Begoña G. Miguel

β-N-methylamino-l-alanine causes neurological and pathological phenotypes mimicking Amyotrophic Lateral Sclerosis (ALS): the first step towards an experimental model for sporadic ALS.

β-N-methylamino-l-alanine (L-BMAA) is a neurotoxic amino acid that has been related to various neurodegenerative diseases. The aim of this work was to analyze the biotoxicity produced by L-BMAA in vivo in rats, trying to elucidate its physiopathological mechanisms and to search for analogies between the found effects and pathologies like Amyotrophic Lateral Sclerosis (ALS). Our data demonstrated that the neurotoxic effects in vivo were dosage-dependent. For evaluating the state of the animals, a neurological evaluation scale was developed as well as a set of functional tests. Ultrastructural cell analysis of spinal motoneurons has revealed alterations both in endoplasmic reticulum and mitochondria. Since GSK3β could play a role in some neuropathological processes, we analyzed the alterations occurring in GSK3β levels in L-BMAA treated rats, we have observed an increase in the active form of GSK3β levels in lumbar spinal cord and motor cerebral cortex. On the other hand, (TAR)-DNA-binding protein 43 (TDP-43) increased in L-BMAA treated animals. Our results indicated that N-acetylaspartate (NAA) declined in animals treated with L-BMAA, and the ratio of N-acetylaspartate/choline (NAA/Cho), N-acetylaspartate/creatine (NAA/Cr) and N-acetylaspartate/choline+creatine (NAA/Cho+Cr) tended to decrease in lumbar spinal cord and motor cortex. This project offers some encouraging results that could help establishing the progress in the development of an animal model of sporadic ALS and L-BMAA could be a useful tool for this purpose.

Analysis of the role of the PAC1 receptor in neutrophil recruitment, acute-phase response, and nitric oxide production in septic shock

Infections caused by Gram‐negative bacteria constitute one of the major causes of septic shock, which results from the inability of the immune system to limit bacterial spread during the ongoing infection. In the last decade, it has been demonstrated that vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase‐activing polypeptide (PACAP) are two endogenous immunopeptides, which together with three G protein‐coupled receptors (VPAC1, VPAC2, and PAC1) exert a significant, therapeutic effect attenuating the deleterious consequences of septic shock by balancing pro‐ and anti‐inflammatory factors. We have recently shown PAC1 receptor involvement in vivo as an anti‐inflammatory receptor, at least in part, by attenuating lipopolysaccharide‐induced production of proinflammatory interleukin‐6. The present study deepens in the protective role of PAC1 receptor in septic shock, elucidating its involvement in the modulation of neutrophil recruitment and in the expression of different molecular sensors such as intercellular adhesion molecule‐1, vascular cell adhesion molecule‐1, fibrinogen, serum amyloid A, and nitric oxide as important, systemic players of the development of septic shock. Our results, using a mice deficient in PAC1 and a PAC1 antagonist, show that VIP and PACAP as well as the PAC1 receptor are involved in neutrophil recruitment in different target organs, in adhesion molecules expression, and in coagulation‐related molecule fibrinogen synthesis. Thus, this study provides some important insights with respect to the involvement of PAC1 into the complexities of sepsis and represents an advantage for the design of more specific drugs complementing standard intensive care therapy in severe sepsis, confirming VIP and PACAP as candidates for multitarget therapy of septic shock.

Involvement of phosphatidylinositol 3-kinase in nuclear translocation of protein kinase C ζ induced by C2-ceramide in rat hepatocytes

In this study we report that protein kinase C ζ (PKC ζ), one of the atypical isoforms of the PKC family located predominantly in cytosol, is redistributed by C2‐ceramide treatment in isolated hepatocytes. PKC ζ increased in membrane and nuclear fractions after 30 min of treatment with C2‐ceramide in a dose‐ and time‐dependent manner. The action of C2‐ceramide was inhibited by wortmannin and LY 294002, indicating that C2‐ceramide‐induced PKC ζ increase in both nucleus and membrane fractions is mediated by phosphatidylinositol 3‐kinase (PI3‐kinase) activation. In addition, a significant translocation of PI3‐kinase to the nucleus was observed after C2‐ceramide treatment.

Insulin action on brain microvessels; Effect on alkaline phosphatase

The effects of insulin on brain alkaline phosphatase activity have been examined. Insulin inhibited the activity of alkaline phosphatase on brain microvessels in in vitro experiments. The inhibition observed was of the non-competitive type. These observations indicate that the hormone is able to induce neurochemical modifications revealed in this case as changes in the phosphate transfer enzymes in brain microvessels.

Role of protein kinase C and mitochondrial permeability transition pore in the neuroprotective effect of ceramide in ischemia-induced cell death

In this study, we investigated the role of protein kinase C (PKC) and mitochondrial permeability transition pore (mPTP) on the effect of ceramide in an in vitro model of ischemia in SH‐SY5Y neuroblastoma cells. In ischemic cell viability studies, a dual effect of ceramide was observed, depending on ceramide concentration. PKC isoforms are involved in the protective effect of low concentrations of ceramide. During ischemia, ceramide treatment leads to an increase in the formation of reactive oxygen species (ROS), which induces a controlled opening of mPTP. This fact prevents mitochondrial Ca2+ overload, which is clearly protective.

