Lifelong nutritional omega-3 deficiency evokes depressive-like state through soluble beta amyloid

Recent evidence pointed out that the prevalence of depression has reached epidemic proportions in last decades. This increase has been linked to many environmental factors, among these the influence of dietary factors has gained great attention. In particular, it has been reported that low n-3 polyunsaturated fatty acid (n-3 PUFA) intake in diet is correlated to the development of depressive and anxiety-like symptoms. Furthermore, maternal malnutrition is a widely accepted risk factor for developing mental illness in later adulthood; among others, depression has been strongly associated to this event. On the other hand, we have previously found that acute intracerebral injection of the soluble beta amyloid 1-42 (Aβ1-42) peptide induces a depressive-like behavior in rats, associated to altered hypothalamic-pituitary-adrenal (HPA) axis activation and reduced cortical serotonin and neurotrophin levels. The aim of the present work was to study the effect of pre- and post-natal (5 weeks post-weaning) exposure to diets differently enriched in n-3, n-6, as well as n-6/n-3 PUFA balanced, on immobility time displayed on the forced swimming test (FST), along with neuroendocrine quantification in offspring rats. Results showed that n-6 PUFA-enriched diet increased depressive- and anxiety-like behaviors, as shown by the elevation in the immobility time in the FST test and self-grooming in the open field test. Those effects were accompanied by reduced cortical serotonin, high plasmatic corticosterone and hypothalamic corticotropin-releasing factor levels. Finally, enhanced plasmatic Aβ1-42 levels after n-6 PUFA diet and reduced plasmatic Aβ1-42 levels after n-3 PUFA were found. Taken together, our data indicate that Aβ1-42 might be crucially involved in behavioral alterations found after n-6 rich PUFA diet and strongly endorse the protective role of n-3 and the detrimental effect of improper n-6 PUFA diet consumption

Early loss of blood-brain barrier integrity precedes NOX2 elevation in the prefrontal cortex of an animal model of psychosis

The social isolation rearing of young adult rats is a model of psychosocial stress and provides a nonpharmacological tool to study alterations reminiscent of symptoms seen in psychosis. We have previously demonstrated that social isolation in rats leads to increased oxidative stress and to cerebral NOX2 elevations. Here, we investigated early alterations in mRNA expression leading to increased NOX2 in the brain. Rats were exposed to a short period of social isolation (1 week) and real-time polymerase chain reaction (PCR) for mRNA expression of genes involved in blood-brain barrier (BBB) formation and integrity (ORLs, Vof 21 and Vof 16, Leng8, Vnr1, and Trank 1 genes) was performed. Real-time PCR experiments, immunohistochemistry, and Western blotting analysis showed an increased expression of these genes and related proteins in isolated rats with respect to control animals. The expression of specific markers of BBB integrity, such as matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), occludin 1, and plasmalemmal vesicle associated protein-1 (PV-1), was also significantly altered after 1 week of social isolation. BBB permeability, evaluated by quantification of Evans blue dye extravasation, as well as interstitial fluid, was significantly increased in rats isolated for 1 week with respect to controls. Isolation-induced BBB disruption was also accompanied by a significant increase of Interleukin 6 (IL-6) expression. Conversely, no differences in NOX2 levels were detected at this time point. Our study demonstrates that BBB disruption precedes NOX2 elevations in the brain. These results provide new insights in the interplay of mechanisms linking psychosocial stress to early oxidative stress in the brain, disruption of the BBB, and the development of mental disorders

The NADPH oxidase NOX2 as a novel biomarker for suicidality: evidence from human post-mortem brain samples

