Lara Macchioni

Botulinum-A Toxin Injections Into the Detrusor Muscle Decrease Nerve Growth Factor Bladder Tissue Levels in Patients With Neurogenic Detrusor Overactivity

PURPOSE We investigated the effects of BTX-A on visceral afferent nerve transmission by measuring bladder tissue NGF levels in patients with neurogenic detrusor overactivity before and after intravesical treatment with BTX-A. We also compared the bladder tissue NGF content with clinical and urodynamic data. MATERIALS AND METHODS A total of 23 patients underwent clinical evaluation and urodynamics with detection of the UDC threshold, maximum pressure and maximum cystometric capacity before, and at the 1 and 3-month followups. Endoscopic bladder wall biopsies were also obtained at the same time points. NGF levels were measured in tissue homogenate by enzyme-linked immunosorbent assay (Promega, Madison, Wisconsin). RESULTS At 1 and 3 months mean catheterization and incontinent episodes were significantly decreased (p <0.05 and <0.001, respectively). On urodynamics we detected a significant increase in the UDC threshold and maximum cystometric capacity, and a significant decrease in UDC maximum pressure at the 1 and 3-month follow-ups compared to baseline (each p <0.001). At the same time points we detected a significant decrease in NGF bladder tissue content (each p <0.02). CONCLUSIONS BTX-A intravesical treatment induces a state of NGF deprivation in bladder tissue that persists at least up to 3 months. As caused by BTX-A, the decrease in acetylcholine release at the presynaptic level may induce a decrease in detrusor contractility and in NGF production by the detrusor muscle. Alternatively BTX-A can decrease the bladder level of neurotransmitters that normally modulate NGF production and release.

Rat Brain Cortex Mitochondria Release Group II Secretory Phospholipase A2 under Reduced Membrane Potential

Activation of brain mitochondrial phospholipase(s) A2 (PLA2) might contribute to cell damage and be involved in neurodegeneration. Despite the potential importance of the phenomenon, the number, identities, and properties of these enzymes are still unknown. Here, we demonstrate that isolated mitochondria from rat brain cortex, incubated in the absence of respiratory substrates, release a Ca2+-dependent PLA2 having biochemical properties characteristic to secreted PLA2 (sPLA2) and immunoreacting with the antibody raised against recombinant type IIA sPLA2 (sPLA2-IIA). Under identical conditions, no release of fumarase in the extramitochondrial medium was observed. The release of sPLA2 from mitochondria decreases when mitochondria are incubated in the presence of respiratory substrates such as ADP, malate, and pyruvate, which causes an increase of transmembrane potential determined by cytofluorimetric analysis using DiOC6(3) as a probe. The treatment of mitochondria with the uncoupler carbonyl cyanide 3-chlorophenylhydrazone slightly enhances sPLA2 release. The increase of sPLA2 specific activity after removal of mitochondrial outer membrane indicates that the enzyme is associated with mitoplasts. The mitochondrial localization of the enzyme has been confirmed by electron microscopy in U-251 astrocytoma cells and by confocal laser microscopy in the same cells and in PC-12 cells, where the structurally similar isoform type V-sPLA2 has mainly nuclear localization. In addition to sPLA2, mitochondria contain another phospholipase A2 that is Ca2+-independent and sensitive to bromoenol lactone, associated with the outer mitochondrial membrane. We hypothesize that, under reduced respiratory rate, brain mitochondria release sPLA2-IIA that might contribute to cell damage.

Long-term effects of silver nanoparticles on reproductive activity of rabbit buck.

Abstract Using the rabbit as an animal model, this study evaluated the long-term effect of silver nanoparticles (NPs) administered intravenously (0.6 mg/kg bw) on reproductive activity and sperm quality. Semen analysis was performed by optical microscopy and sperm motility evaluation by computer assisted sperm analyzer (CASA). Mitochondria oxygen consumption, light and transmission electron microscopy of rabbit testis and ejaculated sperm were also carried out. Throughout the experiment NP-treated rabbits showed higher seminal reactive oxygen species (ROS), less motile sperm, and lower curvilinear velocity and oxygen consumption than control animals. In contrast, libido, serum testosterone, sperm concentration, and semen volume were hardly affected by NPs. Transmission electron microscopy analysis did not show any evident morphological damage in testes; however, Ag NPs are visible in spermatids and ejaculated sperm. These preliminary results show that Ag NPs can reach the testes, compromising sperm motility, sperm speed, and acrosome and mitochondria shape and function.

