ROS produced by NOX promote the neurite growth in a PI3K/Akt independent manner.

Reactive oxygen species (ROS) function as signaling molecules in several physiologic and pathologic processes. In central nervous system, ROS are critical for differentiation, migration, polarization, and neurite growth. These actions are mediated by reversible oxidation of target proteins. On the other hand, PI3K/Akt signaling pathway is susceptible to be modulated by ROS and it has been implicated in neurite growth. In this study, we evaluated the participation of ROS in the neurite growth of cultured rat cerebellar granule neurons (CGN), as well as the possible regulation of the PI3K/Akt pathway by ROS during neurite outgrowth. For this purpose, CGN were treated with cellular or mitochondrial antioxidants, or an NOX inhibitor and neurite growth was evaluated. Moreover, to assess the participation Akt in this process, the p-Akt levels were measured in CGN treated with antioxidants or a NOX inhibitor. The effect of antioxidants on the neurite growth in the presence of a PI3K inhibitor was also measured. We found that cellular antioxidants and the NOX inhibitor decreased the neurite growth, but not the mitochondrial antioxidant. Interestingly, the antioxidants increased the p-Akt levels; however, the effect of antioxidants on neurite growth was no dependent on the Akt activity since the inhibitor of PI3K did not modify the antioxidant action on neurite growth. Our results show that the PI3K/Akt pathway participates in neurite growth and that ROS produced by NOX could function as signals in this process; however, this action is not mediated by a redox regulation of Akt activity.

Tibolone Improves Locomotor Function in a Rat Model of Spinal Cord Injury by Modulating Apoptosis and Autophagy.

Spinal cord injury (SCI) harms patients' health and social and economic well-being. Unfortunately, fully effective therapeutic strategies have yet to be developed to treat this disease, affecting millions worldwide. Apoptosis and autophagy are critical cell death signaling pathways after SCI that should be targeted for early therapeutic interventions to mitigate their adverse effects and promote functional recovery. Tibolone (TIB) is a selective tissue estrogen activity regulator (STEAR) with neuroprotective properties demonstrated in some experimental models. This study aimed to investigate the effect of TIB on apoptotic cell death and autophagy after SCI and verify whether TIB promotes motor function recovery. A moderate contusion SCI was produced at thoracic level 9 (T9) in male Sprague Dawley rats. Subsequently, animals received a daily dose of TIB orally and were sacrificed at 1, 3, 14 or 30 days post-injury. Tissue samples were collected for morphometric and immunofluorescence analysis to identify tissue damage and the percentage of neurons at the injury site. Autophagic (Beclin-1, LC3-I/LC3-II, p62) and apoptotic (Caspase 3) markers were also analyzed via Western blot. Finally, motor function was assessed using the BBB scale. TIB administration significantly increased the amount of preserved tissue (p < 0.05), improved the recovery of motor function (p < 0.001) and modulated the expression of autophagy markers in a time-dependent manner while consistently inhibiting apoptosis (p < 0.05). Therefore, TIB could be a therapeutic alternative for the recovery of motor function after SCI.

Role of NADPH oxidase-2 in the progression of the inflammatory response secondary to striatum excitotoxic damage.

BACKGROUND: During excitotoxic damage, neuronal death results from the increase in intracellular calcium, the induction of oxidative stress, and a subsequent inflammatory response. NADPH oxidases (NOX) are relevant sources of reactive oxygen species (ROS) during excitotoxic damage. NADPH oxidase-2 (NOX-2) has been particularly related to neuronal damage and death, as well as to the resolution of the subsequent inflammatory response. As ROS are crucial components of the regulation of inflammatory response, in this work, we evaluated the role of NOX-2 in the progression of inflammation resulting from glutamate-induced excitotoxic damage of the striatum in an in vivo model. METHODS: The striata of wild-type C57BL/6 J and NOX-2 KO mice (gp91Cybbtm1Din/J) were stereotactically injected with monosodium glutamate either alone or in combination with IL-4 or IL-10. The damage was evaluated in histological sections stained with cresyl violet and Fluoro-Jade B. The enzymatic activity of caspase-3 and NOX were also measured. Additionally, the cytokine profile was identified by ELISA and motor activity was verified by the tests of the cylinder, the adhesive tape removal, and the inverted grid. RESULTS: Our results show a neuroprotective effect in mice with a genetic inhibition of NOX-2, which is partially due to a differential response to excitotoxic damage, characterized by the production of anti-inflammatory cytokines. In NOX-2 KO animals, the excitotoxic condition increased the production of interleukin-4, which could contribute to the production of interleukin-10 that decreased neuronal apoptotic death and the magnitude of striatal injury. Treatment with interleukin-4 and interleukin-10 protected from excitotoxic damage in wild-type animals. CONCLUSIONS: The release of proinflammatory cytokines during the excitotoxic event promotes an additional apoptotic death of neurons that survived the initial damage. During the subsequent inflammatory response to excitotoxic damage, ROS generated by NOX-2 play a decisive role in the extension of the lesion and consequently in the severity of the functional compromise, probably by regulating the anti-inflammatory cytokines production.

