Red-Light-Photosensitized Tyrosine 10 Nitration of ß-Amyloid1-42 Diverts the Protein from Forming Toxic Aggregates.

Several studies have highlighted the presence of nitration damage following neuroinflammation in Alzheimer's disease (AD). Accordingly, post-transcriptional modifications of ß-amyloid (Aß), including peptide nitration, have been explored as a marker of the disease. However, the implications of Aß nitration in terms of aggregation propensity and neurotoxicity are still debated. Here, we show new data obtained using a photoactivatable peroxynitrite generator (BPT-NO) to overcome the limitations associated with chemical nitration methods. We found that the photoactivation of BPT-NO with the highly biocompatible red light selectively induces the nitration of tyrosine 10 of freshly solubilized full-length Aß1-42. Photonitrated Aß1-42 was, therefore, investigated for aggregation states and functions. It resulted that photonitrated Aß1-42 did not aggregate into small oligomers but rather self-assembled into large amorphous aggregates. When tested on neuronal-like SH-SY5Y cells and microglial C57BL/6 BV2 cells, photonitrated Aß1-42 showed to be free of neurotoxicity and able to induce phagocytic microglia cells. We propose that light-controlled nitration of the multiple forms in which Aß occurs (i.e., monomers, oligomers, fibrils) could be a tool to assess in real-time the impact of tyrosine nitration on the amyloidogenic and toxic properties of Aß1-42.

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.

Molecular Connections between DNA Replication and Cell Death in ß-Amyloid-Treated Neurons.

BACKGROUND: Ectopic cell cycle reactivation in neurons is associated with neuronal death in Alzheimer's disease. In cultured rodent neurons, synthetic ß-amyloid (Aß) reproduces the neuronal cell cycle re-entry observed in the Alzheimer's brain, and blockade of the cycle prevents Aß-induced neurodegeneration. DNA polymerase-ß, whose expression is induced by Aß, is responsible for the DNA replication process that ultimately leads to neuronal death, but the molecular mechanism(s) linking DNA replication to neuronal apoptosis are presently unknown. AIM: To explore the role of a conserved checkpoint pathway started by DNA replication stress, namely the ATM-ATR/Claspin/Chk-1 pathway, in switching the neuronal response from DNA replication to apoptosis. METHODS: Experiments were carried out in cultured rat cortical neurons challenged with toxic oligomers of Aß protein. RESULTS: Small inhibitory molecules of ATM/ATR kinase or Chk-1 amplified Aß-induced neuronal DNA replication and apoptosis, as they were permissive to the DNA polymerase-ß activity triggered by Aß oligomers. Claspin, i.e., the adaptor protein between ATM/ATR kinase and the downstream Chk-1, was present on DNA replication forks of neurons early after Aß challenge, and decreased at times coinciding with neuronal apoptosis. The caspase-3/7 inhibitor I maintained overtime the amount of Claspin loaded on DNA replication forks and, concomitantly, reduced neuronal apoptosis by holding neurons in the S phase. Moreover, a short phosphopeptide mimicking the Chk-1-binding motif of Claspin was able to prevent Aß-challenged neurons from entering apoptosis. CONCLUSION: We speculate that, in the Alzheimer's brain, Claspin degradation by intervening factors may precipitate the death of neurons engaged into DNA replication.

ß-amyloid monomers drive up neuronal aerobic glycolysis in response to energy stressors.

