Hugo Peluffo

Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis.

Oxidative stress mediated by nitric oxide (NO) and its toxic metabolite peroxynitrite has previously been associated with motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Degenerating spinal motor neurons in familial and sporadic ALS are typically surrounded by reactive astrocytes expressing the inducible form of NO synthase (iNOS), suggesting that astroglia may have a pathogenic role in ALS. We report here that a brief exposure of spinal cord astrocyte monolayers to peroxynitrite (0.25–1 mM) provoked long‐lasting reactive morphological changes characterized by process‐bearing cells displaying intense glial fibrillary acidic protein and iNOS immunoreactivity. Furthermore, peroxynitrite caused astrocytes to promote apoptosis of embryonic motor neurons subsequently plated on the monolayers. Neuronal death occurred within 24 hr after plating, as evidenced by the presence of degenerating motor neurons positively stained for activated caspase‐3 and nitrotyrosine. Motor neuron death was largely prevented by NOS inhibitors and peroxynitrite scavengers but not by trophic factors that otherwise will support motor neuron survival in the absence of astrocytes. The bacterial lipopolysaccharide, a well‐known inflammatory stimulus that induces iNOS expression in astrocytes, provoked the same effects on astrocytes as peroxynitrite. Thus, spinal cord astrocytes respond to extracellular peroxynitrite by adopting a phenotype that is cytotoxic to motor neurons through peroxynitrite‐dependent mechanisms.

Extracellular ATP and the P2X7 receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis

BackgroundDuring pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X7 receptors. In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X7 receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1G93A astrocytes.MethodsWe evaluated motor neuron survival after co-culture with SOD1G93A or non-transgenic astrocytes pretreated with agents known to modulate ATP release or P2X7 receptor. We also characterized astrocyte proliferation and extracellular ATP degradation.ResultsRepeated stimulation by ATP or the P2X7-selective agonist BzATP caused astrocytes to become neurotoxic, inducing death of motor neurons. Involvement of P2X7 receptor was further confirmed by Brilliant blue G inhibition of ATP and BzATP effects. In SOD1G93A astrocyte cultures, pharmacological inhibition of P2X7 receptor or increased extracellular ATP degradation with the enzyme apyrase was sufficient to completely abolish their toxicity towards motor neurons. SOD1G93A astrocytes also displayed increased ATP-dependent proliferation and a basal increase in extracellular ATP degradation.ConclusionsHere we found that P2X7 receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death. Remarkably, the neurotoxic phenotype of SOD1G93A astrocytes depended upon basal activation the P2X7 receptor. Thus, pharmacological inhibition of P2X7 receptor might reduce neuroinflammation in ALS through astrocytes.

Riluzole promotes survival of rat motoneurons in vitro by stimulating trophic activity produced by spinal astrocyte monolayers.

In the present study we have assessed whether riluzole stimulates the production of trophic activities for motoneurons by spinal astrocyte cultures. Astrocyte monolayers prepared from new-born rats were exposed to vehicle or riluzole (1-10 microM) for 30-36 h, then washed and further incubated without riluzole for 24 h in L15 medium to obtain the astrocyte conditioned media (ACM). Motoneuron-enriched cultures were used to test the ability of the ACM to support motoneuron viability. Astrocyte monolayers exposed to 1 microM riluzole did not show changes in morphology or in DNA or protein synthesis. However, the conditioned medium obtained from astrocyte monolayers after this treatment increased motoneuron survival compared to that from vehicle-treated cultures. A similar effect was found when astrocytes were exposed to a higher riluzole concentration (10 microM) but with greater dilutions of the conditioned medium. This trophic activity was abolished by boiling or after treatment with trypsin. These findings strongly suggest the existence of a new trophic mechanism, through which riluzole may exert motoneuron protection.

Expression of inducible nitric oxide synthase and cyclooxygenase-2 after excitotoxic damage to the immature rat brain.

