Aberrant GSH reductase and NOX activities concur with defective CFTR to pro-oxidative imbalance in cystic fibrosis airways.

Cystic fibrosis (CF) is associated to impaired Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel also causing decreased glutathione (GSH) secretion, defective airway bacterial clearance and inflammation. Here we checked the main ROS-producing and ROS-scavenging enzymes as potential additional factors involved in CF pathogenesis. We found that CFBE41o-cells, expressing F508del CFTR, have increased NADPH oxidase (NOX) activity and expression level, mainly responsible of the increased ROS production, and decreased glutathione reductase (GR) activity, not dependent on GR protein level decrease. Furthermore, defective CFTR proved to cause both extracellular and intracellular GSH level decrease, probably by reducing the amount of extracellular GSH-derived cysteine required for cytosolic GSH synthesis. Importantly, we provide evidence that defective CFTR and NOX/GR activity imbalance both contribute to NADPH and GSH level decrease and ROS overproduction in CF cells.

AMPK is activated early in cerebellar granule cells undergoing apoptosis and influences VADC1 phosphorylation status and activity.

The neurodegeneration of cerebellar granule cells, after low potassium induced apoptosis, is known to be temporally divided into an early and a late phase. Voltage-dependent anion channel-1 (VDAC1) protein, changing from the closed inactive state to the active open state, is central to the switch between the early and late phase. It is also known that: (i) VDAC1 can undergo phosphorylation events and (ii) AMP-activated protein kinase (AMPK), the sensor of cellular stress, may have a role in neuronal homeostasis. In the view of this, the involvement of AMPK activation and its correlation with VDAC1 status and activity has been investigated in the course of cerebellar granule cells apoptosis. The results reported in this study show that an increased level of the phosphorylated, active, isoform of AMPK occurs in the early phase, peaks at 3 h and guarantees an increase in the phosphorylation status of VDCA1, resulting in a reduced activity of this latter. However this situation is transient in nature, since, in the late phase, AMPK activation decreases as well as the level of phosphorylated VDAC1. In a less phosphorylated status, VDAC1 fully recovers its gating activity and drives cells along the death route.

Functional characterization of the oxidative capacity of mitochondria and glycolytic assessment in benthic aquatic organisms.

The metabolism of benthic aquatic invertebrates, populating transitional water ecosystems, is influenced by both physiological and environmental factors, thus involving an adjustment of physiological processes which has a metabolic cost. In order to discover changes in metabolic pathways in response to specific factors, it's firstly necessary characterizing the principal cellular metabolic activities of the small benthic aquatic organisms. We approach here the bioenergetic state issue of two benthic organisms, i.e. Lekanesphaera monodi and Gammarus insensibilis, evidencing that no apparent and statistically significative differences between them in aerobic as well in glycolytic capacities are detected, except for COX activity.

Glucose-6-phosphate tips the balance in modulating apoptosis in cerebellar granule cells.

A metabolic shift from oxidative phosphorylation to glycolysis (i.e. the Warburg effect) occurs in Alzheimer's disease accompanied by an increase of both activity and level of HK-I. The findings reported here demonstrate that in the early phase of apoptosis VDAC1 activity, but not its protein level, progressively decreases, in concomitance with the physical interaction of HK-I with VDAC1. In the late phase of apoptosis, glucose-6-phosphate accumulation in the cell causes the dissociation of the two proteins, the re-opening of the channel and the recovery of VDAC1 function, resulting in a reawakening of the mitochondrial function, thus inevitably leading to cell death.

NH2-truncated human tau induces deregulated mitophagy in neurons by aberrant recruitment of Parkin and UCHL-1: implications in Alzheimer's disease.