ROS as Regulators of Mitochondrial Dynamics in Neurons.

Mitochondrial dynamics is a complex process, which involves the fission and fusion of mitochondrial outer and inner membranes. These processes organize the mitochondrial size and morphology, as well as their localization throughout the cells. In the last two decades, it has become a spotlight due to their importance in the pathophysiological processes, particularly in neurological diseases. It is known that Drp1, mitofusin 1 and 2, and Opa1 constitute the core of proteins that coordinate this intricate and dynamic process. Likewise, changes in the levels of reactive oxygen species (ROS) lead to modifications in the expression and/or activity of the proteins implicated in the mitochondrial dynamics, suggesting an involvement of these molecules in the process. In this review, we discuss the role of ROS in the regulation of fusion/fission in the nervous system, as well as the involvement of mitochondrial dynamics proteins in neurodegenerative diseases.

Endonuclease G takes part in AIF-mediated caspase-independent apoptosis in Mycobacterium bovis-infected bovine macrophages.

Mycobacterium bovis, the causative agent of bovine tuberculosis encodes different virulence mechanisms to survive inside of host cells. One of the possible outcomes in this host-pathogen interaction is cell death. Previous results from our group showed that M. bovis induces a caspase-independent apoptosis in bovine macrophages with the possible participation of apoptosis inducing factor mitochondria associated 1 (AIFM1/AIF), a flavoprotein that functions as a cell-death regulator. However, contribution of other caspase-independent cell death mediators in M. bovis-infected macrophages is not known. In this study, we aimed to further characterize M. bovis-induced apoptosis, addressing Endonuclease G (Endo G) and Poly (ADP-ribose) polymerase 1 (PARP-1). In order to accomplish our objective, we infected bovine macrophages with M. bovis AN5 (MOI 10:1). Analysis of M. bovis-infected nuclear protein extracts by immunoblot, identified a 15- and 43-fold increase in concentration of mitochondrial proteins AIF and Endo G respectively. Interestingly, pretreatment of M. bovis-infected macrophages with cyclosporine A, a mitochondrial permeability transition pore inhibitor, abolished AIF and Endo G nuclear translocation. In addition, it also decreased macrophage DNA fragmentation to baseline and caused a 26.2% increase in bacterial viability. We also demonstrated that PARP-1 protein expression in macrophages did not change during M. bovis infection. Furthermore, pretreatment of M. bovis-infected bovine macrophages with 3-aminobenzamide, a PARP-1 inhibitor, did not change the proportion of macrophage DNA fragmentation. Our results suggest participation of Endo G, but not PARP-1, in M. bovis-induced macrophage apoptosis. To the best of our knowledge this is the first report associating Endo G with caspase-independent apoptosis induced by a member of the Mycobacterium tuberculosis complex.

Effect of comprehensive smoke-free legislation on asthma and coronary disease trends in Spanish primary care patients.

