Robert Nisticò

Involvement of interleukin-1β in the mechanism of human immunodeficiency virus type 1 (HIV-1) recombinant protein gp120-induced apoptosis in the neocortex of rat

The effect of subchronic intracerebroventricular injection of the human immunodeficiency virus type 1 (HIV-1) recombinant protein gp120 (100 ng, given daily for up to seven consecutive days) on interleukin-1beta expression was studied by immunohistochemistry in the brain of adult rats. In comparison to control, bovine serum albumin (300 ng, given intracerebroventricularly for up to seven days) -treated animals (n=6), interleukin-1beta immunoreactivity increased in the brain cortex and hippocampus of rats (n=6) receiving a single injection of the viral protein 24 h before analysis with more substantial increases being observed in these regions of the brain (n=6) after seven days treatment. Double-labelling immunofluorescence experiments support a neuronal and, possibly, a microglial cell origin for gp120-enhanced interleukin-1beta expression. Transmission electron microscopy analysis of brain tissue sections revealed that combination treatments (given intracerebroventricularly daily for seven days) with gp120 (100 ng) and interleukin-1 receptor antagonist (80 ng) or with the interleukin converting enzyme inhibitor II (100 pmol), but not with leupeptin (100 pmol), prevented apoptotic death of rat (n=6/group) brain cortical cells typically elicited by the viral protein. These data demonstrate that gp120 enhances interleukin-1beta expression in the brain and this may be involved in the mechanism underlying apoptosis induced by gp120 in the brain cortex of rat. Further support to this hypothesis comes from the evidence that intracerebroventricular injection of murine recombinant interleukin-1beta (200 U, given daily for seven consecutive days) produces DNA fragmentation in the brain cortex of rat (n=6). Interestingly, the latter treatment enhanced nerve growth factor level in the hippocampus but not in the cerebral cortex and this coincides with a similar effect recently reported in identical brain areas of rats treated likewise with gp120. In conclusion, the present data demonstrate that treatment with gp120 enhances interleukin-1beta expression and this participates in the mechanism of apoptotic cell death in the brain cortex of rat. By contrast, in the hippocampus, gp120-enhanced interleukin-1beta expression elevates nerve growth factor that may prevent or delay apoptosis in this plastic region of the rat brain.

The role of oxidative stress in paraquat-induced neurotoxicity in rats: protection by non peptidyl superoxide dismutase mimetic

Herbicides, including paraquat, may produce neurodegenerative effect when given both peripherally and into the brain though the pathophysiological mechanism is still unknown. Microinfusion of paraquat into the Substantia Nigra (50 microg) produced increased motor activity, jumping and circling opposite to the injection site, associated with ECoG desynchronization, high voltage epileptogenic spikes, and with neuropathological effects. These effects were accompanied by increase of malondialdehyde (MDA) levels in the Substantia Nigra, suggesting that paraquat was able to induce oxidative stress when injected directly into the rat brain. Pre-treatment of rats with M40401, a non peptidyl superoxide dismutase (SOD) mimetic given directly into the Substantia Nigra or i.p. prevented both behavioural, electrocorticogram and neuropathological effects and MDA elevation. Taken together, these results demonstrate that paraquat produces brain damage via abnormal formation of oxygen free radicals and that this effect may be counteracted by novel SOD mimetics.

Inflammation Subverts Hippocampal Synaptic Plasticity in Experimental Multiple Sclerosis

Abnormal use-dependent synaptic plasticity is universally accepted as the main physiological correlate of memory deficits in neurodegenerative disorders. It is unclear whether synaptic plasticity deficits take place during neuroinflammatory diseases, such as multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). In EAE mice, we found significant alterations of synaptic plasticity rules in the hippocampus. When compared to control mice, in fact, hippocampal long-term potentiation (LTP) induction was favored over long-term depression (LTD) in EAE, as shown by a significant rightward shift in the frequency–synaptic response function. Notably, LTP induction was also enhanced in hippocampal slices from control mice following interleukin-1β (IL-1β) perfusion, and both EAE and IL-1β inhibited GABAergic spontaneous inhibitory postsynaptic currents (sIPSC) without affecting glutamatergic transmission and AMPA/NMDA ratio. EAE was also associated with selective loss of GABAergic interneurons and with reduced gamma-frequency oscillations in the CA1 region of the hippocampus. Finally, we provided evidence that microglial activation in the EAE hippocampus was associated with IL-1β expression, and hippocampal slices from control mice incubated with activated microglia displayed alterations of GABAergic transmission similar to those seen in EAE brains, through a mechanism dependent on enhanced IL-1β signaling. These data may yield novel insights into the basis of cognitive deficits in EAE and possibly of MS.

Increased levels of d-aspartate in the hippocampus enhance LTP but do not facilitate cognitive flexibility.

