Rossella Russo

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.

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.

Early upregulation of matrix metalloproteinases following reperfusion triggers neuroinflammatory mediators in brain ischemia in rat.

Abnormal expression of matrix metalloproteinases (MMPs) has been implicated in the pathophysiology of neuroinflammatory processes that accompany most central nervous system disease. In particular, early upregulation of the gelatinases MMP-2 and MMP-9 has been shown to contribute to disruption of the blood-brain barrier and to death of neurons in ischemic stroke. In situ zymography reveals a significant increase in gelatinolytic MMPs activity in the ischemic brain hemisphere after 2-h middle cerebral artery occlusion (MCAo) followed by 2-h reperfusion in rat. Accordingly, gel zymography demonstrates that expression and activity of MMP-2 and MMP-9 are enhanced in cortex and striatum ipsilateral to the ischemic insult. The latter effect appears to be instrumental for development of delayed brain damage since administration of a broad spectrum, highly specific MMPs inhibitor, GM6001, but not by its negative control, results in a significant (50%) reduction in ischemic brain volume. Increased gelatinase activity in the ischemic cortex coincides with elevation (166% vs sham) of mature interleukin-1beta (IL-1beta) after 2-h reperfusion and this does not appear to implicate a caspase-1-dependent processing of pro(31kDa)-IL-1beta to yield mature (17kDa) IL-1beta. More importantly, when administered at a neuroprotective dose GM6001 abolishes the early IL-1beta increase in the ischemic cortex and reduces the cleavage of the cytokine proform supporting the deduction that MMPs may initiate IL-1beta processing. In conclusion, development of tissue damage that follows transient ischemia implicates a crucial interplay between MMPs and mediators of neuroinflammation (e.g., IL-1beta), and this further underscores the therapeutic potential of MMPs inhibitors in the treatment of stroke.

17β-Estradiol prevents retinal ganglion cell loss induced by acute rise of intraocular pressure in rat

Glaucoma, is a progressive optic neuropathy often associated with increased intraocular pressure (IOP) and characterized by progressive death of retinal ganglion cells (RGCs). High acute rise of IOP is a model for retinal ischemia and may represent a model of acute angle closure glaucoma. Here we have used this experimental model in combination with a neurochemical and neuropathological approach to gain more insight in the neuroprotective profile of 17beta-estradiol (E2), a steroid hormone, which has been shown to increase the viability, survival, and differentiation of primary neuronal cultures from different brain areas including amygdala, hypothalamus, and neocortex. Our data demonstrate that systemic administration of E2 significantly reduces RGC loss induced by high IOP in rat. In addition, pretreatment with E2, 30 min before ischemia, minimizes the elevation of glutamate observed during the reperfusion period. These effects seem to be in part mediated by the activation of the estrogen receptor, since a pretreatment with ICI 182-780, a specific estrogen receptor antagonist, partially counteracts the neuroprotection afforded by the estrogen.

Rational basis for the development of coenzyme Q10 as a neurotherapeutic agent for retinal protection.

Glaucoma is a worldwide leading cause of irreversible vision loss characterized by progressive death of retinal ganglion cells (RGCs). In the course of glaucoma, RGC death may be the consequence of energy impairment that triggers secondary excitotoxicity and free radical generation. There is substantial evidence also that a number of free radical scavengers and/or agents that improve mitochondrial function may be useful as therapies to ameliorate cell death in various neurological disorders including glaucoma. Coenzyme Q10 (CoQ10), an essential cofactor of the electron transport chain, has been reported to afford neuroprotection in neurodegenerative diseases, such as Alzheimers and Parkinsons diseases, and its protective effect has been attributed in part to its free radical scavenger ability and to a specific regulation of the mitochondrial permeability transition pore. Using an established animal model of transient retinal ischemia, we have conclusively identified a role for abnormal elevation of extracellular glutamate in the mechanisms underlying RGC death that occurs, at least in part, via activation of the apoptotic program. Under these experimental conditions, N-methyl-D-aspartate (NMDA) and non-NMDA subtype of glutamate receptor antagonists, nitric oxide synthase inhibitors, and CoQ10 afford retinal protection supporting an important role for excitotoxicity in the mechanisms underlying RGC death.

Implication of limonene and linalyl acetate in cytotoxicity induced by bergamot essential oil in human neuroblastoma cells.

Bergamot (Citrus bergamia, Risso et Poiteau) essential oil (BEO) is a widely used plant extract showing anxiolytic, analgesic and neuroprotective effects in rodents; also, BEO activates multiple death pathways in cancer cells. Despite detailed knowledge of its chemical composition, the constituent/s responsible for these pharmacological activities remain largely unknown. Aim of the present study was to identify the components of BEO implicated in cell death. To this end, limonene, linalyl acetate, linalool, γ-terpinene, β-pinene and bergapten were individually tested in human SH-SY5Y neuroblastoma cultures at concentrations comparable with those found in cytotoxic dilutions of BEO. None of the tested compounds elicited cell death. However, significant cytotoxicity was observed when cells were cotreated with limonene and linalyl acetate whereas no other associations were effective. Only cotreatment, but not the single exposure to limonene and linalyl acetate, replicated distinctive morphological and biochemical changes induced by BEO, including caspase-3 activation, PARP cleavage, DNA fragmentation, cell shrinkage, cytoskeletal alterations, together with necrotic and apoptotic cell death. Collectively, our findings suggest a major role for a combined action of these monoterpenes in cancer cell death induced by BEO.