Recent evidence points towards a role of oxidative stress in suicidality. However, few studies were carried out on the sources of reactive oxygen species (ROS) in subjects with suicidal behaviour. We have previously demonstrated that the NADPH oxidase NOX2-derived oxidative stress has a major role in the development of neuropathological alterations observed in an animal model of psychosis. Here, we investigated the possible increase in NOX2 in post mortem brain samples of subjects who died by asphyctic suicide (AS) compared with controls (CTRL) and subjects who died by non-suicidal asphyxia (NSA). We found that NOX2 expression was significantly higher in the cortex of AS subjects than in the other two experimental groups. NOX2 immunostaining was mainly detected in GABAergic neurons, with a minor presence of NOX2-positive-stained cells in glutamatergic and dopaminergic neurons, as well as astrocytes and microglia. A sustained increase in the expression of 8-hydroxy-2’-deoxyguanosine, an indirect marker of oxidative stress, was also detected in the cortex of AS subjects, compared with CTRL and NSA subjects. A significant elevation in cortical interleukin-6 immunoreactivity in AS subjects suggested an involvement of cytokine-associated molecular pathways in NOX2 elevations. Our results suggest that the increase in NOX2-derived oxidative stress in the brain might be involved in the neuropathological pathways leading to suicidal behaviour. These results may open innovative insights in the identification of new pathogenetic and necroscopic biomarkers, predictive for suicidality and potentially useful for suicide prevention

The NADPH oxidase NOX2 as a novel biomarker for suicidality: Evidence from human post mortem brain samples

Recent evidence points towards a role of oxidative stress in suicidality. However, few studies were carried out on the sources of reactive oxygen species (ROS) in subjects with suicidal behaviour. We have previously demonstrated that the NADPH oxidase NOX2-derived oxidative stress has a major role in the development of neuropathological alterations observed in an animal model of psychosis. Here, we investigated the possible increase in NOX2 in post mortem brain samples of subjects who died by asphyctic suicide (AS) compared with controls (CTRL) and subjects who died by non-suicidal asphyxia (NSA). We found that NOX2 expression was significantly higher in the cortex of AS subjects than in the other two experimental groups. NOX2 immunostaining was mainly detected in GABAergic neurons, with a minor presence of NOX2-positive-stained cells in glutamatergic and dopaminergic neurons, as well as astrocytes and microglia. A sustained increase in the expression of 8-hydroxy-2'-deoxyguanosine, an indirect marker of oxidative stress, was also detected in the cortex of AS subjects, compared with CTRL and NSA subjects. A significant elevation in cortical interleukin-6 immunoreactivity in AS subjects suggested an involvement of cytokine-associated molecular pathways in NOX2 elevations. Our results suggest that the increase in NOX2-derived oxidative stress in the brain might be involved in the neuropathological pathways leading to suicidal behaviour. These results may open innovative insights in the identification of new pathogenetic and necroscopic biomarkers, predictive for suicidality and potentially useful for suicide prevention

Soluble beta amyloid evokes alteration in brain norepinephrine levels: role of nitric oxide and interleukin-1

Strong evidence showed neurotoxic properties of beta amyloid (Aβ) and its pivotal role in the Alzheimer's disease (AD) pathogenesis. Beside, experimental data suggest that Aβ may have physiological roles considering that such soluble peptide is produced and secreted during normal cellular activity. There is now suggestive evidence that neurodegenerative conditions, like AD, involve nitric oxide (NO) in their pathogenesis. Nitric oxide also possess potent neuromodulatory actions in brain regions, such as prefrontal cortex (PFC), hippocampus (HIPP), and nucleus accumbens (NAC). In the present study, we evaluated the effect of acute Aβ injection on norepinephrine (NE) content before and after pharmacological manipulations of nitrergic system in above mentioned areas. Moreover, effects of the peptide on NOS activity were evaluated. Our data showed that 2 h after i.c.v. soluble Aβ administration, NE concentrations were significantly increased in the considered areas along with increased iNOS activity. Pre-treatment with NOS inhibitors, 7-Nitroindazole (7-NI), and N6-(1-iminoethyl)-L-lysine-dihydrochloride (L-NIL), reversed Aβ-induced changes. Ultimately, pharmacological block of interleukin1 (IL-1) receptors prevented NE increase in all brain regions. Taken together our findings suggest that NO and IL-1 are critically involved in regional noradrenergic alterations induced by soluble Aβ injection

