Inflammasome-Mediated Neuronal-Microglial Crosstalk: a Therapeutic Substrate for the Familial C9orf72 Variant of Frontotemporal Dementia/Amyotrophic Lateral Sclerosis.

Intronic G4C2 hexanucleotide repeat expansions (HRE) of C9orf72 are the most common cause of familial variants of frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). G4C2 HREs in C9orf72 undergo non-canonical repeat-associated translation, producing dipeptide repeat (DPR) proteins, with various deleterious impacts on cellular homeostasis. While five different DPRs are produced, poly(glycine-arginine) (GR) is amongst the most toxic and is the only DPR to accumulate in the associated clinically relevant anatomical locations of the brain. Previous work has demonstrated the profound effects of a poly (GR) model of C9orf72 FTD/ALS, including motor impairment, memory deficits, neurodegeneration, and neuroinflammation. Neuroinflammation is hypothesized to be a driving factor in the disease course; microglia activation is present prior to symptom onset and persists throughout the disease. Here, using an established mouse model of C9orf72 FTD/ALS, we investigate the contributions of the nod-like receptor pyrin-containing 3 (NLRP3) inflammasome in the pathogenesis of FTD/ALS. We find that inflammasome-mediated neuroinflammation is increased with microglial activation, cleavage of caspase-1, production of IL-1ß, and upregulation of Cxcl10 in the brain of C9orf72 FTD/ALS mice. Excitingly, we find that genetic ablation of Nlrp3 significantly improved survival, protected behavioral deficits, and prevented neurodegeneration suggesting a novel mechanism involving HRE-mediated induction of innate immunity. The findings provide experimental evidence of the integral role of HRE in inflammasome-mediated innate immunity in the C9orf72 variant of FTD/ALS pathogenesis and suggest the NLRP3 inflammasome as a therapeutic target.

Exposure to World Trade Center Dust Exacerbates Cognitive Impairment and Evokes a Central and Peripheral Pro-Inflammatory Transcriptional Profile in an Animal Model of Alzheimer's Disease.

BACKGROUND: The terrorist attacks on September 11, 2001, on the World Trade Center (WTC) led to intense fires and a massive dense cloud of toxic gases and suspended pulverized debris. In the subsequent years, following the attack and cleanup efforts, a cluster of chronic health conditions emerged among First Responders (FR) who were at Ground Zero for prolonged periods and were repeatedly exposed to high levels of WTC particulate matter (WTCPM). Among those are neurological complications which may increase the risk for the development of Alzheimer's disease (AD) later in life. OBJECTIVE: We hypothesize that WTCPM dust exposure affects the immune cross-talking between the periphery and central nervous systems that may induce brain permeability ultimately promoting AD-type phenotype. METHODS: 5XFAD and wild-type mice were intranasally administered with WTCPM dust collected at Ground Zero within 72 h after the attacks. Y-maze assay and novel object recognition behavioral tests were performed for working memory deficits and learning and recognition memory, respectively. Transcriptomic analysis in the blood and hippocampus was performed and confirmed by RT qPCR. RESULTS: Mice exposed to WTCPM dust exhibited a significant impairment in spatial and recognition short and long-term memory. Furthermore, the transcriptomic analysis in the hippocampal formation and blood revealed significant changes in genes related to immune-inflammatory responses, and blood-brain barrier disruption. CONCLUSION: These studies suggest a putative peripheral-brain immune inflammatory cross-talking that may potentiate cognitive decline, identifying for the first time key steps which may be therapeutically targetable in future studies in WTC FR.

Neuronal Pentraxin 1 Promotes Hypoxic-Ischemic Neuronal Injury by Impairing Mitochondrial Biogenesis via Interactions With Active Bax[6A7] and Mitochondrial Hexokinase II.