Autophagy impairment in a mouse model of neuropathic pain

Autophagy is an intracellular membrane trafficking pathway controlling the delivery of cytoplasmic material to the lysosomes for degradation. It plays an important role in cell homeostasis in both normal settings and abnormal, stressful conditions. It is now recognised that an imbalance in the autophagic process can impact basal cell functions and this has recently been implicated in several human diseases, including neurodegeneration and cancer.Here, we investigated the consequences of nerve injury on the autophagic process in a commonly used model of neuropathic pain. The expression and modulation of the main autophagic marker, the microtubule-associated protein 1 light chain 3 (LC3), was evaluated in the L4-L5 cord segment seven days after spinal nerve ligation (SNL). Levels of LC3-II, the autophagosome-associated LC3 form, were markedly higher in the spinal cord ipsilateral to the ligation side, appeared to correlate with the upregulation of the calcium channel subunit α2δ-1 and were not present in mice that underwent sham surgery. However, LC3-I and Beclin 1 expression were only slightly increased. On the contrary, SNL promoted the accumulation of the ubiquitin- and LC3-binding protein p62, which inversely correlates with autophagic activity, thus pointing to a block of autophagosome turnover.Our data showed for the first time that basal autophagy is disrupted in a model of neuropathic pain.

Modulation of pro-survival and death-associated pathways under retinal ischemia/reperfusion: effects of NMDA receptor blockade.

Loss of retinal ganglion cells occurs in a variety of pathological conditions, including central retinal artery occlusion, diabetes and glaucoma. Using an experimental model of retinal ischemia induced by transiently raise the intraocular pressure (IOP), In this study, we report the original observation that ischemic retinal ganglion cells death is associated with the transient deactivation of the pro‐survival kinase Akt and activation of GSK‐3β followed, during reperfusion, by a longer lasting, PI3K‐dependent, activation of Akt and phosphorylation of GSK‐3β. Under these experimental conditions, retinal ischemia induced the expression of Bad, a pro‐apoptotic protein, member of the Bcl‐2 family. The detrimental effects yielded by the ischemic stimulus were minimized by intravitreal administration of the NMDA receptor antagonist, MK801, that reduced the expression of Bad and significantly increased Akt phosphorylation. In conclusion, our present results contribute to unravel the mechanisms underlying retinal damage by high IOP‐induced transient ischemia in rat. In addition, these data implicate the pro‐survival PI3K/Akt pathway and the observed reduced expression of Bad in the neuroprotection afforded by MK801.

Neuroprotection by leptin in a rat model of permanent cerebral ischemia: effects on STAT3 phosphorylation in discrete cells of the brain

In addition to its effects in the hypothalamus to control body weight, leptin is involved in the regulation of neuronal function, development and survival. Recent findings have highlighted the neuroprotective effects of leptin against ischemic brain injury; however, to date, little is known about the role performed by the signal transducer and activator of transcription (STAT)-3, a major mediator of leptin receptor transduction pathway in the brain, in the beneficial effects of the hormone. Our data demonstrate that systemic acute administration of leptin produces neuroprotection in rats subjected to permanent middle cerebral artery occlusion (MCAo), as revealed by a significant reduction of the brain infarct volume and neurological deficit up to 7 days after the induction of ischemia. By combining a subcellular fractionation approach with immunohistofluorescence, we observe that neuroprotection is associated with a cell type-specific modulation of STAT3 phosphorylation in the ischemic cortex. The early enhancement of nuclear phospho-STAT3 induced by leptin in the astrocytes of the ischemic penumbra may contribute to a beneficial effect of these cells on the evolution of tissue damage. In addition, the elevation of phospho-STAT3 induced by leptin in the neurons after 24 h MCAo is associated with an increased expression of tissue inhibitor of matrix metalloproteinases-1 in the cortex, suggesting its possible involvement to the neuroprotection produced by the adipokine.

Identification of novel pharmacological targets to minimize excitotoxic retinal damage.

Excitotoxic neuronal death is a common feature of neurodegenerative and ischemic diseases of the central nervous system (CNS) and of a variety of ocular diseases, including glaucoma. Glaucoma, one of the leading causes of blindness in the world, is characterized by a progressive degeneration of retinal ganglion cells (RGCs) and their axons and is often associated with elevated intraocular pressure (IOP). Retinal ischemia/reperfusion induced by experimental elevation of IOP leads to damage and loss of RGCs. Under these conditions, structural, functional, and biochemical changes implicate the accumulation of extracellular glutamate and activation of the excitotoxic cascade. Beside the activation of associated pathways, death of RGCs is accompanied by impaired endogenous defenses, such as the PI3K/Akt prosurvival pathway. Original neurochemical and pharmacological evidence are discussed here to strengthen the role for excitotoxicity in RGCs death occurring in experimental, angle closure, glaucoma in conjunction with the discovery of novel molecular targets to potentiate endogenous prosurvival defenses in the glaucomatous retina.