Background: To examine the impact of comprehensive smoke-free legislation (SFL) (Law 42/2010) on the incidence and prevalence of adult asthma and coronary disease in primary health care (PHC) patients from three Spanish regions, overall and stratified by sex. Methods: Longitudinal observational study conducted between 2007 and 2013 in the population over 15 years of age assigned to 66 PHC teams in Catalonia, Navarre and the Balearic Islands. Crude rates and age-standardized (truncated: asthma ≥ 16 years and coronary disease ≥ 35 years) incidence and prevalence rates using the direct method based on the European Standard Population were estimated based on data from PHC electronic health records. Joinpoint analysis was used to analyse the trends of age-standardized incidence and prevalence rates. Trends were expressed as annual percentage change and average annual percent change (AAPC). Results: The standardized asthma incidence rate showed a non-significant downward trend and the standardized prevalence rates rose significantly in the three regions. Standardized coronary disease incidence and prevalence rates were considerably higher for men than for women in all regions. The standardized coronary disease incidence rates in Catalonia (AAPC: -8.00%, 95% CI: -10.46; -5.47) and Navarre (AAPC: -3.66%, 95% CI: -4.95;-2.35) showed a significant downward trend from 2007 to 2013, overall and by sex. The standardized coronary disease prevalence trend rate increased significantly in the whole period in Catalonia and the Balearic Islands, although a non-significant downward trend was observed from 2010 in Catalonia. Conclusion: No changes in the trends of adult asthma and coronary disease in PHC Spanish patients were detected after the introduction of comprehensive SFL.

Peroxynitrite is Involved in the Apoptotic Death of Cultured Cerebellar Granule Neurons Induced by Staurosporine, but not by Potassium Deprivation.

Nitric oxide (NO) regulates numerous physiological process and is the main source of reactive nitrogen species (RNS). NO promotes cell survival, but it also induces apoptotic death having been involved in the pathogenesis of several neurodegenerative diseases. NO and superoxide anion react to form peroxynitrite, which accounts for most of the deleterious effects of NO. The mechanisms by which these molecules regulate the apoptotic process are not well understood. In this study, we evaluated the role of NO and peroxynitrite in the apoptotic death of cultured cerebellar granule neurons (CGN), which are known to experience apoptosis by staurosporine (St) or potassium deprivation (K5). We found that CGN treated with the peroxynitrite catalyst, FeTTPs were completely rescued from St-induced death, but not from K5-induced death. On the other hand, the inhibition of the inducible nitric oxide synthase partially protected cell viability in CGN treated with K5, but not with St, while the inhibitor L-NAME further reduced the cell viability in St, but it did not affect K5. Finally, an inhibitor of the soluble guanylate cyclase (sGC) diminished the cell viability in K5, but not in St. Altogether, these results shows that NO promotes cell survival in K5 through sGC-cGMP and promotes cell death by other mechanisms, while in St NO promotes cell survival independently of cGMP and peroxynitrite results critical for St-induced death. Our results suggest that RNS are differentially handled by CGN during cell death depending on the death-inducing conditions.

How does a cadaver model work for testing ultrasound diagnostic capability for rheumatic-like tendon damage?

To establish whether a cadaver model can serve as an effective surrogate for the detection of tendon damage characteristic of rheumatoid arthritis (RA). In addition, we evaluated intraobserver and interobserver agreement in the grading of RA-like tendon tears shown by US, as well as the concordance between the US findings and the surgically induced lesions in the cadaver model. RA-like tendon damage was surgically induced in the tibialis anterior tendon (TAT) and tibialis posterior tendon (TPT) of ten ankle/foot fresh-frozen cadaveric specimens. Of the 20 tendons examined, six were randomly assigned a surgically induced partial tear; six a complete tear; and eight left undamaged. Three rheumatologists, experts in musculoskeletal US, assessed from 1 to 5 the quality of US imaging of the cadaveric models on a Likert scale. Tendons were then categorized as having either no damage, (0); partial tear, (1); or complete tear (2). All 20 tendons were blindly and independently evaluated twice, over two rounds, by each of the three observers. Overall, technical performance was satisfactory for all items in the two rounds (all values over 2.9 in a Likert scale 1-5). Intraobserver and interobserver agreement for US grading of tendon damage was good (mean κ values 0.62 and 0.71, respectively), with greater reliability found in the TAT than the TPT. Concordance between US findings and experimental tendon lesions was acceptable (70-100 %), again greater for the TAT than for the TPT. A cadaver model with surgically created tendon damage can be useful in evaluating US metric properties of RA tendon lesions.

Variability in screening prevention activities in primary care in Spain: a multilevel analysis.