Disarrangement in functions and quality control of mitochondria at synapses are early events in Alzheimer's disease (AD) pathobiology. We reported that a 20-22 kDa NH2-tau fragment mapping between 26 and 230 amino acids of the longest human tau isoform (aka NH2htau): (i) is detectable in cellular and animal AD models, as well in synaptic mitochondria and cerebrospinal fluids (CSF) from human AD subjects; (ii) is neurotoxic in primary hippocampal neurons; (iii) compromises the mitochondrial biology both directly, by inhibiting the ANT-1-dependent ADP/ATP exchange, and indirectly, by impairing their selective autophagic clearance (mitophagy). Here, we show that the extensive Parkin-dependent turnover of mitochondria occurring in NH2htau-expressing post-mitotic neurons plays a pro-death role and that UCHL-1, the cytosolic Ubiquitin-C-terminal hydrolase L1 which directs the physiological remodeling of synapses by controlling ubiquitin homeostasis, critically contributes to mitochondrial and synaptic failure in this in vitro AD model. Pharmacological or genetic suppression of improper mitophagy, either by inhibition of mitochondrial targeting to autophagosomes or by shRNA-mediated silencing of Parkin or UCHL-1 gene expression, restores synaptic and mitochondrial content providing partial but significant protection against the NH2htau-induced neuronal death. Moreover, in mitochondria from human AD synapses, the endogenous NH2htau is stably associated with Parkin and with UCHL-1. Taken together, our studies show a causative link between the excessive mitochondrial turnover and the NH2htau-induced in vitro neuronal death, suggesting that pathogenetic tau truncation may contribute to synaptic deterioration in AD by aberrant recruitment of Parkin and UCHL-1 to mitochondria making them more prone to detrimental autophagic clearance.

Glycolytic enzyme upregulation and numbness of mitochondrial activity characterize the early phase of apoptosis in cerebellar granule cells.

Alzheimer's disease (AD) and cancer proceed via one or more common molecular mechanisms: a metabolic shift from oxidative phosphorylation to glycolysis-corresponding to the activation of the Warburg effect-occurs in both diseases. The findings reported in this paper demonstrate that, in the early phase of apoptosis, glucose metabolism is enhanced, i.e. key proteins which internalize and metabolize glucose-glucose transporter, hexokinase and phosphofructokinase-are up-regulated, in concomitance with a parallel decrease in oxygen consumption by mitochondria and increase of L-lactate accumulation. Reversal of the glycolytic phenotype occurs in the presence of dichloroacetate, inhibitor of the pyruvate dehydrogenase kinase enzyme, which speeds up apoptosis of cerebellar granule cells, reawakening mitochondria and then modulating glycolytic enzymes. Loss of the adaptive advantage afforded by aerobic glycolysis, which occurs in the late phase of apoptosis, exacerbates the pathological processes underlying neurodegeneration, leading inevitably the cell to death. In conclusion, the data propose that both aerobic, i.e. Warburg effect, essentially due to the protective numbness of mitochondria, and anaerobic glycolysis, rather due to the mitochondrial impairment, characterize the entire time frame of apoptosis, from the early to the late phase, which mimics the development of AD.

Thioredoxin/thioredoxin reductase system involvement in cerebellar granule cell apoptosis.

The involvement of thioredoxin/thioredoxin reductase system has been investigated in cerebellar granule cells (CGCs), a cellular system in which neurons are induced in apoptosis by the physiological stimulus of lowering extracellular potassium. Clarifying the sequence of events that occur during apoptosis is a critical issue as it can lead to the identification of those key events that, if blocked, can slow down or reverse the death process. The results reported in this work show that TrxR is involved in the early phase of CGC apoptosis with an increase in activity that coincides with the increased expression of the TrxR1 isoform and guarantees the maintenance of adequate level of Trx in its reduced, active form. However, in late apoptosis, when about 50 % of cells are dead, partial proteolysis of TrxR1 by calpain occurs and the reduction of TrxR1 mRNA, together with the overall decrease in TrxR activity, contribute to increase the levels of the oxidized form of Trx. When the reduced form of Trx is externally added to apoptotic cultures, a significant reduction in cell death is achieved confirming that a well-functioning thioredoxin/thioredoxin reductase system is required for survival of CGCs.

Extracellular ADP prevents neuronal apoptosis via activation of cell antioxidant enzymes and protection of mitochondrial ANT-1.