Mitochondrial dysfunction and effect of antiglycolytic bromopyruvic acid in GL15 glioblastoma cells

Most cancer cells, including GL15 glioblastoma cells, rely on glycolysis for energy supply. The effect of antiglycolytic bromopyruvate on respiratory parameters and viability of GL15 cells was investigated. Bromopyruvate caused Δψm and MTT collapse, ATP decrease, and cell viability loss without involving apoptotic or necrotic pathways. The autophagy marker LC3-II was increased. Δψm decrease was accompanied by reactive oxygen species (ROS) increase and cytochrome c (cyt c) disappearance, suggesting a link between free radical generation and intramitochondrial cyt c degradation. Indeed, the free radical inducer menadione caused a decrease in cyt c that was reversed by N-acetylcysteine. Cyt c is tightly bound to the inner mitochondrial membrane in GL15 cells, which may confer protein peroxidase activity, resulting in auto-oxidation and protein targeting to degradation in the presence of ROS. This process is directed towards impairment of the apoptotic cyt c cascade, although cells are committed to die.

The energy blockers bromopyruvate and lonidamine lead GL15 glioblastoma cells to death by different p53-dependent routes.

The energy metabolism of tumor cells relies on aerobic glycolysis rather than mitochondrial oxidation. This difference between normal and cancer cells provides a biochemical basis for new therapeutic strategies aimed to block the energy power plants of cells. The effects produced by the energy blockers bromopyruvate (3BP) and lonidamine (LND) and the underlying biochemical mechanisms were investigated in GL15 glioblastoma cells. 3BP exerts early effects compared to LND, even though both drugs lead cells to death but by different routes. A dramatic decrease of ATP levels occurred after 1 hour treatment with 3BP, followed by cytochrome c and hexokinase II degradation, and by the decrease of both LC3I/LC3II ratio and p62, markers of an autophagic flux. In addition, Akt(Ser473) and p53(Ser15/Ser315) dephosphorylation occurred. In LND treatment, sustained ATP cellular levels were maintained up to 40 hours. The autophagic response of cells was overcome by apoptosis that was preceded by phosphatidylinositol disappearance and pAkt decrease. This last event favored p53 translocation to mitochondria triggering a p53-dependent apoptotic route, as observed at 48 and 72 hours. Adversely, in 3BP treatment, phospho-p53 dephosphorylation targeted p53 to MDM2-dependent proteolysis, thus channeling cells to irreversible autophagy.

The energy blockers 3-bromopyruvate and lonidamine: effects on bioenergetics of brain mitochondria.

Tumor cells favor abnormal energy production via aerobic glycolysis and show resistance to apoptosis, suggesting the involvement of mitochondrial dysfunction. The differences between normal and cancer cells in their energy metabolism provide a biochemical basis for developing new therapeutic strategies. The energy blocker 3-bromopyruvate (3BP) can eradicate liver cancer in animals without associated toxicity, and is a potent anticancer towards glioblastoma cells. Since mitochondria are 3BP targets, in this work the effects of 3BP on the bioenergetics of normal rat brain mitochondria were investigated in vitro, in comparison with the anticancer agent lonidamine (LND). Whereas LND impaired oxygen consumption dependent on any complex of the respiratory chain, 3BP was inhibitory to malate/pyruvate and succinate (Complexes I and II), but preserved respiration from glycerol-3-phosphate and ascorbate (Complex IV). Accordingly, although electron flow along the respiratory chain and ATP levels were decreased by 3BP in malate/pyruvate- and succinate-fed mitochondria, they were not significantly influenced from glycerol-3-phosphate- or ascorbate-fed mitochondria. LND produced a decrease in electron flow from all substrates tested. No ROS were produced from any substrate, with the exception of 3BP-induced H2O2 release from succinate, which suggests an antimycin-like action of 3BP as an inhibitor of Complex III. We can conclude that 3BP does not abolish completely respiration and ATP synthesis in brain mitochondria, and has a limited effect on ROS production, confirming that this drug may have limited harmful effects on normal cells.

H(2)O(2) disposal in cardiolipin-enriched brain mitochondria is due to increased cytochrome c peroxidase activity.

The mitochondrial electron transport chain is a source of oxygen superoxide anion (O(2)(-)) that is dismutated to H(2)O(2). Although low levels of ROS are physiologically synthesized during respiration, their increase contributes to cell injury. Therefore, an efficient machinery for H(2)O(2) disposal is essential in mitochondria. In this study, the ability of brain mitochondria to acquire cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylserine (PS) in vitro through a fusion process was exploited to investigate lipid effects on ROS. MTT assay, oxygen consumption, and respiratory ratio indicated that the acquired phospholipids did not alter mitochondrial respiration and O(2)(-) production from succinate. However, in CL-enriched mitochondria, H(2)O(2) levels where 27% and 47% of control in the absence and in the presence of antimycin A, respectively, suggesting an increase in H(2)O(2) elimination. Concomitantly, cytochrome c (cyt c) was released outside mitochondria. Since free oxidized cyt c acquired peroxidase activity towards H(2)O(2) upon interaction with CL in vitro, a contribution of cyt c to H(2)O(2) disposal in mitochondria through CL conferred peroxidase activity is plausible. In this model, the accompanying CL peroxidation should weaken cyt c-CL interactions, favouring the detachment and release of the protein. Neither cyt c peroxidase activity was elicited by PS in vitro, nor cyt c release was observed in PS-enriched mitochondria, although H(2)O(2) levels were significantly decreased, suggesting a cyt c-independent role of PS in ROS metabolism in mitochondria.