BACKGROUND: Despite evidence of the benefits of prevention activities, studies have reported only partial integration and great variability of screening in daily clinical practice. The study objectives were: 1) To describe Primary Health Care (PHC) screening for arterial hypertension, dyslipidaemia, obesity, tobacco use, and excessive alcohol consumption in 2008 in 2 regions of Spain, based on electronic health records, and 2) To assess and quantify variability in screening, and identify factors (of patient, general practitioners and PHC team) associated with being screened, that are common throughout the PHC population. METHODS: Multicentre, cross-sectional study of individuals aged ≥ 16 years (N = 468,940) who visited the 426 general practitioners (GPs) in 44 PHC teams in Catalonia and Navarre in 2008. OUTCOMES: screening for hypertension, dyslipidaemia, obesity, tobacco use, and excessive alcohol consumption. Other variables were considered at the individual (sociodemographics, visits, health problems), GP and PHC team (region among others). Individual and contextual factors associated with the odds of being screened and the variance attributable to each level were identified using the SAS PROC GLIMMIX macro. RESULTS: The most prevalent screenings were for dyslipidaemia (64.4%) and hypertension (50.8%); the least prevalent was tobacco use (36.6%). Overall, the odds of being screened were higher for women, older patients, those with more comorbidities, more cardiovascular risk factors, and more frequent office visits, and those assigned to a female GP, a GP with a lower patient load, or a PHC team with a lower percentage of patients older than 65 years. On average, individuals in Navarre were less likely to be screened than those in Catalonia. Hypertension and dyslipidaemia screenings had the least unexplained variability between PHC teams and GPs, respectively, after adjusting for individual and contextual factors. CONCLUSIONS: Of the studied screenings, those for obesity, tobacco, and alcohol use were the least prevalent. Attention to screening, especially for tobacco and alcohol, can be greatly improved in the PHC setting.

Neonatal maternal separation up-regulates protein signalling for cell survival in rat hypothalamus.

We have previously reported that in response to early life stress, such as maternal hyperthyroidism and maternal separation (MS), the rat hypothalamic vasopressinergic system becomes up-regulated, showing enlarged nuclear volume and cell number, with stress hyperresponsivity and high anxiety during adulthood. The detailed signaling pathways involving cell death/survival, modified by adverse experiences in this developmental window remains unknown. Here, we report the effects of MS on cellular density and time-dependent fluctuations of the expression of pro- and anti-apoptotic factors during the development of the hypothalamus. Neonatal male rats were exposed to 3 h-daily MS from postnatal days 2 to 15 (PND 2-15). Cellular density was assessed in the hypothalamus at PND 21 using methylene blue staining, and neuronal nuclear specific protein and glial fibrillary acidic protein immunostaining at PND 36. Expression of factors related to apoptosis and cell survival in the hypothalamus was examined at PND 1, 3, 6, 9, 12, 15, 20 and 43 by Western blot. Rats subjected to MS exhibited greater cell-density and increased neuronal density in all hypothalamic regions assessed. The time course of protein expression in the postnatal brain showed: (1) decreased expression of active caspase 3; (2) increased Bcl-2/Bax ratio; (3) increased activation of ERK1/2, Akt and inactivation of Bad; PND 15 and PND 20 were the most prominent time-points. These data indicate that MS can induce hypothalamic structural reorganization by promoting survival, suppressing cell death pathways, increasing cellular density which may alter the contribution of these modified regions to homeostasis.

Comparing the effects of two neurotoxins in cortical astrocytes obtained from rats of different ages: involvement of oxidative damage.

Oxidative stress has been recognized as a potential mediator of cell death. Astrocytes play an active role in brain physiology responding to harmful stimuli by activating astrogliosis, which in turn has been associated either with survival or degenerative events. The characterization of the mechanistic actions exerted by different toxins in astrocytes is essential to understand the brain function and pathology. As age plays a critical role in degenerative processes, the aim of this study was to determine whether the administration of equimolar concentrations of two neurotoxins evoking different toxic patterns can induce differential effects on primary astrocytes obtained either from newborn or adult rats, with particular emphasis on those events linked to oxidative stress as a potential source of damage. Primary cortical astrocyte cultures derived from rat brains were exposed to 1-methyl-4-phenylpyridinium (MPP+) or beta-amyloid peptide (ß-amyloid). Mitochondrial functionality and cell viability were determined as physiological parameters, whereas lipid and protein oxidation were used as markers of oxidative damage. The results of these experiments pointed towards a higher vulnerability to MPP + over ß-amyloid, on most of the tested markers. Hence, in order to allow a comprehensive evaluation of astrocytic responses against MPP + intoxication, a third astrocyte group was included for dose-response experiments: astrocytes derived from aged rats. The present data indicate that the differences associated with age were mainly found in astrocytes exposed to MPP + (25 and 50 µM) at 1-h treatment. Results are discussed in terms of the differential mechanisms involved in each model.