Research on cerebral glucose metabolism has shown that the aging brain experiences a fall of aerobic glycolysis, and that the age-related loss of aerobic glycolysis may accelerate Alzheimer's disease pathology. In the healthy brain, aerobic glycolysis, namely the use of glucose outside oxidative phosphorylation, may cover energy demand and increase neuronal resilience to stressors at once. Currently, the drivers of aerobic glycolysis in neurons are unknown. We previously demonstrated that synthetic monomers of ß-amyloid protein (Aß) enhance glucose uptake in neurons, and that endogenous Aß is required for depolarization-induced glucose uptake in cultured neurons. In this work, we show that cultured cortical neurons increased aerobic glycolysis in response to the inhibition of oxidative phosphorylation by oligomycin or to a kainate pulse. Such an increase was prevented by blocking the endogenous Aß tone and re-established by the exogenous addition of synthetic Aß monomers. The activity of mitochondria-bound hexokinase-1 appeared to be necessary for monomers-stimulated aerobic glycolysis during oxidative phosphorylation blockade or kainate excitation. Our data suggest that, through Aß release, neurons coordinate glucose uptake with aerobic glycolysis in response to metabolic stressors. The implications of this new finding are that the age-related drop in aerobic glycolysis and the susceptibility to Alzheimer's disease could be linked to factors interfering with release and functions of Aß monomers.

Role for Metallothionein-3 in the Resistance of Human U87 Glioblastoma Cells to Temozolomide.

Metallothioneins (MTs) are metal-binding proteins that are overexpressed in various human cancers and are thought to be associated with resistance to cytotoxic drugs. The knowledge on MT expression, regulation, and function in human gliomas is limited. We found that MT3 mRNA was highly expressed in cell lines derived from grade IV gliomas (i.e., A172 and U87 cells), as compared to grade II astrocytoma cells (i.e., 1321N1). Different from 1321N1, U87 cells were partly resistant to the alkylating drug, temozolomide (TMZ) (100 µM for 96 h), which induced a massive accumulation of U87 into the S and G2 fractions of the cell cycle but not apoptotic death. Silencing of MT3 did not significantly affect U87 cell proliferation and survival, but it delayed G1/S transition and favored the occurrence of apoptosis in TMZ-treated cells. Accordingly, the combination of MT3 silencing and TMZ treatment increased the protein levels of checkpoint kinase-1, which was ultimately responsible for the lasting G1 arrest and death of double treated U87 cells.

Targeting mGlu Receptors for Optimization of Antipsychotic Activity and Disease-Modifying Effect in Schizophrenia.

Metabotropic glutamate (mGlu) receptors are considered as candidate drug targets for the treatment of schizophrenia. These receptors form a family of eight subtypes (mGlu1 to -8), of which mGlu1 and -5 are coupled to Gq/11, and all other subtypes are coupled to Gi/o. Here, we discuss the possibility that selective ligands of individual mGlu receptor subtypes may be effective in controlling the core symptoms of schizophrenia, and, in some cases, may impact mechanisms underlying the progression of the disorder. Recent evidence indicates that activation of mGlu1 receptors inhibits dopamine release in the meso-striatal system. Hence, selective positive allosteric modulators (PAMs) of mGlu1 receptors hold promise for the treatment of positive symptoms of schizophrenia. mGlu5 receptors are widely expressed in the CNS and regulate the activity of cells that are involved in the pathophysiology of schizophrenia, such as cortical GABAergic interneurons and microglial cells. mGlu5 receptor PAMs are under development for the treatment of schizophrenia and cater the potential to act as disease modifiers by restraining neuroinflammation. mGlu2 receptors have attracted considerable interest because they negatively modulate 5-HT2A serotonin receptor signaling in the cerebral cortex. Both mGlu2 receptor PAMs and orthosteric mGlu2/3 receptor agonists display antipsychotic-like activity in animal models, and the latter drugs are inactive in mice lacking mGlu2 receptors. So far, mGlu3 receptors have been left apart as drug targets for schizophrenia. However, activation of mGlu3 receptors boosts mGlu5 receptor signaling, supports neuronal survival, and drives microglial cells toward an antiinflammatory phenotype. This strongly encourages research of mGlu3 receptors in schizophrenia. Finally, preclical studies suggest that mGlu4 receptors might be targeted by novel antipsychotic drugs, whereas studies of mGlu7 and mGlu8 receptors in animal models of psychosis are still at their infancy.

Metabotropic Glutamate Receptors in Glial Cells: A New Potential Target for Neuroprotection?