It is well established that after adult brain damage the enzymes cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS) play an important role in the inflammatory processes and oxidative stress, which are considered to be the leading factors contributing to delayed cell death. The contribution of these enzymes to postnatal brain damage, however, is poorly understood. In our study, excitotoxic lesions were induced by the injection of N‐methyl‐D‐aspartate in the cortex of postnatal day 9 rats. After different survival times ranging from 4 hr to 7 days post‐lesion, brain sections were processed for the immunocytochemical demonstration of COX‐2 and iNOS and double labeling with neuronal, glial and neutrophil markers. First and maximal de novo induction of iNOS and COX‐2 expression was found at 10 hr post‐lesion. Expression of both enzymes started to diminish at 24 hr, reaching basal levels at day 3. iNOS‐expressing cells were mainly identified as infiltrated neutrophils as well as highly ramified protoplasmic astrocytes closely associated with blood vessels. Moreover, scattered iNOS‐positive neurons were found at the lesion borders. In contrast, COX‐2 was mainly observed in reactive microglial cells and neuronal cells. COX‐2‐positive neurons were found within the degenerating area at 10 hr and at the borders of the lesion later on. This study shows that maximal iNOS and COX‐2 expression precedes the period of massive neuronal death observed at 24 hr post‐lesion, and may therefore contribute to the evolution of the inflammatory response and the neurodegenerative process after an excitotoxic lesion to the postnatal brain.

Induction of motor neuron apoptosis by free 3-nitro-l-tyrosine

Peroxynitrite‐dependent tyrosine nitration has been postulated to be involved in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Evidence supporting this supposition includes the appearance of both free and protein‐linked 3‐nitro‐l‐tyrosine (nitrotyrosine) in both sporadic and familial ALS, as well as of increased free nitrotyrosine levels in the spinal cord of transgenic mice expressing ALS‐linked superoxide dismutase mutants at symptom onset. Here we demonstrate that incubation with clinically relevant concentrations of nitrotyrosine induced apoptosis in motor neurons cultured with trophic factors. Nitrotyrosine was bound to proteins, but it was not incorporated into α‐tubulin, as previously demonstrated for other cell types. Neither inhibition of nitric oxide production nor scavenging of superoxide and peroxynitrite prevented increases in cell nitrotyrosine immunoreactivity or motor neuron death, suggesting that these effects are not due to the endogenous formation of reactive nitrogen species. In contrast, some populations of astrocytes incorporated nitrotyrosine into α‐tubulin, but free nitrotyrosine had no effect on the viability and phenotype of astrocytes in culture, as evaluated by glial fibrillary acidic protein immunoreactivity, cell growth and morphology. Co‐culture of motor neurons on astrocyte monolayers delayed, but did not prevent, nitrotyrosine‐induced motor neuron death. These results suggest that free nitrotyrosine may play a role in the induction of motor neuron apoptosis in ALS.

Interleukin-10 and interleukin-10 receptor-I are upregulated in glial cells after an excitotoxic injury to the postnatal rat brain.

Inflammation is an important determinant of the severity and outcome of central nervous system injury. The endogenous anti-inflammatory cytokine interleukin-10 (IL-10) is upregulated in the injured adult central nervous system where it controls and terminates inflammatory processes. The developing brain, however, displays differences in susceptibility to insults and in associated inflammatory responses from the adult brain; the anatomic and temporal patterns of injury-induced IL-10 expression in the immature brain after excitotoxic injury are unknown. We analyzed the spaciotemporal gene and protein expression of IL-10 and its receptor (IL-10RI) in N-methyl-d-aspartate-induced excitotoxic injury in 9-day-old and control rats using quantitative reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. In noninjected control brains, both molecules were expressed mainly in white matter on glial cells and blood vessels; IL-10 was also observed on blood vessels in gray matter and in glial fibrillary acidic protein-positive processes in the hippocampus and near leptomeningeal and ventricle surfaces. In N-methyl-d-aspartate-injected brains, IL-10 gene and protein expression were maximal at 72 hours postinjection; IL-10RI gene and protein expression peaked at 48 hours postinjection. Interleukin-10 and IL-10RI expression in injured areas was mainly found in reactive astrocytes and in microglia/macrophages. The expression patterns of IL-10 and IL-10R suggest possible developmental roles, and their upregulation after injury suggests that this expression may have anti-inflammatory effects in distinct anatomic sites in the immature brain.

Cu/Zn superoxide dismutase expression in the postnatal rat brain following an excitotoxic injury

