Giacinto Bagetta

CXCR4-activated astrocyte glutamate release via TNFalpha: amplification by microglia triggers neurotoxicity.

Astrocytes actively participate in synaptic integration by releasing transmitter (glutamate) via a calcium-regulated, exocytosis-like process. Here we show that this process follows activation of the receptor CXCR4 by the chemokine stromal cell-derived factor 1 (SDF-1). An extraordinary feature of the ensuing signaling cascade is the rapid extracellular release of tumor necrosis factor-α (TNFα). Autocrine/paracrine TNFα-dependent signaling leading to prostaglandin (PG) formation not only controls glutamate release and astrocyte communication, but also causes their derangement when activated microglia cooperate to dramatically enhance release of the cytokine in response to CXCR4 stimulation. We demonstrate that altered glial communication has direct neuropathological consequences and that agents interfering with CXCR4-dependent astrocyte–microglia signaling prevent neuronal apoptosis induced by the HIV-1 coat glycoprotein, gp120IIIB. Our results identify a new pathway for glia–glia and glia–neuron communication that is relevant to both normal brain function and neurodegenerative diseases.

Post-ischemic brain damage: pathophysiology and role of inflammatory mediators

Neuroinflammatory mediators play a crucial role in the pathophysiology of brain ischemia, exerting either deleterious effects on the progression of tissue damage or beneficial roles during recovery and repair. Within hours after the ischemic insult, increased levels of cytokines and chemokines enhance the expression of adhesion molecules on cerebral endothelial cells, facilitating the adhesion and transendothelial migration of circulating neutrophils and monocytes. These cells may accumulate in the capillaries, further impairing cerebral blood flow, or extravasate into the brain parenchyma. Infiltrating leukocytes, as well as resident brain cells, including neurons and glia, may release pro‐inflammatory mediators, such as cytokines, chemokines and oxygen/nitrogen free radicals that contribute to the evolution of tissue damage. Moreover, recent studies have highlighted the involvement of matrix metalloproteinases in the propagation and regulation of neuroinflammatory responses to ischemic brain injury. These enzymes cleave protein components of the extracellular matrix such as collagen, proteoglycan and laminin, but also process a number of cell‐surface and soluble proteins, including receptors and cytokines such as interleukin‐1β. The present work reviewed the role of neuroinflammatory mediators in the pathophysiology of ischemic brain damage and their potential exploitation as drug targets for the treatment of cerebral ischemia.

Calpain-mediated cleavage of Beclin-1 and autophagy deregulation following retinal ischemic injury in vivo

Autophagy is the major intracellular degradation pathway that regulates long-lived proteins and organelles turnover. This process occurs at basal levels in all cells but it is rapidly upregulated in response to starvation and cellular stress. Although being recently implicated in neurodegeneration, it remains still unclear whether autophagy has a detrimental or protective role. In this study, we investigated the dynamics of the autophagic process in retinal tissue that has undergone transient ischemia, an experimental model that recapitulates features of ocular pathologies, including glaucoma, anterior ischemic optic neuropathy and retinal vessels occlusion. Retinal ischemia, induced in adult rats by increasing the intraocular pressure, was characterized by a reduction in the phosphatidylethanolamine-modified form of LC3 (LC3II) and by a significant decrease in Beclin-1. The latter event was associated with a proteolytic cleavage of Beclin-1, leading to the accumulation of a 50-kDa fragment. This event was prevented by intravitreal treatment with the non-competitive N-methyl-D-aspartate antagonist MK801 and calpain inhibitors or by calpain knockdown. Blockade of autophagy by pharmacological inhibition or Beclin-1 silencing in RGC-5 increased cell death, suggesting a pro-survival role of the autophagic process in this neuronal cell type. Altogether, our results provide original evidence for calpain-mediated cleavage of Beclin-1 and deregulation of basal autophagy in the rat retina that has undergone ocular ischemia/reperfusion injury.