Neurodegenerative disorders are characterized by excitotoxicity and neuroinflammation that finally lead to slow neuronal degeneration and death. Although neurons are the principal target, glial cells are important players as they contribute by either exacerbating or dampening the events that lead to neuroinflammation and neuronal damage. A dysfunction of the glutamatergic system is a common event in the pathophysiology of these diseases. Metabotropic glutamate (mGlu) receptors belong to a large family of G protein-coupled receptors largely expressed in neurons as well as in glial cells. They often appear overexpressed in areas involved in neurodegeneration, where they can modulate glutamatergic transmission. Of note, mGlu receptor upregulation may involve microglia or, even more frequently, astrocytes, where their activation causes release of factors potentially able to influence neuronal death. The expression of mGlu receptors has been also reported on oligodendrocytes, a glial cell type specifically involved in the development of multiple sclerosis. Here we will provide a general overview on the possible involvement of mGlu receptors expressed on glial cells in the pathogenesis of different neurodegenerative disorders and the potential use of subtype-selective mGlu receptor ligands as candidate drugs for the treatment of neurodegenerative disorders. Negative allosteric modulators (NAM) of mGlu5 receptors might represent a relevant pharmacological tool to develop new neuroprotective strategies in these diseases. Recent evidence suggests that targeting astrocytes and microglia with positive allosteric modulators (PAM) of mGlu3 receptor or oligodendrocytes with mGlu4 PAMS might represent novel pharmacological approaches for the treatment of neurodegenerative disorders.

Neurobiological links between depression and AD: The role of TGF-ß1 signaling as a new pharmacological target.

In the last several years a large number of studies have demonstrated the neurobiological and clinical continuum between depression and Alzheimer's disease (AD). Depression is a risk factor for the development of AD, and the presence of depressive symptoms significantly increases the conversion of Mild Cognitive Impairment (MCI) into AD. Common pathophysiological events have been identified in depression and AD, including neuroinflammation with an aberrant Tumor Necrosis Factor-α (TNF-α) signaling, and an impairment of Brain-Derived Neurotrophic Factor (BDNF) and Transforming-Growth-Factor-ß1 (TGF-ß1) signaling. TGF-ß1 is an anti-inflammatory cytokine that exerts neuroprotective effects against amyloid-ß (Aß)-induced neurodegeneration, and it has a key role in memory formation and synaptic plasticity. TGF-ß1 plasma levels are reduced in major depressed patients (MDD), correlate with depression severity, and significantly contribute to treatment resistance in MDD. The deficit of Smad-dependent TGF-ß1 signaling is also an early event in AD pathogenesis, which contributes to inflammaging and cognitive decline in AD. A long-term treatment with antidepressants such as selective-serotonin-reuptake inhibitors (SSRIs) is known to reduce the risk of AD in patients with depression and, SSRIs, such as fluoxetine, increase the release of TGF-ß1 from astrocytes and exert relevant neuroprotective effects in experimental models of AD. We propose the TGF-ß1 signaling pathway as a common pharmacological target in depression and AD, and discuss the potential rescue of TGF-ß1 signaling by antidepressants as a way to prevent the transition from depression to AD.

A New Ratiometric Lysosomal Copper(II) Fluorescent Probe To Map a Dynamic Metallome in Live Cells.

We synthesized a new ratiometric fluorescent Cu2+ probe, bearing a morpholine moiety for selective binding to lysosomes and two picolylamine arms for the specific chelation of divalent copper ions. The probe capability to detect lysosomal Cu2+ was demonstrated in human differentiated neuroblastoma cells by confocal microscopy.