Apoptosis in neuronal tissue is an efficient mechanism which contributes to both normal cell development and pathological cell death. The present study explores the effects of extracellular ADP on low [K(+)]-induced apoptosis in rat cerebellar granule cells. ADP, released into the extracellular space in brain by multiple mechanisms, can interact with its receptor or be converted, through the actions of ectoenzymes, to adenosine. The findings reported in this paper demonstrate that ADP inhibits the proapoptotic stimulus supposedly via: i) inhibition of ROS production during early stages of apoptosis, an effect mediated by its interaction with cell receptor/s. This conclusion is validated by the increase in SOD and catalase activities as well as by the GSSG/GSH ratio value decrease, in conjunction with the drop of ROS level and the prevention of the ADP protective effect by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a novel functionally selective antagonist of purine receptor; ii) safeguard of the functionality of the mitochondrial adenine nucleotide-1 translocator (ANT-1), which is early impaired during apoptosis. This effect is mediated by its plausible internalization into cell occurring as such or after its hydrolysis, by means of plasma membrane nucleotide metabolizing enzymes, and resynthesis into the cell. Moreover, the findings that ADP also protects ANT-1 from the toxic action of the two Alzheimer's disease peptides, i.e. Aß1-42 and NH2htau, which are known to be produced in apoptotic cerebellar neurons, further corroborate the molecular mechanism of neuroprotection by ADP, herein proposed.

Mitochondrial respiratory chain Complexes I and IV are impaired by ß-amyloid via direct interaction and through Complex I-dependent ROS production, respectively.

Here we investigate the effect of ß-amyloid on mitochondrial respiratory function, i.e. mitochondrial oxygen consumption and membrane potential generation as well as the individual activities of both the mitochondrial Complexes I-IV, that compose mitochondrial electron transport chain, and the ATP synthase, by using homogenate from cerebellar granule cells, treated with low concentrations of ß-amyloid, and Alzheimer synaptic-enriched brain samples. We found that ß-amyloid caused both a selective defect in Complex I activity associated with an increase (5 fold) of intracellular reactive oxygen species and an impairment of Complex IV likely due to membrane lipid peroxidation. In addition, a 130% increase of the GSSG/GSH ratio was measured in Alzheimer brains with respect to age-matched controls. Knowing the mechanisms of action of ß-amyloid could allow to mitigate or even to interrupt the toxic cascade that leads a cell to death. The results of this study represent an important innovation because they offer the possibility to act at mitochondrial level and on specific sites to protect cells, for example by preventing the interaction of ß-amyloid with the identified targets, by stabilizing or by restoring mitochondrial function or by interfering with the energy metabolism.

Dissecting the molecular mechanism by which NH2htau and Aß1-42 peptides impair mitochondrial ANT-1 in Alzheimer disease.

To find out whether and how the adenine nucleotide translocator-1 (ANT-1) inhibition due to NH2htau and Aß1-42 is due to an interplay between these two Alzheimer's peptides, ROS and ANT-1 thiols, use was made of mersalyl, a reversible alkylating agent of thiol groups that are oriented toward the external hydrophilic phase, to selectively block and protect, in a reversible manner, the -SH groups of ANT-1. The rate of ATP appearance outside mitochondria was measured as the increase in NADPH absorbance which occurs, following external addition of ADP, when ATP is produced by oxidative phosphorylation and exported from mitochondria in the presence of glucose, hexokinase and glucose-6-phosphate dehydrogenase. We found that the mitochondrial superoxide anions, whose production is induced at the level of Complex I by externally added Aß1-42 and whose release from mitochondria is significantly reduced by the addition of the VDAC inhibitor DIDS, modify the thiol group/s present at the active site of mitochondrial ANT-1, impair ANT-1 in a mersalyl-prevented manner and abrogate the toxic effect of NH2htau on ANT-1 when Aß1-42 is already present. A molecular mechanism is proposed in which the pathological Aß-NH2htau interplay on ANT-1 in Alzheimer's neurons involves the thiol redox state of ANT-1 and the Aß1-42-induced ROS increase. This result represents an important innovation because it suggests the possibility of using various strategies to protect cells at the mitochondrial level, by stabilizing or restoring mitochondrial function or by interfering with the energy metabolism providing a promising tool for treating or preventing AD.

A peptide containing residues 26-44 of tau protein impairs mitochondrial oxidative phosphorylation acting at the level of the adenine nucleotide translocator.

