suggesting that membrane‐associated ARs were the relevant sites for the neurotoxic actions of the steroids. Unlike testosterone and nandrolone, toxicity by methandrostenolone and methandrostenolone‐BSA was insensitive to flutamide, but it was prevented by the glucocorticoid receptor (GR) antagonist RU‐486. Methandrostenolone‐BSA was more potent than the parent compound, suggesting that its toxicity relied on the preferential activation of putative membrane‐associated GRs. Consistently with the evidence that membrane‐associated GRs can mediate rapid effects, a brief challenge with methandrostenolone‐BSA was able to promote neuronal toxicity. Activation of putative membrane steroid receptors by nontoxic (nanomolar) concentrations of either nandrolone‐BSA or methandrostenolone‐BSA became sufficient to increase neuronal susceptibility to the apoptotic stimulus provided by β‐amyloid (the main culprit of AD). We speculate that AAS abuse might facilitate the onset or progression of neurodegenerative diseases not usually linked to drug abuse.

Design and synthesis of new trehalose‐conjugated pentapeptides as inhibitors of Aβ(1–42) fibrillogenesis and toxicity

Aggregation of the amyloid Aβ peptide and its accumulation into insoluble deposits (plaques) are believed to be the main cause of neuronal dysfunction associated with Alzheimers disease (AD); small molecules that can interfere with the Aβ amyloid fibril formation are therefore of interest for a potential therapeutic strategy. Three new trehalose‐conjugated peptides of the well known β‐sheet breaker peptide iAβ5p, were synthesized. The disaccharide was covalently attached to different sites of the LPFFD peptide chain, i.e. at the N‐terminus, C‐terminus or at the Asp side chain. CD spectroscopy in different solvents was used to assess changes in the peptide conformation of these compounds. The effects of these glycopeptides on the self‐assembly and morphology of Aβ aggregates were investigated by ThT fluorescence assay and dynamic Scanning Force Microscopy, respectively. All the synthesized compounds were tested as inhibitors of Aβ toxicity toward pure cultures of rat cortical neurons. Copyright

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.

Beta-Amyloid Monomer and Insulin/IGF-1 Signaling in Alzheimer's Disease

Alzheimers disease is the most common form of dementia among older people and is still untreatable. While β-amyloid protein is recognized as the disease determinant with a pivotal role in inducing neuronal loss and dementia, an impaired brain insulin signaling seems to account in part for the cognitive deficit associated with the disease. The origin of this defective signaling is uncertain. Accumulating toxic species of β-amyloid, the so-called oligomers, has been proposed to be responsible for downregulation of neuronal insulin receptors. We have found that the nontoxic form of β-amyloid, the monomer, is able to activate insulin/insulin-like growth factor-1 (IGF-1) receptor signaling and thus behaves as a neuroprotectant agent. Our suggestion is that depletion of β-amyloid monomers, occurring in the preclinical phase of Alzheimers disease, might be the cause of early insulin/IGF-1 signaling disturbances that anticipate cognitive decline.

Ratiometric fluorescence sensing and cellular imaging of Cu2+ by a new water soluble trehalose-naphthalimide based chemosensor

A new turn-on Cu2+ fluorescent sensor (CST) having a trehalose moiety, which confers a relatively large solubility in water, has been synthesized. The chemosensor is therefore suitable for studies in aqueous solution. Full potentiometric and UV-vis characterization evidence that at physiological pH CST forms with Cu2+ a species with a 1:1 stoichiometry allowing for a straightforward correlation between CST response and copper(II) concentration. The presence of the trehalose unit does not negatively affect the selectivity of CST for Cu2+ over a series of metal ions of interest as proven by fluorescence measurements. The novel chemosensor, tested in differentiated neuroblastoma SH-SY5Y cells, is able to detect Cu2+ in the extracellular region, as well as to track copper transfer processes upon cell stimulation induced by cellular depolarization.