Functional partnership between mGlu3 and mGlu5 metabotropic glutamate receptors in the central nervous system.

mGlu5 receptors are involved in mechanisms of activity-dependent synaptic plasticity, and are targeted by drugs developed for the treatment of CNS disorders. We report that mGlu3 receptors, which are traditionally linked to the control of neurotransmitter release, support mGlu5 receptor signaling in neurons and largely contribute to the robust mGlu5 receptor-mediated polyphosphoinositide hydrolysis in the early postnatal life. In cortical pyramidal neurons, mGlu3 receptor activation potentiated mGlu5 receptor-mediated somatic Ca2+ mobilization, and mGlu3 receptor-mediated long-term depression in the prefrontal cortex required the endogenous activation of mGlu5 receptors. The interaction between mGlu3 and mGlu5 receptors was also relevant to mechanisms of neuronal toxicity, with mGlu3 receptors shaping the influence of mGlu5 receptors on excitotoxic neuronal death. These findings shed new light into the complex role played by mGlu receptors in physiology and pathology, and suggest reconsideration of some of the current dogmas in the mGlu receptor field.

Amyloid Beta monomers regulate cyclic adenosine monophosphate response element binding protein functions by activating type-1 insulin-like growth factor receptors in neuronal cells.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder associated with synaptic dysfunction, pathological accumulation of ß-amyloid (Aß), and neuronal loss. The self-association of Aß monomers into soluble oligomers seems to be crucial for the development of neurotoxicity (J. Neurochem., 00, 2007 and 1172). Aß oligomers have been suggested to compromise neuronal functions in AD by reducing the expression levels of the CREB target gene and brain-derived neurotrophic factor (BDNF) (J. Neurosci., 27, 2007 and 2628; Neurobiol. Aging, 36, 2015 and 20406 Mol. Neurodegener., 6, 2011 and 60). We previously reported a broad neuroprotective activity of physiological Aß monomers, involving the activation of type-1 insulin-like growth factor receptors (IGF-IRs) (J. Neurosci., 29, 2009 and 10582, Front Cell Neurosci., 9, 2015 and 297). We now provide evidence that Aß monomers, by activating the IGF-IR-stimulated PI3-K/AKT pathway, induce the activation of CREB in neurons and sustain BDNF transcription and release.

Metabotropic glutamate receptors: the potential for therapeutic applications in Alzheimer's disease.

A dysfunction of glutamate signaling is implicated at several levels in the pathogenesis of Alzheimer's disease. Currently, metabotropic glutamate receptors, which have a wide distribution in the central nervous system and activate a multitude of cell signaling pathways, are pursued as targets for therapeutic intervention in Alzheimer's disease. Research is still limited, but results underscore the relevance of ongoing studies. Here we discuss the latest updates regarding metabotropic glutamate receptors and their role in Alzheimer's disease, as well as promising metabotropic glutamate receptor ligands that have been investigated in preclinical models of Alzheimer's disease.

The underexplored question of ß-amyloid monomers.

Conceived more than 25 years ago, the amyloid cascade hypothesis of Alzheimer's disease has evolved to accommodate new findings, namely different forms of ß-amyloid aggregates and downstream dysfunctions. Yet, the cascade does not mention its very beginning, the ß-amyloid monomer. Here, I will discuss the monomer from a functional evolutionary perspective, highlighting the potential advantages of a native unfolded state that, however, involves an amyloidogenic risk. Finally, I will make a summary of what is known about its functional role in the brain and discuss the implications of its conceivable shortage in the development of Alzheimer's disease.

The impact of metabotropic glutamate receptors into active neurodegenerative processes: A "dark side" in the development of new symptomatic treatments for neurologic and psychiatric disorders.

Metabotropic glutamate (mGlu) receptor ligands are under clinical development for the treatment of CNS disorders with high social and economic burden, such as schizophrenia, major depressive disorder (MDD), and Parkinson's disease (PD), and are promising drug candidates for the treatment of Alzheimer's disease (AD). So far, clinical studies have shown symptomatic effects of mGlu receptor ligands, but it is unknown whether these drugs act as disease modifiers or, at the opposite end, they accelerate disease progression by enhancing neurodegeneration. This is a fundamental issue in the treatment of PD and AD, and is also an emerging theme in the treatment of schizophrenia and MDD, in which neurodegeneration is also present and contribute to disease progression. Moving from in vitro data and preclinical studies, we discuss the potential impact of drugs targeting mGlu2, mGlu3, mGlu4 and mGlu5 receptor ligands on active neurodegeneration associated with AD, PD, schizophrenia, and MDD. We wish to highlight that our final comments on the best drug candidates are not influenced by commercial interests or by previous or ongoing collaborations with drug companies. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

Fluoxetine Prevents Aß1-42-Induced Toxicity via a Paracrine Signaling Mediated by Transforming-Growth-Factor-ß1.