BackgroundIn the nervous system, as in other organs, Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key antioxidant enzyme involved in superoxide detoxification in normal cellular metabolism and after cell injury. Although it has been suggested that immature brain has a different susceptibility to oxidative damage than adult brain, the distribution and cell-specific expression of this enzyme in immature brain and after postnatal brain damage has not been documented.MethodsIn this study, we used immunohistochemistry and western blot to analyze the expression of Cu/Zn SOD in intact immature rat brain and in immature rat brain after an NMDA-induced excitotoxic cortical injury performed at postnatal day 9. Double immunofluorescence labelling was used to identify Cu/Zn SOD-expressing cell populations.ResultsIn intact immature brain, Cu/Zn SOD enzyme was widely expressed at high levels in neurons mainly located in cortical layers II, III and V, in the sub-plate, in the pyriform cortex, in the hippocampus, and in the hypothalamus. Glial fibrillary acidic protein-positive cells only showed Cu/Zn SOD expression in the glia limitans and in scattered cells of the ventricle walls. No expression was detected in interfascicular oligodendroglia, microglia or endothelial cells. Following excitotoxic damage, neuronal Cu/Zn SOD was rapidly downregulated (over 2–4 hours) at the injection site before neurodegeneration signals and TUNEL staining were observed. Later, from 1 day post-lesion onward, an upregulation of Cu/Zn SOD was found due to increased expression in astroglia. A further increase was observed at 3, 5 and 7 days that corresponded to extensive induction of Cu/Zn SOD in highly reactive astrocytes and in the astroglial scar.ConclusionWe show here that, in the intact immature brain, the expression of Cu/Zn SOD was mainly found in neurons. When damage occurs, a strong and very rapid downregulation of this enzyme precedes neuronal degeneration, and is followed by an upregulation of Cu/Zn SOD in astroglial cells.

NLRP3 inflammasome-driven pathways in depression: Clinical and preclinical findings

Over the past three decades, an intricate interaction between immune activation, release of pro-inflammatory cytokines and changes in brain circuits related to mood and behavior has been described. Despite extensive efforts, questions regarding when inflammation becomes detrimental or how we can target the immune system to develop new therapeutic strategies for the treatment of psychiatric disorders remain unresolved. In this context, novel aspects of the neuroinflammatory process activated in response to stressful challenges have recently been documented in major depressive disorder (MDD). The Nod-like receptor pyrin containing 3 inflammasome (NLRP3) is an intracellular multiprotein complex responsible for a number of innate immune processes associated with infection, inflammation and autoimmunity. Recent data have demonstrated that NLRP3 activation appears to bridge the gap between immune activation and metabolic danger signals or stress exposure, which are key factors in the pathogenesis of psychiatric disorders. In this review, we discuss both preclinical and clinical evidence that links the assembly of the NLRP3 complex and the subsequent proteolysis and release of the pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) in chronic stress models and patients with MDD. Importantly, we also focus on the therapeutic potential of targeting the NLRP3 inflammasome complex to improve stress resilience and depressive symptoms.

Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector

BackgroundSuperoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn SOD or the control GFP transgenes 2 hours after intracortical N-methyl-D-aspartate (NMDA) administration, and daily functional evaluation was performed. Moreover, 3 days after, lesion volume, neuronal degeneration and nitrotyrosine immunoreactivity were evaluated.ResultsOverexpression of Cu/Zn SOD transgene after NMDA administration showed improved functional outcome and a reduced lesion volume at 3 days post lesion. In secondary degenerative areas, increased neuronal survival as well as decreased numbers of degenerating neurons and nitrotyrosine immunoreactivity was seen. Interestingly, injection of the NLSCt vector carrying the control GFP transgene also displayed a significant neuroprotective effect but less pronounced.ConclusionWhen the appropriate levels of Cu/Zn SOD are expressed transiently after injury using the non-viral modular protein vector NLSCt a neuroprotective effect is seen. Thus recombinant modular protein vectors may be suitable for in vivo gene therapy, and Cu/Zn SOD should be considered as an interesting therapeutic transgene.

Nonviral gene delivery to the central nervous system based on a novel integrin-targeting multifunctional protein

Successful introduction of therapeutic genes into the central nervous system (CNS) requires the further development of efficient transfer vehicles that avoid viral vector-dependent adverse reactions while maintaining high transfection efficiency. The multifunctional protein 249AL was recently constructed for in vitro gene delivery. Here, we explore the capability of this vector for in vivo gene delivery to the postnatal rat CNS. Significant transgene expression was observed both in the excitotoxically injured and noninjured brain after intracortical injection of the DNA-contaning-249AL vector. In the injured brain, a widespread expression occurred in the entire lesioned area and retrograde transport of the vector toward distant thalamic nuclei and transgene expression were observed. Neurons, astrocytes, microglia, and endothelial cells expressed the transgene. No recruitment of leukocytes, demyelination, interleukin-1beta expression, or increase in astrocyte/microglial activation was observed at 6 days postinjection. In conclusion, the 249AL vector shows promising properties for gene therapy intervention in the CNS, including the targeting of different cell populations.