Mitochondrial dysfunction is a key mechanism of cell death in hypoxic-ischemic brain injury. Neuronal pentraxin 1 (NP1) has been shown to play crucial roles in mitochondria-mediated neuronal death. However, the underlying mechanism(s) of NP1-induced mitochondrial dysfunction in hypoxia-ischemia (HI) remains obscure. Here, we report that NP1 induction following HI and its subsequent localization to mitochondria, leads to disruption of key regulatory proteins for mitochondrial biogenesis. Brain mitochondrial DNA (mtDNA) content and mtDNA-encoded subunit I of complex IV (mtCOX-1) expression was increased post-HI, but not the nuclear DNA-encoded subunit of complex II (nSDH-A). Up-regulation of mitochondrial proteins COXIV and HSP60 further supported enhanced mtDNA function. NP1 interaction with active Bax (Bax6A7) was increased in the brain after HI and in oxygen-glucose deprivation (OGD)-induced neuronal cultures. Importantly, NP1 colocalized with mitochondrial hexokinase II (mtHKII) following OGD leading to HKII dissociation from mitochondria. Knockdown of NP1 or SB216763, a GSK-3 inhibitor, prevented OGD-induced mtHKII dissociation and cellular ATP decrease. NP1 also modulated the expression of mitochondrial transcription factor A (Tfam) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), regulators of mitochondrial biogenesis, following HI. Together, we reveal crucial roles of NP1 in mitochondrial biogenesis involving interactions with Bax[6A7] and mtHKII in HI brain injury.

Discovery and characterization of small-molecule inhibitors of NLRP3 and NLRC4 inflammasomes.

Inflammasomes are macromolecular complexes involved in the host response to external and endogenous danger signals. Inflammasome-mediated sterile inflammation plays a central role in several human conditions such as autoimmune diseases, type-2 diabetes, and neurodegenerative disorders, indicating inflammasomes could be appealing therapeutic targets. Previous work has demonstrated that inhibiting the ATPase activity of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3), disrupts inflammasome assembly and function. However, there is a necessity to find new potent compounds with therapeutic potential. Here we combine computational modeling of the target and virtual screening to discover a group of novel compounds predicted to inhibit NLRP3. We characterized the best compounds and determined their potency, specificity, and ability to inhibit processes downstream from NLRP3 activation. Moreover, we analyzed in mice the competence of a lead candidate to reduce lipopolysaccharide-induced inflammation. We also validated the active pharmacophore shared among all the NLRP3 inhibitors, and through computational docking, we clarify key structural features for compound positioning within the inflammasome ATP-binding site. Our study sets the basis for rational design and optimization of inflammasome-targeting probes and drugs.

Presenilin1 familial Alzheimer disease mutants inactivate EFNB1- and BDNF-dependent neuroprotection against excitotoxicity by affecting neuroprotective complexes of N-methyl-d-aspartate receptor.

Excitotoxicity is thought to play key roles in brain neurodegeneration and stroke. Here we show that neuroprotection against excitotoxicity by trophic factors EFNB1 and brain-derived neurotrophic factor (called here factors) requires de novo formation of 'survival complexes' which are factor-stimulated complexes of N-methyl-d-aspartate receptor with factor receptor and presenilin 1. Absence of presenilin 1 reduces the formation of survival complexes and abolishes neuroprotection. EPH receptor B2- and N-methyl-d-aspartate receptor-derived peptides designed to disrupt formation of survival complexes also decrease the factor-stimulated neuroprotection. Strikingly, factor-dependent neuroprotection and levels of the de novo factor-stimulated survival complexes decrease dramatically in neurons expressing presenilin 1 familial Alzheimer disease mutants. Mouse neurons and brains expressing presenilin 1 familial Alzheimer disease mutants contain increased amounts of constitutive presenilin 1-N-methyl-d-aspartate receptor complexes unresponsive to factors. Interestingly, the stability of the familial Alzheimer disease presenilin 1-N-methyl-d-aspartate receptor complexes differs from that of wild type complexes and neurons of mutant-expressing brains are more vulnerable to cerebral ischaemia than neurons of wild type brains. Furthermore, N-methyl-d-aspartate receptor-mediated excitatory post-synaptic currents at CA1 synapses are altered by presenilin 1 familial Alzheimer disease mutants. Importantly, high levels of presenilin 1-N-methyl-d-aspartate receptor complexes are also found in post-mortem brains of Alzheimer disease patients expressing presenilin 1 familial Alzheimer disease mutants. Together, our data identify a novel presenilin 1-dependent neuroprotective mechanism against excitotoxicity and indicate a pathway by which presenilin 1 familial Alzheimer disease mutants decrease factor-depended neuroprotection against excitotoxicity and ischaemia in the absence of Alzheimer disease neuropathological hallmarks which may form downstream of neuronal damage. These findings have implications for the pathogenic effects of familial Alzheimer disease mutants and therapeutic strategies.

Presenilin 1 is necessary for neuronal, but not glial, EGFR expression and neuroprotection via γ-secretase-independent transcriptional mechanisms.

