Alfredo Cagnotto

Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis.

Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)–expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R–expressing inflammatory cells.

Functionalization of liposomes with ApoE-derived peptides at different density affects cellular uptake and drug transport across a blood-brain barrier model

A promising strategy to enhance blood-brain barrier penetration by drugs is the functionalization of nanocarriers with uptake-facilitating ligands. We studied the cellular uptake, by cultured RBE4 brain capillary endothelial cells, of nanoliposomes (NLs) covalently coupled with monomer or tandem dimer of apolipoprotein E (ApoE)-derived peptides (residues 141-150), at various densities. NLs without functionalization did not show either relevant membrane accumulation or cellular uptake, as monitored by confocal microscopy and quantified by fluorescence-activated cell sorting. Functionalization with peptides mediated an efficient NLs uptake that increased with peptide density; NLs carrying monomeric peptide performed the best. Moreover, we studied the ability of ApoE-NLs to enhance the transport of a drug payload through a RBE4 cell monolayer. The permeability of a tritiated curcumin derivative was enhanced after its entrapment into ApoE-NLs, in particular those functionalized with the dimer (+83% with respect to free drug, P < 0.01). Thus, these NLs appear particularly suitable for implementing further strategies for drug brain targeting.

Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity

Pharmacological blockade of NR2B-containing N-methyl-d-aspartate receptors (NMDARs) during epileptogenesis reduces neurodegeneration provoked in the rodent hippocampus by status epilepticus. The functional consequences of NMDAR activation are crucially influenced by their synaptic vs extrasynaptic localization, and both NMDAR function and localization are dependent on the presence of the NR2B subunit and its phosphorylation state. We investigated whether changes in NR2B subunit phosphorylation, and alterations in its neuronal membrane localization and cellular expression occur during epileptogenesis, and if these changes are involved in neuronal cell loss. We also explored NR2B subunit changes both in the acute phase of status epilepticus and in the chronic phase of spontaneous seizures which encompass the epileptogenesis phase. Levels of Tyr1472 phosphorylated NR2B subunit decreased in the post-synaptic membranes from rat hippocampus during epileptogenesis induced by electrical status epilepticus. This effect was concomitant with a reduced interaction between NR2B and post-synaptic density (PSD)-95 protein, and was associated with decreased CREB phosphorylation. This evidence suggests an extra-synaptic localization of NR2B subunit in epileptogenesis. Accordingly, electron microscopy showed increased NR2B both in extra-synaptic and pre-synaptic neuronal compartments, and a concomitant decrease of this subunit in PSD, thus indicating a shift in NR2B membrane localization. De novo expression of NR2B in activated astrocytes was also found in epileptogenesis indicating ectopic receptor expression in glia. The NR2B phosphorylation changes detected at completion of status epilepticus, and interictally in the chronic phase of spontaneous seizures, are predictive of receptor translocation from synaptic to extrasynaptic sites. Pharmacological blockade of NR2B-containing NMDARs by ifenprodil administration during epileptogenesis significantly reduced pyramidal cell loss in the hippocampus, showing that the observed post-translational and cellular changes of NR2B subunit contribute to excitotoxicity. Therefore, pharmacological targeting of misplaced NR2B-containing NMDARs, or prevention of these NMDAR changes, should be considered to block excitotoxicity which develops after various pro-epileptogenic brain injuries.

Synthesis of New Arylpiperazinylalkylthiobenzimidazole, Benzothiazole, or Benzoxazole Derivatives as Potent and Selective 5-HT1A Serotonin Receptor Ligands†

A series of new compounds containing a benzimidazole, benzothiazole, or benzoxazole nucleus linked to an arylpiperazine by different thioalkyl chains was prepared. They were tested in radioligand binding experiments to evaluate their affinity for 5-HT 1A and 5-HT 2A serotonergic, alpha 1 adrenergic, D1, and D2 dopaminergic receptors. Many of tested compounds showed an interesting binding profile; in particular, 36 displayed very high 5-HT 1A receptor affinity and selectivity over all the other investigated receptors. Selected compounds, evaluated in functional assays, showed antagonistic or partial agonistic activity at 5-HT 1A receptor. An extensive conformational research using both NMR and modeling techniques indicated that extended conformations predominated in vacuum, in solution and during interactions with 5-HT 1A receptor. Finally, the elaborated binding mode of selected compounds at 5-HT 1A receptor was used to explain the influence of spacer length on ligands affinity.