Rational modulation of the innate immune system for neuroprotection in ischemic stroke

The innate immune system plays a dualistic role in the evolution of ischemic brain damage and has also been implicated in ischemic tolerance produced by different conditioning stimuli. Early after ischemia, perivascular astrocytes release cytokines and activate metalloproteases (MMPs) that contribute to blood–brain barrier (BBB) disruption and vasogenic oedema; whereas at later stages, they provide extracellular glutamate uptake, BBB regeneration and neurotrophic factors release. Similarly, early activation of microglia contributes to ischemic brain injury via the production of inflammatory cytokines, including tumor necrosis factor (TNF) and interleukin (IL)-1, reactive oxygen and nitrogen species and proteases. Nevertheless, microglia also contributes to the resolution of inflammation, by releasing IL-10 and tumor growth factor (TGF)-β, and to the late reparative processes by phagocytic activity and growth factors production. Indeed, after ischemia, microglia/macrophages differentiate toward several phenotypes: the M1 pro-inflammatory phenotype is classically activated via toll-like receptors or interferon-γ, whereas M2 phenotypes are alternatively activated by regulatory mediators, such as ILs 4, 10, 13, or TGF-β. Thus, immune cells exert a dualistic role on the evolution of ischemic brain damage, since the classic phenotypes promote injury, whereas alternatively activated M2 macrophages or N2 neutrophils prompt tissue remodeling and repair. Moreover, a subdued activation of the immune system has been involved in ischemic tolerance, since different preconditioning stimuli act via modulation of inflammatory mediators, including toll-like receptors and cytokine signaling pathways. This further underscores that the immuno-modulatory approach for the treatment of ischemic stroke should be aimed at blocking the detrimental effects, while promoting the beneficial responses of the immune reaction.

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.

Retinal damage caused by high intraocular pressure-induced transient ischemia is prevented by coenzyme Q10 in rat.

Recent studies support a role for excitotoxicity in the development of retinal ganglion cell (RGC) damage in subjects suffering from glaucoma. Coenzyme Q10 (CoQ10), an essential cofactor of the electron transport chain, has been reported to afford neuroprotection, preventing the formation of the mitochondrial permeability transition pore. Using an established animal model of retinal ischemia/reperfusion here, we show that synaptic glutamate increases at 130min from beginning of reperfusion and delayed apoptosis in the RGC layer is seen at 24h. Intraocular administration of CoQ10 minimizes glutamate increase and affords neuroprotection, suggesting that oxidative stress and energy failure might be implicated in the mechanisms of RGC death.

Ventral tegmental area: site through which dopamine D2-receptor agonists evoke behavioural and electrocortical sleep in rats

1 In freely moving rats the effects on behaviour and electrocortical (ECoG) spectrum power of some dopamine agonists, i.e. apomorphine and (+)−3PPP, given directly into different areas of the rat brain were studied. In particular, dopamine agonists were microinfused in the ventral tegmental area (VTA) and substantia nigra (SN) or into the caudate nucleus, n. accumbens and prefrontal cortex. The ECoG spectrum power effects were continuously analysed by means of a computerized Berg‐Fourier analyser as total spectrum power and power in preselected frequency bands. 2 Apomorphine and (+)−3PPP (0.01, 0.1 and 1.0 nmol) given bilaterally into the VTA produced behavioural and ECoG sleep in a dose‐dependent fashion. A statistically significant (P < 0.01) increase in ECoG total spectrum power with a predominant increase in the lower frequency bands (0.25–3, 3–6 and 6–9 Hz) occurred. No behavioural and ECoG changes were evoked by the same doses of apomorphine bilaterally microinfused into the SN or into the caudate nucleus or by (+)−3PPP (1.0 nml) microinjected into the n. accumbens or applied onto the prefrontal cortex. 3 Behavioural and ECoG sleep was also induced in rats after systemic administration of apomorphine (263 nmol kg−1, i.p.). 4 The behavioural and ECoG spectrum power effects of apomorphine (1.0 nmol) bilaterally microinfused into the VTA were prevented by a previous microinjection into the same site of (−)‐sulpiride (9.8 nmol). Similarly, behavioural and ECoG effects evoked by (+)−3PPP (0.1 nmol) given bilaterally into the VTA, were completely antagonized by a previous injection into the same site of haloperidol (16 pmol given 10 min before). In contrast, pretreatment with SCH 23390 (50 μg kg−1, s.c.), a selective antagonist at dopamine D1‐receptors, was unable to antagonize the behavioural and ECoG spectrum power effects of (+)−3PPP. 5 Soporific effects induced by systemic administration of apomorphine were antagonized by (−)‐sulpiride (9.8 nmol) given bilaterally into the VTA 10 min before, whereas, yohimbine (1.3 nmol), (an antagonist at α2‐adrenoceptors) bilaterally microinfused into the VTA, was ineffective in this respect. 6 The present experiments provide evidence suggesting that stimulation of dopamine D2‐receptors located at the cell body level and/or the dendrites of dopaminergic neurones in the VTA may represent the mechanism through which apomorphine or (+)−3PPP exert their soporific effects in rats.