Epidermal growth factor receptor (EGFR) plays pivotal roles in cell proliferation, differentiation, and tissue development, while EGFs protect neurons from toxic insults by binding EGFR and stimulating survival signaling. Furthermore, recent evidence implicates this receptor in neurometabolic disorders like Alzheimer disease and aging. Here we show that absence of presenilin 1 (PS1) results in dramatic decrease (>95%) of neuronal EGFR and that PS1-null (PS1(-/-)) brains have reduced amounts of this receptor. PS1(-/-) cortical neurons contain little EGFR and show no epidermal growth factor-induced survival signaling or protection against excitotoxicity, but exogenous EGFR rescues both functions even in absence of PS1. EGFR mRNA is greatly reduced (>95%) in PS1(-/-) neurons, and PS1(-/-) brains contain decreased amounts of this mRNA, although PS1 affects the stability of neither EGFR nor its mRNA. Exogenous PS1 increases neuronal EGFR mRNA, while down-regulation of PS1 decreases this mRNA. These effects are neuron specific, as PS1 affects the EGFR of neither glial nor fibroblast cells. In addition, PS1 controls EGFR through novel mechanisms shared with neither γ-secretase nor PS2. Our data reveal that PS1 functions as a positive transcriptional regulator of neuronal EGFR controlling its expression in a cell-specific manner. Severe downregulation of EGFR may contribute to developmental abnormalities and lethal phenotype found in PS1, but not PS2, null mice. Furthermore, PS1 may affect neuroprotection and Alzheimer disease by controlling survival signaling of neuronal EGFR.

Genetic deletion of neuronal pentraxin 1 expression prevents brain injury in a neonatal mouse model of cerebral hypoxia-ischemia.

Neonatal hypoxic-ischemic (HI) brain injury is a leading cause of mortality and morbidity in infants and children for which there is no promising therapy at present. Previously, we reported induction of neuronal pentraxin 1 (NP1), a novel neuronal protein of the long-pentraxin family, following HI injury in neonatal brain. Here, we report that genetic deletion of NP1 expression prevents HI injury in neonatal brain. Elevated expression of NP1 was observed in neurons, not in astrocytes, of the ipsilateral cortical layers (I-IV) and in the hippocampal CA1 and CA3 areas of WT brains following hypoxia-ischemia; brain areas that developed infarcts (at 24-48 h), showed significantly increased numbers of TUNEL-(+) cells and tissue loss (at 7 days). In contrast, NP1-KO mice showed no evidence of brain infarction and tissue loss after HI. The immunofluorescence staining of brain sections with mitochondrial protein COX IV and subcellular fractionation analysis showed increased accumulation of NP1 in mitochondria, pro-death protein Bax activation and NP1 co-localization with activated caspase-3 in WT, but not in the NP1-KO brains; corroborating NP1 interactions with the mitochondria-derived pro-death pathways. Disruption of NP1 translocation to mitochondria by NP1-siRNA in primary cortical cultures significantly reduced ischemic neuronal death. NP1 was immunoprecipitated with activated Bax [6A7] proteins; HI caused increased interactions of NP1 with Bax, thereby, facilitating Bax translocation to mitochondrial and neuronal death. To further delineate the specificity of NPs, we found that NP1 but not the NP2 induction is specifically involved in brain injury mechanisms and that knockdown of NP1 only results in neuroprotection. Furthermore, live in vivo T2-weighted magnetic resonance imaging (MRI) including fractional anisotropy (FA) mapping showed no sign of delayed brain injury or tissue loss in the NP1-KO mice as compared to the WT at different post-HI periods (4-24 weeks) examined; indicating a long-term neuroprotective efficacy of NP1 gene deletion. Collectively, our results demonstrate a novel mechanism of neuronal death and predict that inhibition of NP1 expression is a promising strategy to prevent hypoxic-ischemic injury in immature brain.

Anxiolytic action of pterostilbene: involvement of hippocampal ERK phosphorylation.

Pterostilbene, a natural analog of resveratrol, has diverse health-beneficial properties. However, the neurological activities of this compound are largely unexplored. Here, we report that pterostilbene shows anxiolytic-like actions by down-regulating phosphorylated levels of extracellular regulated kinases in the hippocampus of mice. Adult male mice administered pterostilbene (1-10 mg/kg, p. o.) were subjected to the elevated plus maze test. Pterostilbene manifested anxiolytic activity at 1 and 2 mg/kg doses, demonstrated by increases in % permanence time and number of open arm entries. The locomotor activity of the animals was unaffected at all doses. Western blot analysis revealed a decrease in both extracellular regulated kinase 1 and extracellular regulated kinase 2 phosphorylation in hippocampal homogenates from mice treated with 1 and 2 mg/kg pterostilbene. Moreover, pterostilbene was detected in the plasma and brains of mice following single oral administration. Anxiolytic activity was not observed at the higher doses (5 and 10 mg/kg). However, no impairment of motor function was observed either, suggesting a favorable safety index for the compound. These results suggest that pterostilbene has the potential for therapeutic drug development for anxiety disorders.