The Molecular Assembly of Amyloid Aβ Controls Its Neurotoxicity and Binding to Cellular Proteins

Accumulation of β-sheet-rich peptide (Aβ) is strongly associated with Alzheimers disease, characterized by reduction in synapse density, structural alterations of dendritic spines, modification of synaptic protein expression, loss of long-term potentiation and neuronal cell death. Aβ species are potent neurotoxins, however the molecular mechanism responsible for Aβ toxicity is still unknown. Numerous mechanisms of toxicity were proposed, although there is no agreement about their relative importance in disease pathogenesis. Here, the toxicity of Aβ 1–40 and Aβ 1–42 monomers, oligomers or fibrils, was evaluated using the N2a cell line. A structure-function relationship between peptide aggregation state and toxic properties was established. Moreover, we demonstrated that Aβ toxic species cross the plasma membrane, accumulate in cells and bind to a variety of internal proteins, especially on the cytoskeleton and in the endoplasmatic reticulum (ER). Based on these data we suggest that numerous proteins act as Aβ receptors in N2a cells, triggering a multi factorial toxicity.

Discovery of a new class of potential multifunctional atypical antipsychotic agents targeting dopamine D3 and serotonin 5-HT1A and 5-HT2A receptors: design, synthesis, and effects on behavior.

Dopamine D(3) antagonism combined with serotonin 5-HT(1A) and 5-HT(2A) receptor occupancy may represent a novel paradigm for developing innovative antipsychotics. The unique pharmacological features of 5i are a high affinity for dopamine D(3), serotonin 5-HT(1A) and 5-HT(2A) receptors, together with a low affinity for dopamine D(2) receptors (to minimize extrapyramidal side effects), serotonin 5-HT(2C) receptors (to reduce the risk of obesity under chronic treatment), and for hERG channels (to reduce incidence of torsade des pointes). Pharmacological and biochemical data, including specific c-fos expression in mesocorticolimbic areas, confirmed an atypical antipsychotic profile of 5i in vivo, characterized by the absence of catalepsy at antipsychotic dose.

Functionalization with ApoE-derived peptides enhances the interaction with brain capillary endothelial cells of nanoliposomes binding amyloid-beta peptide

Nanoliposomes containing phosphatidic acid or cardiolipin are able to target in vitro with very high affinity amyloid-β (Aβ), a peptide whose overproduction and progressive aggregation in the brain play a central role in the pathogenesis of Alzheimers disease. However, the presence of the blood-brain barrier (BBB) severely limits the penetration of either drugs or drug vehicles (nanoparticles) to the brain. Therefore, there is a need to develop and design approaches specifically driving nanoparticles to brain in a better and effective way. The aim of the present investigation is the search of a strategy promoting the interaction of liposomes containing acidic phospholipids with brain capillary endothelial cells, as a first step toward their passage across the BBB. We describe the preparation and physical characterization of nano-sized liposomes decorated with peptides derived from apolipoprotein E and characterize their interaction with human immortalized brain capillary cells cultured in vitro (hCMEC/D3). For this purpose, we synthesized two ApoE-derived peptides (the fragment 141-150 or its tandem dimer) containing a cysteine residue at the C-terminus and decorated NL by exploiting the cysteine reaction with a maleimide-group on the nanoparticle surface. NL without ApoE functionalization did not show either relevant membrane accumulation or cellular uptake, as monitored by confocal microscopy using fluorescently labeled nanoliposomes or quantifying the cell-associated radioactivity of isotopically labeled nanoliposomes. The uptake of nanoliposomes by cell monolayers was enhanced by ApoE-peptide-functionalization, and was higher with the fragment 141-150 than with its tandem dimer. The best performance was displayed by nanoliposomes containing phosphatidic acid and decorated with the ApoE fragment 141-150. Moreover, we show that the functionalization of liposomes containing acidic phospholipids with the ApoE fragment 141-150 scarcely affects their reported ability to bind Aβ peptide in vitro. These are important and promising features for the possibility to use these nanoliposomes for the targeting of Aβ in the brain districts.