Selective reuptake inhibitors (SSRIs), such as fluoxetine and sertraline, increase circulating Transforming-Growth-Factor-ß1 (TGF-ß1) levels in depressed patients, and are currently studied for their neuroprotective properties in Alzheimer's disease. TGF-ß1 is an anti-inflammatory cytokine that exerts neuroprotective effects against ß-amyloid (Aß)-induced neurodegeneration. In the present work, the SSRI, fluoxetine, was tested for the ability to protect cortical neurons against 1 µM oligomeric Aß1-42-induced toxicity. At therapeutic concentrations (100 nM-1 µM), fluoxetine significantly prevented Aß-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 Aß toxicity. The effect was lost in the presence of a neutralizing antibody against TGF-ß1 in the conditioned medium, or when the specific inhibitor of type-1 TGF-ß1 receptor, SB431542, was added to pure neuronal cultures. Accordingly, a 24 h treatment of cortical astrocytes with fluoxetine promoted the release of active TGF-ß1 in the culture media through the conversion of latent TGF-ß1 to mature TGF-ß1. Unlike fluoxetine, both serotonin and sertraline did not stimulate the astrocyte release of active TGF-ß1. We conclude that fluoxetine is neuroprotective against Aß toxicity via a paracrine signaling mediated by TGF-ß1, which does not result from a simplistic SERT blockade.

Ac-LPFFD-Th: A Trehalose-Conjugated Peptidomimetic as a Strong Suppressor of Amyloid-ß Oligomer Formation and Cytotoxicity.

The inhibition of amyloid formation is a promising therapeutic approach for the treatment of neurodegenerative diseases. Peptide-based inhibitors, which have been widely investigated, are generally derived from original amyloid sequences. Most interestingly, trehalose, a nonreducing disaccharide of α-glucose, is effective in preventing the aggregation of numerous proteins. We have determined that the development of hybrid compounds could provide new molecules with improved properties that might synergically increase the potency of their single moieties. In this work, the ability of Ac-LPFFD-Th, a C-terminally trehalose-conjugated derivative, to slow down the Aß aggregation process was investigated by means of different biophysical techniques, including thioflavin T fluorescence, dynamic light scattering, ESI-MS, and NMR spectroscopy. Moreover, we demonstrate that Ac-LPFFD-Th modifies the aggregation features of Aß and protects neurons from Aß oligomers' toxic insult.

The antineoplastic drug flavopiridol reverses memory impairment induced by Amyloid-ß1-42 oligomers in mice.

The ectopic re-activation of cell cycle in neurons is an early event in the pathogenesis of Alzheimer's disease (AD), which could lead to synaptic failure and ensuing cognitive deficits before frank neuronal death. Cytostatic drugs that act as cyclin-dependent kinase (CDK) inhibitors have been poorly investigated in animal models of AD. In the present study, we examined the effects of flavopiridol, an inhibitor of CDKs currently used as antineoplastic drug, against cell cycle reactivation and memory loss induced by intracerebroventricular injection of Aß1-42 oligomers in CD1 mice. Cycling neurons, scored as NeuN-positive cells expressing cyclin A, were found both in the frontal cortex and in the hippocampus of Aß-injected mice, paralleling memory deficits. Starting from three days after Aß injection, flavopiridol (0.5, 1 and 3mg/kg) was intraperitoneally injected daily, for eleven days. Here we show that a treatment with flavopiridol (0.5 and 1mg/kg) was able to rescue the loss of memory induced by Aß1-42, and to prevent the occurrence of ectopic cell-cycle events in the mouse frontal cortex and hippocampus. This is the first evidence that a cytostatic drug can prevent cognitive deficits in a non-transgenic animal model of AD.

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.