Genetic deletion of NP1 prevents hypoxic-ischemic neuronal death via reducing AMPA receptor synaptic localization in hippocampal neurons.

BACKGROUND: Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) to excitatory synapses is critical to their synaptic functions. Previously, we have shown induction of neuronal pentraxin 1 (NP1) and its colocalization with AMPAR subunit GluR1 in hypoxic-ischemic (HI) brain injury. However, the role of NP1 in mediating GluR1 surface expression, trafficking, and clustering at synapses in HI neuronal death is unclear. METHODS AND RESULTS: Primary hippocampal neurons, isolated from wild-type (WT) and NP1-knockout (C57BL/6 background) mice at DIV 12 to 14 were exposed to 2 to 8 hours of oxygen glucose deprivation (OGD)-in vitro conditions that mimic human stroke. OGD exposure resulted in time-dependent induction of NP1 (∼4-fold), enhanced redistribution of AMAP GluR1 receptors at excitatory synapses, and increased neuronal death. We observed a significant increase in surface and synaptic GluR1 clusters that colocalized with PSD-95 on dendrites with a simultaneous decrease in internalized GluR1. Surface cross-linking with BS(3) showed enhanced membrane insertions of GluR1, and increased phosphorylation at Ser-845 further supported enhanced surface availability of GluR1 after OGD. NP1 protein colocalized with GluR1 and PSD-95, and OGD significantly increased their synaptic coclustering. Most strikingly, the genetic deletion of NP1 resulted in decreases in surface GluR1 cluster density, synaptic localization, phospho-GluR1 (Ser-845) levels, and neuronal death after OGD compared with WT neurons. AMPA (50 µmol/L) induced NP1 and significant cell death in WT but not in NP1-/- neurons. CONCLUSIONS: Our results indicate that NP1 plays a key role in synaptic clustering of GluR1, suggesting that targeting NP1 might be a practical approach to preventing ischemic brain damage.

Critical role of neuronal pentraxin 1 in mitochondria-mediated hypoxic-ischemic neuronal injury.

Developing brain is highly susceptible to hypoxic-ischemic (HI) injury leading to severe neurological disabilities in surviving infants and children. Previously, we have reported induction of neuronal pentraxin 1 (NP1), a novel neuronal protein of long-pentraxin family, following HI neuronal injury. Here, we investigated how this specific signal is propagated to cause the HI neuronal death. We used wild-type (WT) and NP1 knockout (NP1-KO) mouse hippocampal cultures, modeled in vitro following exposure to oxygen glucose deprivation (OGD), and in vivo neonatal (P9-10) mouse model of HI brain injury. Our results show induction of NP1 in primary hippocampal neurons following OGD exposure (4-8 h) and in the ipsilateral hippocampal CA1 and CA3 regions at 24-48 h post-HI compared to the contralateral side. We also found increased PTEN activity concurrent with OGD time-dependent (4-8 h) dephosphorylation of Akt (Ser473) and GSK-3ß (Ser9). OGD also caused a time-dependent decrease in the phosphorylation of Bad (Ser136), and Bax protein levels. Immunofluorescence staining and subcellular fractionation analyses revealed increased mitochondrial translocation of Bad and Bax proteins from cytoplasm following OGD (4 h) and simultaneously increased release of Cyt C from mitochondria followed by activation of caspase-3. NP1 protein was immunoprecipitated with Bad and Bax proteins; OGD caused increased interactions of NP1 with Bad and Bax, thereby, facilitating their mitochondrial translocation and dissipation of mitochondrial membrane potential (ΔΨ(m)). This NP1 induction preceded the increased mitochondrial release of cytochrome C (Cyt C) into the cytosol, activation of caspase-3 and OGD time-dependent cell death in WT primary hippocampal neurons. In contrast, in NP1-KO neurons there was no translocation of Bad and Bax from cytosol to the mitochondria, and no evidence of ΔΨ(m) loss, increased Cyt C release and caspase-3 activation following OGD; which resulted in significantly reduced neuronal death. Our results indicate a regulatory role of NP1 in Bad/Bax-dependent mitochondrial release of Cyt C and caspase-3 activation. Together our findings demonstrate a novel mechanism by which NP1 regulates mitochondria-driven hippocampal cell death; suggesting NP1 as a potential therapeutic target against HI brain injury in neonates.