Involvement of mitochondria on neuroprotective effect of sphingosine-1-phosphate in cell death in an in vitro model of brain ischemia.

Sphingosine-1-phosphate (S1P) has been demonstrated to be an important regulator of cell death and survival. Although it has been suggested that the sphingolipid may act as a neuroprotector in the cell apoptosis induced by traumatic brain injury, the mechanisms involved in this action are unknown. In this study, the relationship between S1P and neuroprotective effect was studied in an in vitro model of ischemia, maintaining SH-SY5Y human neuroblastoma cells under oxygen-glucose deprivation (OGD). When cells were treated with 1 microM S1P simultaneously with OGD and recovery, cell viability increases in a dose-response manner. S1P treatment reduces significantly both necrosis and apoptosis cell death. On the other hand, the treatment with specific PKC epsilon (V1-2), prevents S1P protective effect of OGD/recovery-induced necrosis. Moreover, S1P treatment provokes the translocation of PKC epsilon to the mitochondria. From these results, it is reasonable to assume that S1P protection from necrosis is mediated by PKC epsilon. We also studied the action of S1P on mitochondrial inner membrane potential and mitochondrial Ca(2+) levels during ischemia. In this regard, we must point out that S1P treatment reduces the OGD-induced membrane depolarization and also reduces the increase of Ca(2+) in mitochondria during OGD. Results also indicate that mitochondria from OGD treated cells have significantly less ability to resist swelling on Ca(2+) loading than those obtained in presence of oxygen and glucose. Nevertheless, when S1P was added, this resistance increases considerably. These findings suggest that S1P may have a potential role as a neuroprotective agent in brain injury.

PAF-induced activation of polyphosphoinositide-hydrolyzing phospholipase C in cerebral cortex

The action of platelet-activating factor (PAF) on phosphoinositide hydrolysis was studied in rat brain slices. PAF produced a significant increase of 32P incorporation into phosphoinositides and phosphatidic acid (PA), in a dose- and time-dependent manner. Concomitantly, an increase of inositol phosphates and diacylglycerol (DAG) production was observed. Both inositol bisphosphate (IP2) and inositol trisphosphate (IP3) were detected as early as 5 s and they returned immediately to basal levels; concomitantly, formation of inositol monophosphate (IP) was detected. These findings demonstrated that PAF causes a rapid hydrolysis of polyphosphoinositides in cerebral cortex by a phospholipase C-dependent mechanism followed by subsequent resynthesis.

Effect of β-N-methylamino-L-alanine on oxidative stress of liver and kidney in rat.

β-N-methylamino-(L)-alanine (L)-BMAA) is a neurotoxic amino acid, found in the majority of cyanbacterial genera tested. Evidence for implication of (L)-BMAA in neurodegenerative disorders, like amyotrophic lateral sclerosis (ALS), relies on bioaccumulation and biomagnification from symbiotic cyanobacteria. The involvement of (L)-BMAA in oxidative stress was demonstrated in several studies in the central nervous system. In the present study, we investigated the effect of (L)-BMAA on the oxidative stress responses of liver and kidney in rats treated by intraperitoneal administration with this amino acid. Oxidative stress was demonstrated by the quantification of lipid peroxidation, the measurement of both catalase and glutathione peroxidase activities, as well as the quantification of glutathione (GSH) levels and the total antioxidant capacity. It was observed that (L)-BMAA caused a significant increase in the degree of lipid peroxidation and catalase activity in both organs. A significant increase in glutathione peroxidase activity was obtained only in liver, whereas glutathione levels were also increased in both organs. The total antioxidant capacity decreased in liver and increased in kidney. These results suggest that the oxidative stress was higher in liver than in kidney, and might be crucial for (L)-BMAA toxicological action.

Increase of phosphoinositide hydrolysis and diacylglycerol production by PAF in isolated rat liver nuclei.

When isolated rat liver nuclei were treated with platelet-activating factor (PAF), a rapid increase in the mass of diacylglycerol (DAG) occurred. This effect was dose- and time-dependent. The maximum effect was observed after 1 min of 10(-7) M PAF treatment. A concomitant decrease of polyphosphoinositides and phosphatidic acid (PA) levels was observed. PAF-induced DAG accumulation was inhibited by the treatment with WEB 2086 or PCA-4248, specific PAF-receptor antagonists. This result may suggest that PAF exerts its action in the nucleus through specific nuclear PAF binding sites. The findings described herein are due to the activation of phospholipase C, as the results from experiments using U73122, a phospholipase C inhibitor, indicate. These are the first data on the action of

Sphingosylphosphorylcholine increases calcium concentration in isolated brain nuclei.

Sphingosylphosphorylcholine (SPC) caused a rapid increase of Ca2+ concentration in isolated brain nuclei. This effect was prevented by nimodipine, an inhibitor of L-type Ca2+ channels, and by thapsigargin, an inhibitor of Ca(2+)-ATPase. Neither heparin nor U73122 modified this effect, suggesting that phospholipase C activation and inositol 1,4,5-trisphosphate (IP3) production are not involved. Results also indicated that SPC-induced increase in Ca2+ concentration is not protein kinase C-dependent.