Cell signaling pathways in the mechanisms of neuroprotection afforded by bergamot essential oil against NMDA-induced cell death in vitro

The effects of bergamot essential oil (BEO; Citrus bergamia, Risso) on excitotoxic neuronal damage was investigated in vitro.

BRAIN REGIONAL AND CELLULAR LOCALIZATION OF GELATINASE ACTIVITY IN RAT THAT HAVE UNDERGONE TRANSIENT MIDDLE CEREBRAL ARTERY OCCLUSION

Matrix metalloproteinases (MMPs) have been implicated in the pathophysiology of ischemic stroke. In particular, the gelatinases MMP-2 and MMP-9 contribute to disruption of the blood-brain barrier and hemorrhagic transformation following ischemic injury. In addition to extracellular matrix degradation, MMPs may directly regulate neuronal cell death through mechanisms that are not completely understood. Here we describe the spatio-temporal distribution of activated MMP-2 and MMP-9 in the brain of rats subjected to 2 h middle cerebral artery occlusion (MCAo) followed by different periods of reperfusion (15 min, 2 h, 6 h and 22 h). By in situ zymography we have observed that gelatinases become activated 15 min and 2 h after the beginning of reperfusion in the ischemic core and penumbra, respectively. In situ zymography signal broadly co-localized with NeuN-positive cells, thus suggesting that proteolysis mainly occurs in neurons. Gelatinolytic activity was mainly detected in cell nuclei, marginally appearing in the cytosol only at later stages following the insult; we did not detect variations in gelatinolysis in the extracellular matrix. Finally, we report that pharmacological inhibition of MMPs by N-[(2R)-2-(hydroxamidocarbonyl-methyl)-4-methylpenthanoyl]-L-tryptophan methylamide (GM6001) significantly reduces brain infarct volume induced by transient MCAo. Taken together our data underscore the crucial role of gelatinases during the early stages of reperfusion and further extend previous observations documenting the detrimental role of these enzymes in the pathophysiology of brain ischemia.

p73-alpha is capable of inducing scotin and ER stress

p73, like its family member p53, can induce programmed cell death following DNA damage. Here, we report that this mechanism also involves endoplasmic reticulum (ER) stress and the transactivation of scotin, a protein identified recently as a p53 target able to induce ER stress. By using Tet-On inducible cell lines (Saos 2 osteosarcoma cells that lack p53), we observed that TAp73α elicits significant alterations in the morphology of the ER system, namely in the fine subcellular localization of calnexin. We found that both TAp73α and p53 are strong inducers of scotin. On the other hand, the transcriptionally deficient short isoforms ΔNp73α did not upregulate the steady-state mRNA level of scotin, as evaluated by real-time RT–PCR. Following the induction of scotin, ER staining with calnexin showed evidence of morphological alteration, with variations in the intracellular concentration of free calcium, visualized by fluo-3 staining. The induction of ER stress by p73 was further supported by the transcriptional induction of Gadd 153, a transcription factor induced under ER stress conditions. In conclusion, the data reported indicate the ability of TAp73α and p53 (not ΔNp73α) to elicit scotin transactivation and ER stress. This molecular mechanism might contribute to the effector events inducing apoptosis downstream of p73.