Fraping: A computational tool for detecting slight differences in fluorescence recovery after photobleaching (FRAP) data for actin polymerization analysis.

Fluorescence recovery after photobleaching (FRAP) is a laser method of light microscopy to evaluate the rapid movement of fluorescent molecules. To have a more reliable approach to analyze data from FRAP, we designed Fraping, a free access R library to data analysis obtained from FRAP. Unlike other programs, Fraping has a new form of analyzing curves of FRAP using statistical analysis based on the average curve difference. To evaluate our library, we analyzed the differences of actin polymerization in real time between dendrites and secondary neurites of cultured neuron transfected with LifeAct to track F-actin changes of neurites. We found that Fraping provided greater sensitivity than the conventional model using mobile fraction analysis. Likewise, this approach allowed us to normalize the fluorescence to the size area of interest and adjust data curves choosing the best parametric model. In addition, this library was supplemented with data simulation to have a more significant enrichment for the analysis behavior. We concluded that Fraping is a method that reduces bias when analyzing two data groups as compared with the conventional methods. This method also allows the users to choose a more suitable analysis approach according to their requirements. RESEARCH HIGHLIGHTS: Fraping is a new programming tool to analyze FRAP data to normalize fluorescence recovery curves. The conventional method uses one-point analysis, and the new one compares all the points to define the similarity of the fluorescence recovery.

Infection by Brazilian and Dutch swine hepatitis E virus strains induces haematological changes in Macaca fascicularis.

BACKGROUND: Hepatitis E virus (HEV) has been described as an emerging pathogen in Brazil and seems to be widely disseminated among swine herds. An autochthonous human case of acute hepatitis E was recently reported. To obtain a better understanding of the phenotypic profiles of both human and swine HEV strains, a experimental study was conducted using the animal model, Macaca fascicularis. METHODS: Six cynomolgus monkeys (Macaca fascicularis) were inoculated intravenously with swine HEV genotype 3 that was isolated from naturally and experimentally infected pigs in Brazil and the Netherlands. Two other monkeys were inoculated with HEV genotype 3 that was recovered from Brazilian and Argentinean patients with locally acquired acute and fulminant hepatitis E. The haematological, biochemical, and virological parameters of all animals were monitored for 67 days. RESULTS: Subclinical hepatitis was observed in all monkeys after inoculation with HEV genotype 3 that was recovered from the infected swine and human patients. HEV RNA was detected in the serum and/or faeces of 6 out of the 8 cynomolgus monkeys between 5 and 53 days after inoculation. The mild inflammation of liver tissues and elevations of discrete liver enzymes were observed. Seroconversions to anti-HEV IgM and/or IgG were detected in 7 animals. Reactivities to anti-HEV IgA were also detected in the salivary samples of 3 animals. Interestingly, all of the infected monkeys showed severe lymphopenia and a trend toward monocytosis, which coincided with elevations in alanine aminotransferase and antibody titres. CONCLUSIONS: The ability of HEV to cross the species barrier was confirmed for both the swine (Brazilian and Dutch) and human (Argentinean) strains, thus reinforcing the zoonotic risk of hepatitis E in South America. Cynomolgus monkeys that were infected with HEV genotype 3 developed subclinical hepatitis that was associated with haematological changes. Haematological approaches should be considered in future studies of HEV infection.

Effectiveness of the trivalent influenza vaccine in Navarre, Spain, 2010-2011: a population-based test-negative case-control study.

