Luigi A. De Girolamo

Monoamine oxidase-A modulates apoptotic cell death induced by staurosporine in human neuroblastoma cells

Monoamine oxidases (MAOs) are mitochondrial enzymes which control the levels of neurotransmitters in the brain and dietary amines in peripheral tissues via oxidative deamination. MAO has also been implicated in cell signalling. In this study, we describe the MAO‐A isoform as functional in apoptosis induced by staurosporine (STS) in human dopaminergic neuroblastoma cells (SH‐SY5Y). Increased levels of MAO‐A activity were induced by STS, accompanied by increased MAO‐A protein and activation of the initiator of the intrinsic pathway, caspase 9, and the executioner caspase 3. MAO‐A mRNA levels were unaffected by STS, suggesting that changes in MAO‐A protein are due to post‐transcriptional events. Two unrelated MAO‐A inhibitors reduced caspase activation. STS treatment resulted in sustained activation of the mitogen‐activated protein kinase pathway enzymes extracellular regulated kinase, c‐jun terminal kinase and p38, and depletion of the anti‐apoptotic protein Bcl‐2. These changes were significantly reversed by MAO inhibition. Production of reactive oxygen species was increased following STS exposure, which was blocked by both MAO inhibition and the antioxidant N‐acetylcysteine. Therefore our data provide evidence that MAO‐A, through its production of reactive oxygen species as a by‐product of its catalytic activity on the mitochondrial surface, is recruited by the cell to enhance apoptotic signalling.

Protection from MPTP‐induced neurotoxicity in differentiating mouse N2a neuroblastoma cells

We have shown previously that subcytotoxic concentrations of MPTP (1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine) inhibit axon outgrowth and are associated with increased neurofilament heavy chain (NF‐H) phosphorylation in differentiating mouse N2a neuroblastoma cells while higher doses (> 100 µm) cause cell death. In this work we assessed the ability of potential neuroprotective agents to alleviate both MPTP‐induced cell death (cytotoxicity) and MPTP‐induced NF‐H phosphorylation/reduction in axon outgrowth (neurotoxicity) in N2a cells induced to differentiate by dbcAMP. The neurotoxic effects of MPTP occurred in the absence of significant alterations in energy status or mitochondrial membrane potential. The hormone oestradiol (100 µm) reduced the cytotoxic effect of MPTP, but blocked di‐butyryl cyclic AMP (dbcAMP)‐induced differentiation, i.e. axon outgrowth. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the monoamine osidase (MAO) inhibitors deprenyl and, to a lesser extent, clorgyline. Alleviation of both neurotoxicity and cytotoxicity was also achieved by conditioned medium derived from rat C6 glioma cells. In contrast, whilst the p38 MAP kinase inhibitor, SB202190, protected cells against MPTP‐induced neurotoxicity, it could not maintain cell viability at high MPTP exposures. In each case neuroprotection involved maintenance of the differentiating phenotype linked with attenuation of NF‐H hyper‐phosphorylation; the latter may represent a mechanism by which neuronal cells can moderate MPTP‐induced neurotoxicity. The use of a simplified neuronal cell model, which expresses subtle biochemical changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents.

Alterations in the mitochondrial proteome of neuroblastoma cells in response to complex 1 inhibition.

Increasing evidence points to mitochondrial dysfunction in Parkinsons disease (PD) associated with complex I dysfunction, but the exact pathways which lead to cell death have not been resolved. 2D-gel electrophoresis profiles of isolated mitochondria from neuroblastoma cells treated with subcytotoxic concentrations of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a well-characterized complex I inhibitor, were assessed to identify associated targets. Up to 27 differentially expressed proteins were observed, of which 16 were identified using peptide mass fingerprinting. Changes in protein levels were validated by immunoprobing 1D blots, confirming increases in heat shock cognate 71 kDa (Hsc70), 60 kDa heat shock protein (Hsp60), fumarase, glutamate oxaloacetate transaminase 2, ATP synthase subunit d, and voltage-dependent anion-channel 1 (VDAC1). Immunoprobing of 2D blots revealed isoform changes in Hsc70, Hsp60, and VDAC1. Subcytotoxic concentrations of MPTP modulated a host of mitochondrial proteins including chaperones, metabolic enzymes, oxidative phosphorylation-related proteins, an inner mitochondrial protein (mitofilin), and an outer mitochondrial membrane protein (VDAC1). Early changes in chaperones suggest a regulated link between complex 1 inhibition and protein folding. VDAC1, a multifunctional protein, may have a key role in signaling between mitochondria and the rest of the cell prior to cell death. Our work provides new important information of relevance to PD.