Evidence that cytosolic phospholipase A2 is down-regulated by protein kinase C in intact human platelets stimulated with fluoroaluminate

We reported that protein kinase C (PKC) inhibitors increase the release of arachidonic acid induced by fluoroaluminate (AlF4 −), an unspecific G‐protein activator, in intact human platelets. Now we demonstrate that this effect is independent of the extracellular Ca2+ concentration and that AlF4 −‐induced release of AA is abolished by BAPTA, an intracellular Ca2+ chelator, even in the presence of GF 109203X, a specific and potent PKC inhibitor. This compound also blocks the liberation of the secretory phospholipase A2 in the extracellular medium, indicating that this enzyme is not involved in the potentiation of arachidonic acid by PKC inhibitors. On the other hand, the latter effect is completely abolished by treatment of platelets with AACOCF3, a specific inhibitor of cytosolic phospholipase A2 (cPLA2). These observations indicate that cPLA2 is responsible for the AlF4 −‐induced release of arachidonic acid by a mechanism that is down‐regulated by PKC.

Enteric glial cells counteract Clostridium difficile Toxin B through a NADPH oxidase/ROS/JNK/caspase-3 axis, without involving mitochondrial pathways

Enteric glial cells (EGCs) are components of the intestinal epithelial barrier essential for regulating the enteric nervous system. Clostridium difficile is the most common cause of antibiotic-associated colitis, toxin B (TcdB) being the major virulence factor, due to its ability to breach the intestinal epithelial barrier and to act on other cell types. Here we investigated TcdB effects on EGCs and the activated molecular mechanisms. Already at 2 hours, TcdB triggered ROS formation originating from NADPH-oxidase, as demonstrated by their reduction in the presence of the NADPH-oxidase inhibitor ML171. Although EGCs mitochondria support almost completely the cellular ATP need, TcdB exerted weak effects on EGCs in terms of ATP and mitochondrial functionality, mitochondrial ROS production occurring as a late event. ROS activated the JNK signalling and overexpression of the proapoptotic Bim not followed by cytochrome c or AIF release to activate the downstream apoptotic cascade. EGCs underwent DNA fragmentation through activation of the ROS/JNK/caspase-3 axis, evidenced by the ability of ML171, N-acetylcysteine, and the JNK inhibitor SP600125 to inhibit caspase-3 or to contrast apoptosis. Therefore, TcdB aggressiveness towards EGCs is mainly restricted to the cytosolic compartment, which represents a peculiar feature, since TcdB primarily influences mitochondria in other cellular types.

Lethal digenic mutations in the K+ channels Kir4.1 (KCNJ10) and SLACK (KCNT1) associated with severe-disabling seizures and neurodevelopmental delay

A 2-yr-old boy presented profound developmental delay, failure to thrive, ataxia, hypotonia, and tonic-clonic seizures that caused the death of the patient. Targeted and whole exome sequencing revealed two heterozygous missense variants: a novel mutation in the KCNJ10 gene that encodes for the inward-rectifying K+ channel Kir4.1 and another previously characterized mutation in KCNT1 that encodes for the Na+-activated K+ channel known as Slo2.2 or SLACK. The objectives of this study were to perform the clinical and genetic characterization of the proband and his family and to examine the functional consequence of the Kir4.1 mutation. The mutant and wild-type KCNJ10 constructs were generated and heterologously expressed in Xenopus laevis oocytes, and whole cell K+ currents were measured using the two-electrode voltage-clamp technique. The KCNJ10 mutation c.652C>T resulted in a p.L218F substitution at a highly conserved residue site. Wild-type KCNJ10 expression yielded robust Kir current, whereas currents from oocytes expressing the mutation were reduced, remarkably. Western Blot analysis revealed reduced protein expression by the mutation. Kir5.1 subunits display selective heteromultimerization with Kir4.1 constituting channels with unique kinetics. The effect of the mutation on Kir4.1/5.1 channel activity was twofold: a reduction in current amplitudes and an increase in the pH-dependent inhibition. We thus report a novel loss-of-function mutation in Kir4.1 found in a patient with a coexisting mutation in SLACK channels that results in a fatal disease.NEW & NOTEWORTHY We present and characterize a novel mutation in KCNJ10 Unlike previously reported EAST/SeSAME patients, our patient was heterozygous, and contrary to previous studies, mimicking the heterozygous state by coexpression resulted in loss of channel function. We report in the same patient co-occurrence of a KCNT1 mutation resulting in a more severe phenotype. This study provides new insights into the phenotypic spectrum and to the genotype-phenotype correlations associated with EAST/SeSAME and MMFSI.