Hypoxia Signaling in Parkinson's Disease: There Is Use in Asking "What HIF?"

Hypoxia is a condition characterized by insufficient tissue oxygenation, which results in impaired oxidative energy production. A reduction in cellular oxygen levels induces the stabilization of hypoxia inducible factor α (HIF-1α), master regulator of the molecular response to hypoxia, involved in maintaining cellular homeostasis and driving hypoxic adaptation through the control of gene expression. Due to its high energy requirement, the brain is particularly vulnerable to oxygen shortage. Thus, hypoxic injury can cause significant metabolic changes in neural cell populations, which are associated with neurodegeneration. Recent evidence suggests that regulating HIF-1α may ameliorate the cellular damage in neurodegenerative diseases. Indeed, the hypoxia/HIF-1α signaling pathway has been associated to several processes linked to Parkinson's disease (PD) including gene mutations, risk factors and molecular pathways such as mitochondrial dysfunction, oxidative stress and protein degradation impairment. This review will explore the impact of hypoxia and HIF-1α signaling on these specific molecular pathways that influence PD development and will evaluate different novel neuroprotective strategies involving HIF-1α stabilization.

A co-culture nanofibre scaffold model of neural cell degeneration in relevance to Parkinson's disease.

Current therapeutic strategies for Parkinson's disease (PD) aim to delay progression or replace damaged neurons by restoring the original neuronal structures. The poor regenerative capacity of neural tissue highlights the need for the development of cellular environments to model the pathogenesis of PD. In the current work, we have characterised the growth, survival and response to PD mimetics of human SH-SY5Y neuroblastoma and U-87MG glioblastoma cell lines cultured on polyacrylonitrile (PAN) and Jeffamine® doped polyacrylonitrile (PJ) nano-scaffolds. Differentiation induced by a range of agents was evaluated by immunoassays of neural protein biomarkers. PAN and PJ nanofibre scaffolds provided suitable three-dimensional (3D) environment to support the growth, differentiation and network formation of dopaminergic neuron- and astrocyte-like cell populations, respectively. The scaffolds selectively supported the survival and differentiation of both cell populations with prolonged neuronal survival when exposed to PD mimetics in the presence of astrocytes in a co-culture model. Such 3D nanoscaffold-based assays could aid our understanding of the molecular basis of PD mimetic-induced Parkinsonism and the discovery of neuroprotective agents.

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 ß-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 ß-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.

Bisphenol A increases BeWo trophoblast survival in stress-induced paradigms through regulation of oxidative stress and apoptosis.

Bisphenol A (BPA) is ubiquitous in the environment and is reported to be present at high concentrations in placental tissue, where its presence raises concerns over its potential to disrupt placental function. This report investigates how BPA interferes with the survival of human choriocarcinoma BeWo cells (a model of placental trophoblasts) under stress-induced paradigms reminiscent of pathways activated in placental development. These include conditions that promote oxidative stress (glutathione depletion) and apoptosis (serum withdrawal) or mimic hypoxia (HIF-1α accumulation via dimethyloxalylglycine treatment). Treatment of BeWo cells with BPA during stress-induced paradigms led to a consistent and significant increase in cell viability, with a concomitant increase in glutathione levels and a reduction in apoptosis. Assessment of the antioxidant capacity of BPA revealed its ability to quench reactive oxygen species and reduce the levels generated during glutathione and serum depletion. BPA was also able to reduce the activation of the antioxidant response element (ARE) through mediation of its activators, nuclear factor erythroid related factor family members (Nrf's). Indeed, the expression and nuclear translocation of Nrf2 (an important ARE activator) were impaired by BPA, while Nrf1 and Nrf3 expression levels were increased. Furthermore, BPA increased the levels of the anti-apoptotic proteins (Bcl-2 and Hsp70) and decreased HIF-1α levels during stress-induced conditions. Together, these results indicate that BPA inhibits trophoblast cell death under conditions of cellular stress. This could have implications on placental trophoblasts during development.

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 host's 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.

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 caspase-mediated 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.

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

Increasing evidence points to mitochondrial dysfunction in Parkinson's 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.

Assessment of the direct and indirect effects of MPP+ and dopamine on the human proteasome: implications for Parkinson's disease aetiology.

Mitochondrial impairment, glutathione depletion and oxidative stress have been implicated in the pathogenesis of Parkinson's disease (PD), linked recently to proteasomal dysfunction. Our study analysed how these factors influence the various activities of the proteasome in human SH-SY5Y neuroblastoma cells treated with the PD mimetics MPP+ (a complex 1 inhibitor) or dopamine. Treatment with these toxins led to dose- and time-dependent reductions in ATP and glutathione and also chymotrypsin-like and post-acidic like activities; trypsin-like activity was unaffected. Antioxidants blocked the effects of dopamine, but not MPP+, suggesting that oxidative stress was more important in the dopamine-mediated effects. With MPP+, ATP depletion was a prerequisite for loss of proteasomal activity. Thus in a dopaminergic neuron with complex 1 dysfunction both oxidative stress and ATP depletion will contribute independently to loss of proteasomal function. We show for the first time that addition of MPP+ or dopamine to purified samples of the human 20S proteasome also reduced proteasomal activities; with dopamine being most damaging. As with toxin-treated cells, chymotrypsin-like activity was most sensitive and trypsin-like activity the least sensitive. The observed differential sensitivity of the various proteasomal activities to PD mimetics is novel and its significance needs further study in human cells.

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.

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.

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 microM 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.