BACKGROUND: Some studies have evaluated vaccine effectiveness in preventing outpatient influenza while others have analysed its effectiveness in preventing hospitalizations. This study evaluates the effectiveness of the trivalent influenza vaccine in preventing outpatient illness and hospitalizations from laboratory-confirmed influenza in the 2010-2011 season. METHODS: We conducted a nested case-control study in the population covered by the general practitioner sentinel network for influenza surveillance in Navarre, Spain. Patients with influenza-like illness in hospitals and primary health care were swabbed for influenza testing. Influenza vaccination status and other covariates were obtained from health care databases. Using logistic regression, the vaccination status of laboratory-confirmed influenza cases was compared with that of test-negative controls, adjusting for age, sex, comorbidity, outpatient visits in the previous 12 months, health care setting, time between symptom onset and swabbing, period and A(H1N1)pdm09 vaccination. Effectiveness was calculated as (1-odds ratio)x100. RESULTS: The 303 confirmed influenza cases (88% for A(H1N1)pdm09 influenza) were compared with the 286 influenza test-negative controls. The percentage of persons vaccinated against influenza was 4.3% and 15.7%, respectively (p<0.001). The adjusted estimate of effectiveness was 67% (95% CI: 24%, 86%) for all patients and 64% (95% CI: 8%, 86%) in those with an indication for vaccination (persons age 60 or older or with major chronic conditions). Having received both the 2010-2011 seasonal influenza vaccine and the 2009-2010 pandemic influenza vaccine provided 87% protection (95% CI: 30%, 98%) as compared to those not vaccinated. CONCLUSION: The 2010-2011 seasonal influenza vaccine had a moderate protective effect in preventing laboratory-confirmed influenza.

Txnip expression promotes JNK-mediated neuronal death in response to reactive oxygen species.

TXNIP is a protein sensitive to oxidant conditions whose expression is related to the progression of death in cancer, diabetes, ischemia, and neurodegenerative diseases, among others. Because of this, many studies propose TXNIP as a therapeutic target in several diseases. Exposure of cerebellar granule neurons to staurosporine or low potassium leads to apoptotic death. Both conditions generate an early production of reactive oxygen species (ROS) that induces the activation of the ASK1 pathway and the apoptotic machinery. In these models, it has been shown an increase in TXNIP protein mediated by ROS. Here, we evaluated the molecular mechanisms involved in the regulation of the Txnip expression during neuronal death, as well as the role of the protein in the progression of cell death induced by these two apoptotic conditions. In cultured cerebellar granule neurons, we observed that low potassium and staurosporine induced an early increase in ROS that correlated with an increase in Txnip mRNA. When we evaluated the promoter of the gene, we found that the JASPAR-reported FOXO1/3 transcription factor motifs are close to the transcription start site (TSS). We then verified through the Chromatin immunoprecipitation technique (ChIP) that FOXO3 interacts with the Txnip promoter after 1 h of low potassium treatment. We also detected FOXO3 nuclear translocation by low potassium and staurosporine treatments. Finally, by using shRNA in the neuroblastoma MSN cell line, we found that Txnip downregulation decreased neuronal death induced by staurosporine stimulus. Together, these results suggest that ROS promotes the expression of Txnip through the activation of the FOXO3 transcription factor mediated by Akt inhibition. We also demonstrated that TXNIP is necessary for neuronal death progression.

Corrigendum: Txnip expression promotes JNK-mediated neuronal death in response to reactive oxygen species.

[This corrects the article DOI: 10.3389/fnmol.2023.1210962.].

Factors associated with continued adherence to influenza vaccination in the elderly.

OBJECTIVE: We aimed to analyze the factors influencing continued adherence to influenza vaccination in elderly persons vaccinated in the preceding season. METHODS: Using a population-based vaccination registry, we evaluated the proportion of persons vaccinated against influenza in Navarre, Spain, in the 2010-11 season among non-institutionalized persons aged 65 years or over who had been vaccinated in the 2009-10 season. Logistic regression was used to analyze the influence of sociodemographic, clinical and health care factors. RESULTS: Of the 64,245 persons vaccinated against influenza in the 2009-10 season, 87% were vaccinated in the 2010-11 season. Continued adherence to vaccination increased with the number of physician visits per year. It was lower in women, in the 65-69 and ≥ 95 year age-groups, in those hospitalized or diagnosed with any major chronic condition in the previous year, and in persons with hematological cancer or dementia. Health districts and physicians with higher coverage in the previous season continued to have higher adherence in the following season. CONCLUSIONS: People vaccinated against influenza in one season tend to be vaccinated in the following one. Sociodemographic, clinical and health care factors have a moderate effect on the continuity of vaccination, with the most important factor being the treating physician.

