Search

The rate of mitosis of cancer cells is significantly higher than normal primary cells with increased metabolic needs, which in turn enhances the generation of reactive oxygen species (ROS) production. Higher ROS production is known to increase cancer cell dependence on ROS scavenging systems to counteract the increased ROS. Therapeutic options which selectively modulate the levels of intracellular ROS in cancers are likely candidates for drug discovery. Docetaxel (DTX) has demonstrated antitumor activity in preclinical and clinical studies. It is thought that DTX induces cell death through excessive ROS production and increased Ca 2+ entry. The Ca 2+ permeable TRPM2 channel is activated by ROS. Selenium (Se) has been previously used to stimulate apoptosis for the treatment of glioblastoma cells resistant to DTX. However, the potential mechanism(s) of the additive effect of DTX on TRPM2 channels in cancer cells remains unclear. The aim of this study was to evaluate the effect of combination therapy of DTX and Se on activation of TRPM2 in DBTRG glioblastoma cells. DBTRG cells were divided into four treatment groups: control, DTX (10 nM for 10 h), Se (1 μM for 10 h), and DTX+Se. Our study showed that apoptosis (Annexin V and propidium iodide), mitochondrial membrane depolarization (JC1), and ROS production levels were increased in DBTRG cells following treatment with Se and DTX respectively. Cell number and viability, and the levels of apoptosis, JC1, ROS, and [Ca 2+ ] i , induced by DTX, were further increased following addition of Se. We also observed an additive increase in the activation of the NAD-dependent DNA repair enzyme poly (ADP-ribose) polymerase-1 (PARP-1) activity, which was accompanied by a decline in its essential substrate NAD + . As well, the Se- and DTX-induced increases in intracellular Ca 2+ florescence intensity were decreased following treatment with the TRPM2 antagonist N-(p-amylcinnamoyl) anthranilic acid (ACA). Therefore, combination therapy with Se and DTX may represent an effective strategy for the treatment of glioblastoma cells and may be associated with TRPM2-mediated increases in oxidative stress and [Ca 2+ ] i .

Quinolinic acid (QUIN) is a neurotoxin, gliotoxin, and proinflammatory molecule involved in the pathogenesis of several neurological diseases. Myxobacteria have been known as a rich source of secondary metabolites with diverse structures and mode of actions. In this study, we examined the potential neuroprotective effects of myxobacterial extracts on QUIN-induced excitotoxicity in primary human neurons. For this purpose, primary cultures of human neurons were pre-incubated with myxobacterial extracts and subsequently treated with QUIN at a pathophysiological concentration of 550 nM. The results showed that some myxobacterial extracts can significantly attenuate formation of reactive oxygen species (ROS), nitric oxide (NO) production, and extracellular lactate dehydrogenase (LDH) activity of human neurons. Moreover, myxobacterial extracts were also able to reduce neuronal nitric oxide synthase (nNOS) activity. Some extracts prevented cell death by reducing the activation of poly (ADP-ribose) polymerase (PARP1) by QUIN, therefore by maintaining NAD + levels. In addition, myxobacterial extracts ameliorated oxidative stress by increasing the intracellular levels of glutathione after treatment with QUIN. The results showed that extracts of Stigmatella sp. UTMC 4072 and Archangium sp. UTMC 4070 and were the most effective in reducing QUIN-induced excitotoxicity in primary human neurons. Due to their antioxidative activity, myxobacterial extracts represent an underexplored source of potential new drugs for the treatment of neurodegenerative diseases.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the progressive loss of cholinergic neurons. Amyloid beta is a misfolded protein that represents one of the key pathological hallmarks of AD. Numerous studies have shown that Aβ 1-42 induces oxidative damage, neuroinflammation, and apoptosis, leading to cognitive decline in AD. Recently, fibroblast growth factor 21 (FGF21) has been suggested to be a potential regulator of oxidative stress in mammalian cells. FGF21 has been shown to improve insulin sensitivity, reduce hyperglycemia, increase adipose tissue glucose uptake and lipolysis, and decrease body fat and weight loss by enhancing energy expenditure. In this study, we investigated the effect of FGF21 Aβ 1-42 toxicity in SH-SY5Y neuroblastoma cells. Our data shows that FGF21 significantly decreased Aβ 1- 42-induced toxic effects and repressed oxidative stress and apoptosis in cells exposed to Aβ 1-42 peptide. Our investigation also confirmed that FGF21 pretreatment favorably affects HSP90/TLR4/NF-κB signaling pathway. Therefore, FGF21 represents a viable therapeutic strategy to abrogate Aβ 1-42 -induced cellular inflammation and apoptotic death in the SH-SY5Y neuroblastoma cells.