Negative DNA supercoiling induces genome-wide Cas9 off-target activity

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites

Mechanism-Based Screen Establishes Signalling Framework for DNA Damage-Associated G1 Checkpoint Response

DNA damage activates checkpoint controls which block progression of cells through the division cycle. Several different checkpoints exist that control transit at different positions in the cell cycle. A role for checkpoint activation in providing resistance of cells to genotoxic anticancer therapy, including chemotherapy and ionizing radiation, is widely recognized. Although the core molecular functions that execute different damage activated checkpoints are known, the signals that control checkpoint activation are far from understood. We used a kinome-spanning RNA interference screen to delineate signalling required for radiation-mediated retinoblastoma protein activation, the recognized executor of G1 checkpoint control. Our results corroborate the involvement of the p53 tumour suppressor (TP53) and its downstream targets p21CIP1/WAF1 but infer lack of involvement of canonical double strand break (DSB) recognition known for its role in activating TP53 in damaged cells. Instead our results predict signalling involving the known TP53 phosphorylating kinase PRPK/TP53RK and the JNK/p38MAPK activating kinase STK4/MST1, both hitherto unrecognised for their contribution to DNA damage G1 checkpoint signalling. Our results further predict a network topology whereby induction of p21CIP1/WAF1 is required but not sufficient to elicit checkpoint activation. Our experiments document a role of the kinases identified in radiation protection proposing their pharmacological inhibition as a potential strategy to increase radiation sensitivity in proliferating cancer cells

Cyclin-Cyclin-dependent Kinase Regulatory Response Is Linked to Substrate Recognition*

Cyclin/cyclin-dependent kinase (CDK) complexes are critical regulators of cellular proliferation.A complex network of regulatory mechanisms has evolved to control their activity, including activating and inactivating phosphorylation of the catalytic CDK subunit and inhibition through specific regulatory proteins. Primate herpesviruses, including the oncogenic Kaposi sarcoma herpesvirus, encode cyclin D homologues. Viral cyclins have diverged from their cellular progenitor in that they elicit holoenzyme activity independent of activating phosphorylation by the CDK-activating kinase and resistant to inhibition by CDK inhibitors. Using sequence comparison and site-directed mutagenesis, we performed molecular analysis of the cellular cyclin D and the Kaposi sarcoma herpesvirus-cyclin to delineate the molecular mechanisms behind their different behavior. This provides evidence that a surface recognized for its involvement in the docking of CIP/KIP inhibitors is required and sufficient to modulate cyclin-CDK response to a range of regulatory cues, including INK4 sensitivity and CDK-activating kinase dependence. Importantly, amino acids in this region are critically linked to substrate selection, suggesting that a mutational drift in this surface simultaneously affects function and regulation. Together our work provides novel insight into the molecular mechanisms governing cyclin-CDK function and regulation and defines the biological forces that may have driven evolution of viral cyclins

Early Loss of Blood-Brain Barrier Integrity Precedes NOX2 Elevation in the Prefrontal Cortex of an Animal Model of Psychosis