Reactive oxygen species are related to ionic fluxes and volume decrease in apoptotic cerebellar granule neurons: role of NOX enzymes.

Reactive oxygen species (ROS) are produced early during apoptosis of cerebellar granule neurons induced by low potassium (K5) and staurosporine (Sts). In addition, K5 and Sts activate NADPH oxidases (NOX). Recently, we described that K5 and Sts induce apoptotic volume decrease (AVD) at a time when ROS generation and NOX activity occur. In the present study, we evaluated the relationship between ROS generation and ionic fluxes during AVD. Here, we showed that K5- and Sts-induced AVD was inhibited by antioxidants and that direct ROS production induced AVD. Moreover, NOX inhibitors eliminated AVD induced by both K5 and Sts. Sts, but not K5, failed to induce AVD in cerebellar granule neurons from NOX2 knockout mice. These findings suggest that K5- and Sts-induced AVD is largely mediated by ROS produced by NOX. On the other hand, we also found that the blockage of ionic fluxes involved in AVD inhibited both ROS generation and NOX activity. These findings suggest that ROS generation and NOX activity are involved in ionic fluxes activation, which in turn could maintain ROS generation by activating NOX, leading to a self-amplifying cycle.

Role of cytoskeleton proteins in the morphological changes during apoptotic cell death of cerebellar granule neurons.

Cytoskeleton proteins are substrates for proteases and further apoptotic death. We evaluated the participation of cytoskeleton in morphological changes during cell death induced by two apoptotic conditions, potassium deprivation (K5) and staurosporine, in cerebellar granule neurons (CGC). We found that K5 induced somatic damage, but neurites were relatively preserved, which corresponded to the reorganization of actin and α-tubulin in neurites. Staurosporine (STS) induced an early alteration of neurites with reorganization of cytoskeleton proteins in somas. Caspase inhibitor ZVAD totally inhibited STS-induced α-tubulin reorganization and partially blocked STS-induced actin reorganization. α-tubulin and actin reorganization induced by K5 was affected by ZVAD. Calpain inhibitor (IC1) did not affect α-tubulin or actin reorganization induced by STS, K5 or ionomycin. Neither ZVAD, nor IC1 changed α-tubulin or actin levels upon K5 treatment. STS increased α-tubulin and actin levels, but neither ZVAD nor IC1 changed α-tubulin levels upon STS treatment. In contrast, ZVAD reduced the STS-induced increase of actin. These results suggest that CGC cytoskeleton proteins undergo a differential expression and reorganization depending on the apoptotic condition.

Oxidation of biomolecules in the apoptotic death of cerebellar granule neurons induced by potassium deprivation.

Cerebellar granule neurons (CGN) cultured in a 25 mM KCl medium (K25) die apoptotically when they are transferred to a medium containing 5 mM KCl (K5). It has been previously shown that apoptotic death of CGN induced by K5 is mediated by an increase in the levels of reactive oxygen species (ROS). ROS may participate in the apoptotic program either as signaling molecules or as effectors by causing oxidative damage to lipids, DNA and proteins. In this study we evaluated ROS production in CGN treated with K5 for different periods of time and evaluated a possible correlation between ROS production and oxidation of DNA proteins and two lipid peroxidation products, conjugated dienes and malondialdehyde. Under these conditions, we found two episodes of ROS generation, one at an early time (4 h) and another at a later time point (18-24 h). We also identified two peaks in the formation of conjugated dienes, the initial and transient by-products of lipoperoxidation. The first one occurred after 4 h of K5 treatment and the other was observed after 18 h, both of them correlated with the formation of ROS. In contrast, we found significant levels of the late product of lipidperoxidation, malondialdehyde only after 18 h of treatment. Besides, we did not find significant levels of DNA and protein oxidation products that could be correlated with the observed ROS production. These results support the idea that ROS produced early by K5 treatment could act primarily as a signal of the apoptotic cell death and that ROS produced later could be mainly a product of the cell death that could contribute directly to this process.