4'-Demethylnobiletin, a bioactive metabolite of nobiletin enhancing PKA/ERK/CREB signaling, rescues learning impairment associated with NMDA receptor antagonism via stimulation of the ERK cascade.

The biochemical and pharmacological activities of nobiletin, including neurotrophic and memory-enhancing action, in both in vitro and in vivo systems are well established. However, whether its metabolites do have such beneficial effects like nobiletin remains to be examined. Here we, for the first time, report that 2-(4-hydroxy-3-methoxyphenyl)-5,6,7,8-tetramethoxychromen-4-one (4'-demethylnobiletin), a major metabolite of nobiletin identified in the urine of rats and mice, stimulates the phosphorylation of ERK and CREB and enhances CRE-mediated transcription by activating a PKA/MEK/ERK pathway, like nobiletin, in cultured hippocampal neurons. Since NMDA receptor-mediated ERK signaling is involved in memory processing, including associative memories, we also examined whether 4'-demethylnobiletin, by activating ERK signaling, could restore learning impairment. Chronic intraperitoneal (ip) treatment of the mice with 10 or 50 mg of 4'-demethylnobiletin/kg rescued the NMDA receptor antagonist MK-801-induced learning impairment, accompanied by improvement of the MK-801-induced decrease in the level of ERK phosphorylation in the hippocampus of the animals. Consistently, 4'-demethylnobiletin also restored MK-801-induced inhibition of NMDA-stimulated phosphorylation of not only ERK but also PKA substrates in cultured rat hippocampal neurons. Moreover, we actually detected 4'-demethylnobiletin in the brain of mice following acute ip administration, demonstrating that the metabolite can cross the blood-brain barrier to reach the brain and thereby exert its effects to reverse learning impairment. Therefore, these results suggest that 4'-demethylnobiletin, a bioactive metabolite of nobiletin, may serve as a potential therapeutic agent, at least, for memory disorders associated with a dysregulated NMDA receptor ERK signaling, like nobiletin.

A novel diol-derivative of chalcone produced by bioconversion, 3-(2,3-dihydroxyphenyl)-1-phenylpropan-1-one, activates PKA/MEK/ERK signaling and antagonizes Abeta-inhibition of the cascade in cultured rat CNS neurons.

Chalcone compounds have been widely studied for their anti-inflammatory, anti-pyretic, anti-invasive and anti-proliferative activities in various cell lines. However, their effects on the central nervous system (CNS) are still largely unexplored. We have recently developed a bioconversion system using a recombinant Escherichia coli that enables us to produce chemical compounds that are naturally rare and usually difficult to chemically synthesize. One such compound is 3-(2,3-dihydroxyphenyl)-1-phenylpropan-1-one, a novel chalcone-diol. Here we show, for the first time, that the chalcone-diol enhanced the phosphorylation of extracellular signal-regulated kinase (ERK) in a time- and concentration-dependent manner in cultured cortical neurons. Also, this chalcone-diol increased intracellular cyclic AMP (cAMP) concentration, thereby enhancing phosphorylation of ERK and cAMP-response element-binding protein (CREB), and CRE-mediated transcription via the cAMP-dependent protein kinase (PKA)/mitogen-activated protein kinase/ERK kinase (MEK) pathway in cultured rat hippocampal neurons. Recent studies have demonstrated that PKA/CREB-dependent signaling, which is required for long-term potentiation, is inhibited by sublethal concentrations of amyloid beta-peptide (Abeta) in cultured hippocampal neurons. After treatment with the chalcone-diol at 50 muM prior to treatment with a sublethal concentration of Abeta(1-42), the Abeta(1-42)-induced inhibition of phosphorylation of PKA substrates and CREB was prevented in cultured hippocampal neurons, indicating the potential for protection against the Abeta-induced impairment of PKA/CREB signaling observed in Alzheimer's disease. Therefore, these results suggest that our present study provides a new approach for discovering novel lead compounds for the treatment of neurodegenerative CNS diseases associated with impaired PKA/CREB signaling, including Alzheimer's disease.