The social isolation rearing of young adult rats is a model of psychosocial stress and provides a nonpharmacological tool to study alterations reminiscent of symptoms seen in psychosis. We have previously demonstrated that social isolation in rats leads to increased oxidative stress and to cerebral NOX2 elevations. Here, we investigated early alterations in mRNA expression leading to increased NOX2 in the brain. Rats were exposed to a short period of social isolation (1 week) and real-time polymerase chain reaction (PCR) for mRNA expression of genes involved in blood-brain barrier (BBB) formation and integrity (ORLs, Vof 21 and Vof 16, Leng8, Vnr1, and Trank 1 genes) was performed. Real-time PCR experiments, immunohistochemistry, and Western blotting analysis showed an increased expression of these genes and related proteins in isolated rats with respect to control animals. The expression of specific markers of BBB integrity, such as matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), occludin 1, and plasmalemmal vesicle associated protein-1 (PV-1), was also significantly altered after 1 week of social isolation. BBB permeability, evaluated by quantification of Evans blue dye extravasation, as well as interstitial fluid, was significantly increased in rats isolated for 1 week with respect to controls. Isolation-induced BBB disruption was also accompanied by a significant increase of Interleukin 6 (IL-6) expression. Conversely, no differences in NOX2 levels were detected at this time point. Our study demonstrates that BBB disruption precedes NOX2 elevations in the brain. These results provide new insights in the interplay of mechanisms linking psychosocial stress to early oxidative stress in the brain, disruption of the BBB, and the development of mental disorders.The social isolation rearing of young adult rats is a model of psychosocial stress and provides a nonpharmacological tool to study alterations reminiscent of symptoms seen in psychosis. We have previously demonstrated that social isolation in rats leads to increased oxidative stress and to cerebral NOX2 elevations. Here, we investigated early alterations in mRNA expression leading to increased NOX2 in the brain. Rats were exposed to a short period of social isolation (1 week) and real-time polymerase chain reaction (PCR) for mRNA expression of genes involved in blood-brain barrier (BBB) formation and integrity (ORLs, Vof 21 and Vof 16, Leng8, Vnr1, and Trank 1 genes) was performed. Real-time PCR experiments, immunohistochemistry, and Western blotting analysis showed an increased expression of these genes and related proteins in isolated rats with respect to control animals. The expression of specific markers of BBB integrity, such as matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), occludin 1, and plasmalemmal vesicle associated protein-1 (PV-1), was also significantly altered after 1 week of social isolation. BBB permeability, evaluated by quantification of Evans blue dye extravasation, as well as interstitial fluid, was significantly increased in rats isolated for 1 week with respect to controls. Isolation-induced BBB disruption was also accompanied by a significant increase of Interleukin 6 (IL-6) expression. Conversely, no differences in NOX2 levels were detected at this time point. Our study demonstrates that BBB disruption precedes NOX2 elevations in the brain. These results provide new insights in the interplay of mechanisms linking psychosocial stress to early oxidative stress in the brain, disruption of the BBB, and the development of mental disorders

Celecoxib Prevents Cognitive Impairment and Neuroinflammation in Soluble Amyloid β-treated Rats

Recent findings suggest that soluble forms of amyloid-β (sAβ) peptide contribute to synaptic and cognitive dysfunctions in early stages of Alzheimer's disease (AD). On the other hand, neuroinflammation and cyclooxygenase-2 (COX-2) enzyme have gained increased interest as key factors involved early in AD, although the signaling pathways and pathophysiologic mechanisms underlying a link between sAβ-induced neurotoxicity and inflammation are still unclear. Here, we investigated the effects of selective COX-2 enzyme inhibition on neuropathological alterations induced by sAβ administration in rats. To this purpose, animals received an intracerebroventricular (icv) injection of predominantly monomeric forms of sAβ and, 7 days after, behavioral as well as biochemical parameters and neurotransmitter alterations were evaluated. During this period, rats also received a sub-chronic treatment with celecoxib. Biochemical results demonstrated that icv sAβ injection significantly increased both COX-2 and pro-inflammatory cytokines expression in the hippocampus (Hipp) of treated rats. In addition, the number of hypertrophic microglial cells and astrocytes were upregulated in sAβ-treated group. Interestingly, rats treated with sAβ showed long-term memory deficits, as confirmed by a significant reduction of discrimination index in the novel object recognition test, along with reduced brain-derived neurotrophic factor expression and increased noradrenaline levels in the Hipp. Systemic administration of celecoxib prevented behavioral dysfunctions, as well as biochemical and neurotransmitter alterations. In conclusion, our results suggest that sAβ neurotoxicity might be associated to COX-2-mediated inflammatory pathways and that early treatment with selective COX-2 inhibitor might provide potential remedies to counterbalance the sAβ-induced effects