The role of tissue transglutaminase in 1-methyl-4-phenylpyridinium (MPP + )-induced toxicity in differentiated human SH-SY5Y neuroblastoma cells

Tissue transglutaminase (TG2) can induce post-translational modification of proteins, resulting in protein cross-linking or incorporation of polyamines into substrates, and can also function as a signal transducing G protein. The role of TG2 in the formation of insoluble cross-links has led to its implication in some neurodegenerative conditions. Exposure of pre-differentiated SH-SY5Y cells to the Parkinsonian neurotoxin 1-methyl-4-phenylpyridinium ion (MPP(+)) resulted in significant dose-dependent reductions in TG2 protein levels, measured by probing Western blots with a TG2-specific antibody. Transglutaminase (TG) transamidating activity, on the other hand, monitored by incorporation of a polyamine pseudo-substrate into cellular proteins, was increased. Inhibitors of TG (putrescine) and TG2 (R283) exacerbated MPP(+) toxicity, suggesting that activation of TG2 may promote a survival response in this toxicity paradigm.

Monoamine oxidase-A knockdown in human neuroblastoma cells reveals protection against mitochondrial toxins

The study examined how the mitochondrial enzyme monoamine oxidase‐A (MAO‐A), which produces hydrogen peroxide as a catalytic by‐product, influences death and survival mechanisms. Targeted microRNA (miRNA) was used to stably knock down MAO‐A mRNA, protein, and catalytic activity by 60–70% in SH‐SY5Y human neuroblastoma cells. The effects of MAO‐A knockdown (KD) on ATP, oxidative stress, electron transport chain, and survival following exposure to mitochondrial toxins were assessed. In control cells, complex I inhibition resulted in caspasemediated cell death linked with ROS production and reduced ATP, followed by up‐regulation of MAO‐A mRNA, protein, and enzyme activity levels. Inhibition of complex III and IV resulted in a similar increase in MAO‐A expression, while up‐regulation of MAO‐A was lower following complex II inhibition. MAO‐A KD decreased basal reactive oxygen species levels by 50% and increased levels of ATP and reduced glutathione and Bcl‐2. MAO‐A KD specifically increased the activity of complex I but had no effect on complex II‐IV activities. Furthermore, MAO‐A KD protected against inhibitors of complex I, III, and IV. In summary, endogenous MAO‐A levels influence mitochondrial function, notably complex I activity, and MAO‐A may be a target for protection against neurodegenerative conditions that involve oxidative stress and mitochondrial dysfunction as underlying pathogenic factors.—Fitzgerald, J. C., Ugun‐Klusek, A., Allen, G., De Girolamo, L. A., Hargreaves, I., Ufer, C., Abramov, A. Y., Billett, E. E. Monoamine oxidase‐A knockdown in human neuroblastoma cells reveals protection against mitochondrial toxins. FASEB J. 28, 218–229 (2014). www.fasebj.org

Characterization of outer membrane vesicles from a neonatal meningitic strain of Cronobacter sakazakii

Cronobacter sakazakii is associated with severe and often fatal cases of infant meningitis and necrotizing enterocolitis. The form of meningitis differs from that due to Neisseria meningitidis and Streptococcus spp., in that it is highly invasive and destructive towards human brain cells. However, there is relatively little understanding of the cytopathogenic interaction of C. sakazakii with host cells which results in stimulation of an inflammatory immune response. The production of Cronobacter outer membrane vesicles (OMV) and their potential pathogenic functions have not yet been elucidated. This study is the first to show that C. sakazakii produce OMV, which may play a role in the activation of cytopathogenic and host cell responses on human intestinal epithelial cells. Cronobacter sakazakii strain 767 was used which had been isolated from a fatal outbreak of neonatal meningitis and necrotizing enterocolitis. Cronobacter sakazakii OMV were internalized by Caco-2 cells, increased cell proliferation and stimulated the hosts innate proinflammatory response without inducing overt toxicity. A total of 18 OMV-associated proteins were identified by mass spectrometry and their potential pathogenicity roles were evaluated. Collectively, these data indicate that C. sakazakii OMV could play a role in pathogenesis by delivering bacterial toxins into host epithelial cells, driving proliferative and proinflammatory responses.

Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine on Differentiating Mouse N2a Neuroblastoma Cells

Abstract : The effect of the neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) was investigated in mouse N2a neuroblastoma cells, induced to differentiate by serum withdrawal and addition of dibutyryl cyclic AMP, over a 24‐h period. Addition of MPTP (10 μM) during differentiation caused a change in cell morphology characterised by an inhibition of axon outgrowth, in the absence of cell death. Biochemical characterisation by western blotting revealed that MPTP had no significant effects on the levels of actin, α‐tubulin, or total heavy‐chain neurofilament (NF‐H). However, NF‐H phosphorylation appeared to increase following MPTP treatment when blots were probed with the phosphorylation state‐specific antibodies RMd09 and Ta51. In addition, indirect immunofluorescence analysis revealed an accumulation of phosphorylated NF‐H in the cell perikaryon, suggesting that altered NF‐H distribution was associated with the observed effects of MPTP on cell morphology. These changes may represent a useful in vitro marker of MPTP neurotoxicity within a simple differentiating neuronal cell model system.

Role of extracellular-regulated kinase and c-Jun NH2-terminal kinase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurofilament phosphorylation.

The neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) causes selective degeneration of dopaminergic neurons in which the c‐Jun NH2‐terminal kinase (JNK) signalling cascade has been implicated. We have employed a differentiated mouse neuroblastoma N2a cell model to investigate the involvement of JNK and extracellular‐regulated kinase (ERK) in MPTP‐mediated toxicity and their role in neurofilament heavy chain (NF‐H) phosphorylation. Acute treatment with a cytotoxic MPTP concentration (5 mM) caused rapid and sustained JNK phosphorylation and ERK dephosphorylation, accompanied by cell death. In contrast, subcytotoxic concentrations of 10 μM MPTP resulted in lower, transient JNK activation in the presence of sustained ERK activity. This resulted in an aberrant increase in a phosphorylation‐dependent NF‐H epitope, perikaryal accumulation of NF‐H, and loss of axon‐like processes, prior to cell death. Inhibition of MEK kinase, using PD98059, showed that MEK 1/2 or the downstream kinase, ERK, is required for N2a cell differentiation, NF‐H phosphorylation and survival. Indeed, MPTP‐induced cell death was exacerbated by the presence of PD98059. However, in the presence of MPTP, reducing JNK activity by using an upstream specific mixed‐lineage kinase inhibitor (CEP‐11004) significantly attenuated aberrant NF‐H phosphorylation and perikaryal NF‐H accumulation and maintained axon‐like processes, in addition to attenuating cell death. This study reports a switch in the predominant kinase involved in NF phosphorylation in a neuronal cell model and may have implications for the formation of inclusions. Our studies provide further evidence that modulation of the JNK pathway could have a role in alleviating neuronal cell death.

Carnosine scavenging of glucolipotoxic free radicals enhances insulin secretion and glucose uptake

The worldwide prevalence of diabetes has risen to 8.5% among adults, which represents a staggering rise in prevalence from 4.7% in 1980. Whilst some treatments work by increasing insulin secretion, over time their effectiveness decreases. We aim to increase insulin secretion by developing strategies that work through mechanisms independent of current treatment options. Isolated CD1 mouse islets, INS-1 pancreatic β-cells, or C2C12 mouse myotubes were incubated in standard tissue culture media, or media supplemented with 28 mM glucose, 200 μM palmitic acid, and 200 μM oleic acid as a cellular model of diabetic glucolipotoxicity. Intracellular reactive species content was assayed using 2′,7′-dichlorofluorescein diacetate dye, inducible nitric oxide synthase levels determined by Western blot, 3-nitrotyrosine and 4-hydrpxnonenal both assayed by ELISA, insulin secretion quantified using ELISA or radioimmunoassay, and glucose uptake determined through 2-deoxy glucose 6 phosphate luminescence. Our data indicate that carnosine, a histidine containing dipeptide available through the diet, is an effective scavenger of each of the aforementioned reactive species. This results in doubling of insulin secretion from isolated mouse islets or INS-1 β-cells. Crucially, carnosine also reverses glucolipotoxic inhibition of insulin secretion and enhances glucose uptake into skeletal muscle cells. Thus, carnosine, or non-hydrolysable carnosine analogs, may represent a new class of therapeutic agent to fight type 2 diabetes.