Having confirmed that adenovirus-mediated overexpression of NH(2)-tau fragment lacking the first 25 aminoacids evokes a potent neurotoxic effect, sustained by protracted stimulation of NMDA receptors, in primary neuronal cultures we investigated whether and how chemically synthesized NH(2)-derived tau peptides, i.e. NH(2)-26-44 and NH(2)-1-25 fragments, affect mitochondrial function. We tested both fragments on each step of the processes leading to ATP synthesis via oxidative phosphorylation: i) electron flow via the respiratory chain from physiological substrates to oxygen with the activity of each individual complex of the respiratory chain investigated in some detail, ii) membrane potential generation arising from externally added succinate and iii) the activity of both the adenine nucleotide translocator and iv) ATP synthase. Oxidative phosphorylation is not affected by NH(2)-1-25 tau fragment, but dramatically impaired by NH(2)-26-44 tau fragment. Both cytochrome c oxidase and the adenine nucleotide translocator are targets of NH(2)-26-44 tau fragment, but adenine nucleotide translocator is the unique mitochondrial target responsible for impairment of oxidative phosphorylation by the NH(2)-26-44 tau fragment, which then exerts deleterious effects on cellular availability of ATP synthesized into mitochondria.

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols.

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.

Non-radioactive detection of five common microsatellite markers for ATP7B gene in Wilson disease patients.

Haplotype analysis using microsatellite markers is a useful indicator of specific mutations and is often exploited as the first large-scale screening technique to carry out the molecular characterization of the disease gene in probands from a specific population. However, the methodologies available are still cumbersome and require the use of either radioactive compounds or specialized equipment suitable to follow fluorescent dyes. Both these techniques may not be available for newly developing clinical laboratories. We have set up a sensitive and easy-to-use protocol to characterize five closely spaced, highly polymorphic microsatellite polymorphisms (CA repeats) that span the Wilson disease (WD) region, i.e. D13S316, D13S133, D13S301, D13S314, D13S315. The technique described here for the analysis of the WD gene microsatellite system relies on the quick detection method of silver staining, avoiding the use of toxic or sophisticated equipment. This approach could be the method of choice to implement molecular genetic testing in clinical laboratories, even those not especially equipped for DNA analysis and in particular in newly developed molecular genetics centers in countries whose population has not yet been characterized for WD-causing ATP7B gene mutations.

Glutamate neurotoxicity, oxidative stress and mitochondria.

The excitatory neurotransmitter glutamate plays a major role in determining certain neurological disorders. This situation, referred to as 'glutamate neurotoxicity' (GNT), is characterized by an increasing damage of cell components, including mitochondria, leading to cell death. In the death process, reactive oxygen species (ROS) are generated. The present study describes the state of art in the field of GNT with a special emphasis on the oxidative stress and mitochondria. In particular, we report how ROS are generated and how they affect mitochondrial function in GNT. The relationship between ROS generation and cytochrome c release is described in detail, with the released cytochrome c playing a role in the cell defense mechanism against neurotoxicity.

Cytochrome c is released from mitochondria in a reactive oxygen species (ROS)-dependent fashion and can operate as a ROS scavenger and as a respiratory substrate in cerebellar neurons undergoing excitotoxic death.

In rat cerebellar granule cells both reactive oxygen species production and release of cytochrome c take place during glutamate toxicity. This investigation was aimed (i) to ascertain whether and how these two processes are related and (ii) to gain insight into the role played by the released cytochrome c in the onset of neurotoxicity. Cytochrome c release takes place owing to the generation of reactive oxygen species both in glutamate-treated cerebellar granule cells and in sister control cultures incubated in the presence of the reactive oxygen species-generating system consisting of xanthine plus xanthine oxidase. In the early phase of neurotoxicity (30-min glutamate exposure) about 40% of the maximum (as measured at 3 h of glutamate exposure) cytochrome c release was found to occur in cerebellar granule cells from mitochondria that were essentially coupled and intact and that had a negligible production of oxygen free radicals. Contrarily, mitochondria from cells treated with glutamate for 3 h were mostly uncoupled and produced reactive oxygen species at a high rate. The cytosolic fraction containing the released cytochrome c was able to transfer electrons from superoxide anion to molecular oxygen via the respiratory chain and was found to partially prevent glutamate toxicity when added externally to cerebellar neurons undergoing necrosis. In the light of these findings, we propose that in the early phase of neurotoxicity, cytochrome c release can be part of a cellular and mitochondrial defense mechanism against oxidative stress.

Characterization of the CYP21 gene 5' flanking region in patients affected by 21-OH deficiency.