In the present study, we demonstrate a direct role for d-aspartate in regulating hippocampal synaptic plasticity. These evidences were obtained using two different experimental strategies which enabled a non-physiological increase of endogenous d-aspartate levels in the mouse hippocampus: a genetic approach based on the targeted deletion of d-aspartate oxidase gene and another based on the oral administration of d-aspartate. Overall, our results indicate that increased d-aspartate content does not affect basal properties of synaptic transmission but enhances long-term potentiation in hippocampal slices from both genetic and pharmacological animal models. Besides electrophysiological data, behavioral analysis suggests that altered levels of d-aspartate in the hippocampus do not perturb basal spatial learning and memory abilities, but may selectively interfere with the dynamic NMDAR-dependent processes underlying cognitive flexibility.

Evidence that the HIV-1 coat protein gp120 causes neuronal apoptosis in the neocortex of rat via a mechanism involving CXCR4 chemokine receptor.

The HIV-1 coat protein, gp120 (100 ng given intracerebroventricularly (i.c.v.) daily for seven consecutive days) causes DNA fragmentation in the brain neocortex of rat. In neocortical cells bearing ultrastructural features typical of apoptosis, electron microscopy revealed specific immunopositivity for neurofilament cytoskeletal proteins, suggesting the neuronal nature of dying cells. Neuronal apoptosis by gp120 implicates CXCR4 chemokine receptors; in fact, in rats receiving a single daily, non-neurotoxic, dose of SDF-1alpha (0.25 pmoles given i.c.v. for 7 days before gp120), the natural ligand of CXCR4 receptor, apoptosis was significantly hindered. The mechanism of SDF-1alpha protection involves inhibition of gp120-enhanced expression of IL-1beta, a cytokine implicated in the mechanisms of apoptosis induced by the viral protein in the neocortex of rat.

Paraquat-and Rotenone-Induced Models of Parkinson's Disease

Parkinsons disease (PD) is a neurodegenerative disorder mainly characterized by a loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. In recent years, several new genes and environmental factors have been implicated in PD, and their impact on DA neuronal cell death is slowly emerging. However, PD etiology remains unknown, whereas its pathogenesis begins to be clarified as a multifactorial cascade of deleterious factors. Recent epidemiological studies have linked exposure to environmental agents, including pesticides, with an increased risk of developing the disease. As a result, over the last two decades the ‘environmental hypothesis’ of PD has gained considerable interest. This speculates that agricultural chemicals in the environment, by producing selective dopaminergic cell death, can contribute to the development of the disease. However, a causal role for pesticides in the etiology of PD has yet to be definitively established. Importantly, most insights into PD pathogenesis came from investigations performed in experimental models of PD, especially those produced by neurotoxins. This review presents data obtained in our laboratories along with current views on the neurotoxic actions induced by the two most popular parkinsonian pesticide neurotoxins, namely paraquat and rotenone. Although confined to these two chemicals, mechanistic studies underlying dopaminergic cell death are of the utmost importance to identify new drug targets for the treatment of PD.

The γ-secretase modulator CHF5074 restores memory and hippocampal synaptic plasticity in plaque-free Tg2576 mice.

Abnormal amyloid-β (Aβ) production and deposition is believed to represent one of the main causes of Alzheimers disease (AD). γ-Secretase is the enzymatic complex responsible for Aβ generation from its precursor protein. Inhibition or modulation of γ-secretase represents an attractive therapeutic approach. CHF5074 is a new γ-secretase modulator that has been shown to inhibit brain plaque deposition and to attenuate memory deficit in adult AD transgenic mice after chronic treatment. To date, it is not known whether the positive behavioral effects of this compound also occur in young transgenic mice without plaque deposition. Here, we evaluated the effects of acute and subchronic treatment with CHF5074 on contextual and recognition memory and on hippocampal synaptic plasticity in plaque-free Tg2576 mice. We found that at 5 months of age, contextual memory impairment was significantly attenuated after acute subcutaneous administration of 30 mg/kg CHF5074. At 6 months of age, recognition memory impairment was fully reversed after a 4-week oral treatment in the diet (≈60 mg/kg/day). These cognitive effects were associated with a reversal of long-term potentiation (LTP) impairment in the hippocampus. A significant reduction in brain intraneuronal AβPP/Aβ levels and hyperphosphorylated tau, but no change in soluble or oligomeric Aβ levels was detected in Tg2576 mice showing functional recovery following CHF5074 treatment. We conclude that the beneficial effects of CHF5074 treatment in young transgenic mice occurred at a stage that precedes plaque formation and were associated with a reduction in intraneuronal AβPP/Aβ and hyperphosphorylated tau.