Multifunctional Liposomes Reduce Brain β-Amyloid Burden and Ameliorate Memory Impairment in Alzheimer's Disease Mouse Models

Alzheimers disease is characterized by the accumulation and deposition of plaques of β-amyloid (Aβ) peptide in the brain. Given its pivotal role, new therapies targeting Aβ are in demand. We rationally designed liposomes targeting the brain and promoting the disaggregation of Aβ assemblies and evaluated their efficiency in reducing the Aβ burden in Alzheimers disease mouse models. Liposomes were bifunctionalized with a peptide derived from the apolipoprotein-E receptor-binding domain for blood–brain barrier targeting and with phosphatidic acid for Aβ binding. Bifunctionalized liposomes display the unique ability to hinder the formation of, and disaggregate, Aβ assemblies in vitro (EM experiments). Administration of bifunctionalized liposomes to APP/presenilin 1 transgenic mice (aged 10 months) for 3 weeks (three injections per week) decreased total brain-insoluble Aβ1–42 (−33%), assessed by ELISA, and the number and total area of plaques (−34%) detected histologically. Also, brain Aβ oligomers were reduced (−70.5%), as assessed by SDS-PAGE. Plaque reduction was confirmed in APP23 transgenic mice (aged 15 months) either histologically or by PET imaging with [11C]Pittsburgh compound B (PIB). The reduction of brain Aβ was associated with its increase in liver (+18%) and spleen (+20%). Notably, the novel-object recognition test showed that the treatment ameliorated mouse impaired memory. Finally, liposomes reached the brain in an intact form, as determined by confocal microscopy experiments with fluorescently labeled liposomes. These data suggest that bifunctionalized liposomes destabilize brain Aβ aggregates and promote peptide removal across the blood–brain barrier and its peripheral clearance. This all-in-one multitask therapeutic device can be considered as a candidate for the treatment of Alzheimers disease.

[3H](+)-Pentazocine binding to rat brain σ1 receptors

Abstract [3H](+)-Pentazocine binding has been characterized in the rat brain. It binds to a single population of binding sites with affinity of about 7 nM and density of 280 fmol/mg protein. [3H](+)-Pentazocine binding is not enriched in the crude synaptic membrane, being about 1 6 of what we found in the crude membrane preparation. The binding, like that for other σ ligands, was enriched in the microsomal and nuclear fractions. The inhibition by haloperidol, proadifen and d-fenfluramine was the same in the crude synaptic membrane, nuclear and microsomal fractions, suggesting that [3H](+)-pentazocine binds to a homogeneous protein in the different subcellular fractions. Our pharmacological characterization using 45 different drugs suggests that the [3H](+)-pentazocine binding site in rat brain differs from other σ ligands, like N-propyl-3-(3-hydroxyphenyl)piperidine ([3H](+)-3PPP), N,N′-di(o-tolyl)guanidine ([3H]DTG) and (+)-N-allylnormetazocine ([3H](+)-SKF10,047). [3H](+)-Pentazocine binding in rat brain is inhibited by σ compounds and some cytochrome P450 ligands, like proadifen and 1-[2-[bis(4-fluorophenyl) methoxy]ethyl]-4-[3-phenylpropyl] piperazine (GBR 12909), although with considerably lower potency than reported for other σ ligands. Other inhibitors are some serotonin uptake blockers or their metabolites and phenylalkylamines.

Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aβ aggregation features and cross the blood-brain-barrier: implications for therapy of Alzheimer disease.

Targeting amyloid-β peptide (Aβ) within the brain is a strategy actively sought for therapy of Alzheimers disease (AD). We investigated the ability of liposomes bi-functionalized with phosphatidic acid and with a modified ApoE-derived peptide (mApoE-PA-LIP) to affect Aβ aggregation/disaggregation features and to cross in vitro and in vivo the blood-brain barrier (BBB). Surface plasmon resonance showed that bi-functionalized liposomes strongly bind Aβ (kD=0.6 μM), while Thioflavin-T and SDS-PAGE/WB assays show that liposomes inhibit peptide aggregation (70% inhibition after 72 h) and trigger the disaggregation of preformed aggregates (60% decrease after 120 h incubation). Moreover, experiments with dually radiolabelled LIP suggest that bi-functionalization enhances the passage of radioactivity across the BBB either in vitro (permeability=2.5×10(-5) cm/min, 5-fold higher with respect to mono-functionalized liposomes) or in vivo in healthy mice. Taken together, our results suggest that mApoE-PA-LIP are valuable nanodevices with a potential applicability in vivo for the treatment of AD. From the clinical editor: Bi-functionalized liposomes with phosphatidic acid and a modified ApoE-derived peptide were demonstrated to influence Aβ aggregation/disaggregation as a potential treatment in an Alzheimers model. The liposomes were able to cross the blood-brain barrier in vitro and in vivo. Similar liposomes may become clinically valuable nanodevices with a potential applicability for the treatment of Alzheimers disease.