Search

Monoamine oxidase (MAO) enzymes play a central role in the pathogenesis of Alzheimer's disease (AD) and MAO inhibitors (MAOIs) are antidepressant drugs currently studied for their neuroprotective properties in neurodegenerative disorders. In the present work MAOIs such as tranylcypromine [trans-(+)-2-phenylcyclopropanamine, TCP] and its amide derivatives, TCP butyramide (TCP-But) and TCP acetamide (TCP-Ac), were tested for their ability to protect cortical neurons challenged with synthetic amyloid-β (Aβ)-(1-42) oligomers (100 nM) for 48 h. TCP significantly prevented Aβ-induced neuronal death in a concentration-dependent fashion and was maximally protective only at 10 µM. TCP-But was maximally protective in mixed neuronal cultures at 1 µM, a lower concentration compared to TCP, whereas the new derivative, TCP-Ac, was more efficacious than TCP and TCP-But and significantly protected cortical neurons against Aβ toxicity at nanomolar concentrations (100 nM). Experiments carried out with the Thioflavin-T (Th-T) fluorescence assay for fibril formation showed that TCP and its amide derivatives influenced the early events of the Aβ aggregation process in a concentration-dependent manner. TCP-Ac was more effective than TCP-But and TCP in slowing down the Aβ(1-42) aggregates formation through a lengthening at the lag phase. In our experimental model co-incubation of Aβ(1-42) oligomers with TCP-Ac was able to almost completely prevent Aβ-induced neurodegeneration. These results suggest that inhibition of Aβ oligomer-mediated aggregation significantly contributes to the overall neuroprotective activity of TCP-Ac and also raise the possibility that TCP, and in particular the new compound TCP-Ac, might represent new pharmacological tools to yield neuroprotection in AD., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Fingolimod (FTY720), a novel drug approved for the treatment of relapsing-remitting multiple sclerosis, activates different sphingosine-1-phosphate receptor (S1PR) subtypes. Its primary mechanism of action is to reduce the egress of T lymphocytes from secondary lymphoid organs, thus restraining neuroinflammation and autoimmunity. However, recent evidence suggests that the action of FTY720 involves S1PRs expressed by cells resident in the CNS, including neurons. Here, we examined the effect of FTY720, its active metabolite, FTY720-P, and sphingosine-1-phosphate (S1P) on neuronal viability using a classical in vitro model of excitotoxic neuronal death. Mixed cultures of mouse cortical cells were challenged with toxic concentrations of N-methyl-d-aspartate (NMDA) for 10 min, and neuronal death was assessed 20 h later. FTY720, FTY720-P, and S1P were all neuroprotective when applied 18-20 h prior to the NMDA pulse. Neuroprotection was attenuated by pertussis toxin, and inhibited by the selective type-1 S1PR (S1P1R) antagonist, W146, and by inhibitors of the mitogen associated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PtdIns-3-K) pathways. Both FTY720 and FTY720-P retained their protective activity in pure cultures of mouse or rat cortical neurons. These data offer the first direct demonstration that FTY720 and its active metabolite protect neurons against excitotoxic death., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2011 Wiley-Liss, Inc.)

The 42-aa-long beta-amyloid protein--Abeta(1-42)--is thought to play a central role in the pathogenesis of Alzheimer's 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 Abeta peptides (Lambert et al., 1998) indicate that self-association of Abeta(1-42) monomers into soluble oligomers is required for neurotoxicity. The function of monomeric Abeta(1-42) is unknown. The evidence that Abeta(1-42) is present in the brain and CSF of normal individuals suggests that the peptide is physiologically active (Shoji, 2002). Here we show that synthetic Abeta(1-42) 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 Abeta(1-42) 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 Abeta(1-42) carrying the Arctic mutation (E22G) associated with familiar AD (Nilsberth et al., 2001) were not neuroprotective. We suggest that pathological aggregation of Abeta(1-42) may also cause neurodegeneration by depriving neurons of the protective activity of Abeta(1-42) monomers. This "loss-of-function" hypothesis of neuronal death should be taken into consideration when designing therapies aimed at reducing Abeta burden.

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.

The mechanism whereby a reactivation of cell cycle in neurons causes cell death is beginning to be identified. In cellular models of Alzheimer's disease, activation of a non-canonical pathway of DNA replication contributes to neuronal death. This pathway involves the repair enzyme DNA polymerase-beta, which is highly expressed in neurons of the Alzheimer's brain at early stages of the disease. Loading of DNA polymerase-beta into the replication forks generates a death signal, which involves the tumor suppressor p53. The increasing knowledge of the main actors of the unscheduled DNA replication in neurons will pave the way for novel therapeutic interventions in Alzheimer's disease and other neurodegenerative disorders.

Expression of the Wnt antagonist Dickkopf-1 (DKK1) is induced during neurodegenerative processes associated with Alzheimer's Disease and brain ischemia. However, little is known about DKK1-mediated effects on neurons. We now describe that, in cultured neurons, DKK1 is able to inhibit canonical Wnt signaling, as assessed by TCF reporter assay and analysis of beta-catenin levels, and to elicit cell death associated with loss of BCL-2 expression, induction of BAX, and TAU hyperphosphorylation. Local infusion of DKK1 in rats caused neuronal cell death and astrocytosis in the CA1 region of the hippocampus and death of cholinergic neurons in the nucleus basalis magnocellularis. Both effects were reversed by systemic administration of lithium ions, which rescue the Wnt pathway by inhibiting glycogen synthase kinase-3beta. The demonstration that DKK1 inhibits Wnt signaling in neurons and causes neuronal death supports the hypothesis that inhibition of the canonical Wnt pathway contributes to the pathophysiology of neurodegenerative disorders.