In order to test the hypothesis that mutations in the 5' non-coding region of CYP21 gene could contribute to the various spectrum of disease presentation due to 21-OH deficiency, the 400bp nucleotide sequence upstream of the ATG codon of CYP21 gene has been characterized in 28 CAH patients who have previously been genotyped by screening for the ten most frequent CYP21 mutations. Six specific sequence variations (-4C-->T, -73C-->T, -295T-->C, -294A-->C, -283A-->G, -281T-->G) have been identified in this region of CYP21 gene in 3 out of 28 21-OH deficient patients for whom the coding region mutations have been previously identified. Three of these mutations, -295T-->C, -294A-->C, -283A-->G, are apparently generated by a gene-conversion event, thus giving first evidence that this mechanism also applies to the 5' untranslated region of CYP21 gene in 21-OH deficiency. Four other sequence changes, identified at nucleotide position -279, -331, -350 and -353, could be referred to as normal since they are present also in healthy subjects. It may not be excluded that some of the newly-identified single nucleotide changes in the regulatory region could have a modulatory effect on the CYP21 gene transcriptional activity thus affecting the clinical outcome.

Early release and subsequent caspase-mediated degradation of cytochrome c in apoptotic cerebellar granule cells.

Cytochrome c (cyt c) release was investigated in cerebellar granule cells used as an in vitro neuronal model of apoptosis. We have found that cyt c is released into the cytoplasm as an intact, functionally active protein, that this event occurs early, in the commitment phase of the apoptotic process, and that after accumulation, this protein is progressively degraded. Degradation, but not release, is fully blocked by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylchetone (z-VAD-fmk). On the basis of previous findings obtained in the same neuronal population undergoing excitotoxic death, it is hypothesized that release of cyt c may be part of a cellular attempt to maintain production of ATP via cytochrome oxidase, which is reduced by cytosolic NADH in a cytochrome b5-soluble cyt c-mediated fashion.

Characterisation of CAH alleles with non-radioactive DNA single strand conformation polymorphism analysis of the CYP21 gene.

The major cause of congenital adrenal hyperplasia (CAH), a common recessive genetic disease, is the deficiency of steroid 21-hydroxylase (21OH), a microsomal enzyme encoded by the CYP21 gene. Although several CAH causing mutations have been identified in the CYP21 gene of patients with 21OH deficiency, genotyping of the 21OH locus is quite complex because of the high frequency of gene conversion and the presence of multiple mutations on single CAH alleles. In order to perform the complete characterisation of the CYP21 gene coding region more simply, we developed a highly sensitive, non-radioactive method allowing DNA single strand conformation polymorphism (DNA-SSCP) analysis. This method was applied to the characterisation of all the exons and intron-exon junctions of the CYP21 gene in five patients affected by the simple virilising form and one affected by the salt wasting form. In all samples showing SSCP signals, direct sequence analysis showed the presence of more than one single sequence variant. In particular, four mutations which are already known to cause the disease, 16 polymorphisms, and one newly identified C to T transition at position 849 were detected. A random sequence analysis, performed on 31 out of 81 exons showing a normal SSCP pattern, shows the method to be highly sensitive: no sequence variant was detected, thus confirming the validity of this non-radioactive DNA-SSCP analysis in characterising the CYP21 gene in patients with steroid 21OH deficiency. Notwithstanding the complete characterisation of all exons and exon/intron junctions of the CYP21 gene, no complete genotype/phenotype correlation was found in the panel of patients analysed, thus suggesting that characterisation of CAH alleles must be extended to outside the coding region of the CYP21 gene, most probably into the promoter region.

Detection of microsatellites by ethidium bromide staining. The analysis of an STR system in the human phenylalanine hydroxylase gene.

The analysis of short tandem repeat (STR) systems usually relies on polyacrylamide gel electrophoresis analysis followed by visualization with silver staining or autoradiography. Both these techniques may not be suitable for clinical laboratories. We developed a simple procedure based on the visualization of STR alleles by ethidium bromide staining. The 4-bp STR system analysed is located in the human phenylalanine hydroxylase gene. Alleles differing by 4 bp are clearly separated independently of the size of the amplified fragments and homozygous samples are easily identified by comparison of the relative intensity of the electrophoretic bands. This method could be applied to the analysis of other STR systems located in different genetic loci by carefully changing the electrophoretic conditions.