Changes in mGlu5 receptor-dependent synaptic plasticity and coupling to homer proteins in the hippocampus of Ube3A hemizygous mice modeling angelman syndrome

Angelman syndrome (AS) is caused by the loss of Ube3A, an ubiquitin ligase that commits specific proteins to proteasomal degradation. How this defect causes autism and other pathological phenotypes associated with AS is unknown. Long-term depression (LTD) of excitatory synaptic transmission mediated by type 5 metabotropic glutamate (mGlu5) receptors was enhanced in hippocampal slices of Ube3Am−/p+ mice, which model AS. No changes were found in NMDA-dependent LTD induced by low-frequency stimulation. mGlu5 receptor-dependent LTD in AS mice was sensitive to the protein synthesis inhibitor anisomycin, and relied on the same signaling pathways as in wild-type mice, e.g., the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycine pathway, and protein tyrosine phosphatase. Neither the stimulation of MAPK and PI3K nor the increase in Arc (activity-regulated cytoskeleton-associated protein) levels in response to mGlu5 receptor activation were abnormal in hippocampal slices from AS mice compared with wild-type mice. mGlu5 receptor expression and mGlu1/5 receptor-mediated polyphosphoinositide hydrolysis were also unchanged in the hippocampus of AS mice. In contrast, AS mice showed a reduced expression of the short Homer protein isoform Homer 1a, and an increased coupling of mGlu5 receptors to Homer 1b/c proteins in the hippocampus. These findings support the link between Homer proteins and monogenic autism, and lay the groundwork for the use of mGlu5 receptor antagonists in AS.

The contribution of oxidative stress in apoptosis of human-cultured astroglial cells induced by supernatants of HIV-1-infected macrophages

Apoptosis of neurons and astrocytes has been found in patientsundergoing AIDS dementia complex. We demonstrated that supernatantsfrom human primary macrophages (M/M) infected by HIV‐1 lead humanastroglial cells to oxidative stress, as shown by elevated levels ofmalondialdehyde, and then to apoptosis. Electron microscopy ofastrocytes shortly incubated with HIV‐1‐infected M/M supernatantsshowed apoptotic blebbing, cytoplasmic loss, and chromatincondensation. Apoptosis was antagonized by pretreating astrocytes withthe nonpeptidic superoxide dismutase (SOD) mimetic M40401 but notwith anti‐HIV‐1 compounds, thus showing that apoptosis of astrocytesdriven by HIV‐1‐infected M/M supernatants is mainly mediated byabnormal production of superoxide anions without relationship to HIV‐1replication in such cells. Overall results support the role ofoxidative stress mediated by HIV‐1‐infected M/M as one of the leadingcauses of neurodegeneration in patients with HIV‐1 and suggest the useof nonpeptidic SOD mimetics to counteract HIV‐1‐related neurologicaldisorders.

Stress dynamically regulates behavior and glutamatergic gene expression in hippocampus by opening a window of epigenetic plasticity

Significance Chronic stress alters the hippocampal responses to familiar and novel stressors, behaviorally, physiologically, and epigenetically. In the aftermath of chronic stress in WT mice and in mice with a BDNF loss-of-function allele without any applied stress, there is a window of plasticity that allows familiar and novel experiences to alter anxiety- and depressive-like behaviors, reflected also in electrophysiological changes in the dentate gyrus (DG) in vitro. A consistent biomarker of mood-related behaviors in DG is reduced type 2 metabotropic glutamate (mGlu2), which regulates the release of glutamate. Within this window, familiar stress rapidly and epigenetically up-regulates mGlu2 by a P300-driven histone H3 lysine 27 acetylation and improves mood behaviors. This transient epigenetic plasticity may be useful for treatment of stress-related disorders where dysregulaton of glutamate is involved. Excitatory amino acids play a key role in both adaptive and deleterious effects of stressors on the brain, and dysregulated glutamate homeostasis has been associated with psychiatric and neurological disorders. Here, we elucidate mechanisms of epigenetic plasticity in the hippocampus in the interactions between a history of chronic stress and familiar and novel acute stressors that alter expression of anxiety- and depressive-like behaviors. We demonstrate that acute restraint and acute forced swim stressors induce differential effects on these behaviors in naive mice and in mice with a history of chronic-restraint stress (CRS). They reveal a key role for epigenetic up- and down-regulation of the putative presynaptic type 2 metabotropic glutamate (mGlu2) receptors and the postsynaptic NR1/NMDA receptors in the hippocampus and particularly in the dentate gyrus (DG), a region of active neurogenesis and a target of antidepressant treatment. We show changes in DG long-term potentiation (LTP) that parallel behavioral responses, with habituation to the same acute restraint stressor and sensitization to a novel forced-swim stressor. In WT mice after CRS and in unstressed mice with a BDNF loss-of-function allele (BDNF Val66Met), we show that the epigenetic activator of histone acetylation, P300, plays a pivotal role in the dynamic up- and down-regulation of mGlu2 in hippocampus via histone-3-lysine-27-acetylation (H3K27Ac) when acute stressors are applied. These hippocampal responses reveal a window of epigenetic plasticity that may be useful for treatment of disorders in which glutamatergic transmission is dysregulated.