Cultured neurons exposed to synthetic beta-amyloid (Abeta) fragments reenter the cell cycle and initiate a pathway of DNA replication that involves the repair enzyme DNA polymerase-beta (DNA pol-beta) before undergoing apoptotic death. In this study, by performing coimmunoprecipitation experiments on cross-linked nucleoprotein fragments from Abeta-treated neurons, we demonstrate that DNA pol-beta coimmunoprecipitates with cell division cycle 45 (Cdc45) and with DNA primase in short nucleoprotein fragments. This indicates that DNA pol-beta is loaded into neuronal DNA replication forks after Abeta treatment. In response to Abeta the canonical DNA-synthesizing enzyme DNA pol-delta also was loaded into neuronal replication forks, but at later times than DNA pol-beta. Methoxyamine, an inhibitor of the apurinic/apyrimidinic endonuclease that allows for the recruitment of DNA pol-beta during the process of base excision repair (BER), failed to affect coimmunoprecipitation between DNA pol-beta and Cdc45, indicating that DNA pol-beta loading to the replication forks is independent of DNA breaks. However, methoxyamine reduced DNA replication and ensuing apoptosis in neurons exposed to Abeta, suggesting that an efficient BER process allows DNA replication to proceed up to the threshold for death. These data demonstrate that DNA pol-beta is an essential component of the DNA replication machinery in Abeta-treated neurons and additionally support the hypothesis of a close association of cell cycle events with neuronal death in Alzheimer's disease (AD). Accordingly, by investigating the neuronal expression of DNA pol-beta, along with phosphorylated retinoblastoma protein and neurofibrillary changes in AD brain, we show an early involvement of DNA pol-beta in the pathogenesis of AD.

Neurospheres from adult mouse subventricular zone (SVZ) were grown in suspension cultures for 12-15 days. Neurospheres consisted mainly of neural precursor cells (NPCs) immunoreactive for nestin and also contained nestin-negative precursors. We used these neurospheres to determine the effects of synthetic beta-amyloid fragments (both betaAP(1-42) and betaAP(25-35)) on NPC proliferation, differentiation and survival. We show that neurospheres exposed to 25 microM betaAP(25-35) or betaAP(1-42) for 24 h (a toxic condition for mature neurons) did not undergo apoptosis. Instead, betaAP(25-35) orientated nestin-negative precursors towards nestin-positive NPCs and turned nestin-positive NPCs into neuroblasts. Intracerebroventricular infusion of full-length betaAP(1-42) increased the population of PSA-NCAM-positive cells in the SVZ, without affecting proliferation. We conclude that betaAP influences the fate of progenitor cells, driving their differentiation towards a neuronal lineage.

Prolonged exposure of cultured cortical neurons to the residue 25-35 fragment of beta-amyloid protein, in the presence of dizocilpine, an antagonist of the N-methyl-D-aspartate receptor, and of 6,7-dinitroquinoxaline-2,3-dione, an antagonist of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors, resulted in the expression of the proapoptotic protein Bax and neuronal death. Beta-amyloid protein(25-35)-induced neuronal death was substantially attenuated by the sigma1 receptor agonist 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate. The neuroprotective action of 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate was mimicked by the sigma1 ligand methyl (1S,2R)-2-[1-adamantyl(methyl)amino]methyl-1-phenylcyclopropanecarboxylate and was antagonized by the sigma1 receptor antagonist N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)-phenyl]-ethylamine monohydrochloride. These results suggest that sigma1 receptor agonists might function as neuroprotectant agents in Alzheimer's disease.

Nicergoline, a drug used for the treatment of Alzheimer's disease and other types of dementia, was tested for its ability to protect neurons against beta-amyloid toxicity. Pure cultures of rat cortical neurons were challenged with a toxic fragment of beta-amyloid peptide (betaAP(25-35)) and toxicity was assessed after 24 h. Micromolar concentrations of nicergoline or its metabolite, MDL, attenuated betaAP(25-35)-induced neuronal death, whereas MMDL (another metabolite of nicergoline), the alpha1-adrenergic receptor antagonist, prazosin, or the serotonin 5HT-2 receptor antagonist, methysergide, were inactive. Nicergoline increased the basal levels of Bcl-2 and reduced the increase in Bax levels induced by beta-amyloid, indicating that the drug inhibits the execution of an apoptotic program in cortical neurons. In mixed cultures of rat cortical cells containing both neurons and astrocytes, nicergoline and MDL were more efficacious than in pure neuronal cultures in reducing beta-amyloid neurotoxicity. Experiments carried out in pure cultures of astrocytes showed that a component of neuroprotection was mediated by a mechanism of glial-neuronal interaction. The conditioned medium of cultured astrocytes treated with nicergoline or MDL for 72-96 h (collected 24 h after drug withdrawal) was neuroprotective when transferred to pure neuronal cultures challenged with beta-amyloid. In cultured astrocytes, nicergoline increased the intracellular levels of transforming-growth factor-beta and glial-derived neurotrophic factor, two trophic factors that are known to protect neurons against beta-amyloid toxicity. These results raise the possibility that nicergoline reduces neurodegeneration in the Alzheimer's brain.