Environmental toxins produced by cyanobacteria and dinoflagellates have increasingly become a public health concern due to their ability to damage several tissues in humans. In particular, emerging evidence has called attention to the neurodegenerative effects of the cyanobacterial toxin β-N-methylamino-L-alanine (BMAA). Furthermore, other toxins such as anatoxin, saxitoxin, microcystin, nodularin and ciguatoxin also have a different range of effects on human tissues, including hepatotoxicity, neurotoxicity and gastrointestinal irritation. However, the vast majority of known environmental toxins have not yet been examined in the context of neurodegenerative disease. This review aims to investigate whether neurotoxic mechanisms can be demonstrated in all aforementioned toxins, and whether there exists a link to neurodegeneration. Management of toxin exposure and potential neuroprotective compounds is also discussed. Collectively, all aforementioned microbial toxins are likely to exert some form of neuronal damage, with many of their modes of action consistent with neurodegeneration. This is important in advancing our current understanding of the cytotoxic potential of environmental toxins upon human brain function, particularly in the context of age-related neurodegenerative disease.

Introduction: Nicotinamide adenine dinucleotide (NAD + ) is an essential pyridine nucleotide that serves as a key hydride transfer coenzyme for several oxidoreductases. It is also the substrate for intracellular secondary messenger signalling by CD38 glycohydrolases, DNA repair by poly(adenosine diphosphate ribose) polymerase, and epigenetic regulation of gene expression by a class of histone deacetylase enzymes known as sirtuins. The measurement of NAD + and its related metabolites (hereafter, the NAD + metabolome) represents an important indicator of cellular function., Objectives: A study was performed to develop a sensitive, selective, robust, reproducible, and rapid method for the concurrent quantitative determination of intracellular levels of the NAD + metabolome in glial and oocyte cell extracts using liquid chromatography coupled to mass spectrometry (LC/MS/MS)., Methods: The metabolites were separated on a versatile amino column using a dual HILIC-RP gradient with heated electrospray (HESI) tandem mass spectrometry detection in mixed polarity multiple reaction monitoring mode., Results: Quantification of 17 metabolites in the NAD + metabolome in U251 human astroglioma cells could be achieved. Changes in NAD + metabolism in U251 cell line, and murine oocytes under different culture conditions were also investigated., Conclusion: This method can be used as a sensitive profiling tool, tailoring chromatography for metabolites that express significant pathophysiological changes in several disease conditions and is indispensable for targeted analysis.

Toxins produced by cyanobacteria and dinoflagellates have increasingly become a public health concern due to their degenerative effects on mammalian tissue and cells. In particular, emerging evidence has called attention to the neurodegenerative effects of the cyanobacterial toxin β-N-methylamino-L-alanine (BMAA). Other toxins such as the neurotoxins saxitoxin and ciguatoxin, as well as the hepatotoxic microcystin, have been previously shown to have a range of effects upon the nervous system. However, the capacity of these toxins to cause neurodegeneration in human cells has not, to our knowledge, been previously investigated. This study aimed to examine the cytotoxic effects of BMAA, microcystin-LR (MC-LR), saxitoxin (STX) and ciguatoxin (CTX-1B) on primary adult human astrocytes. We also demonstrated that α-lipoate attenuated MC-LR toxicity in primary astrocytes and characterised changes in gene expression which could potentially be caused by these toxins in primary astrocytes. Herein, we are the first to show that all of these toxins are capable of causing physiological changes consistent with neurodegeneration in glial cells, via oxidative stress and excitotoxicity, leading to a reduction in cell proliferation culminating in cell death. In addition, MC-LR toxicity was reduced significantly in astrocytes-treated α-lipoic acid. While there were no significant changes in gene expression, many of the probes that were altered were associated with neurodegenerative disease pathogenesis. Overall, this is important in advancing our current understanding of the mechanism of toxicity of MC-LR on human brain function in vitro, particularly in the context of neurodegeneration.

The kynurenine (KYN) pathway (KP) is a major degradative pathway of the amino acid, L-tryptophan (TRP), that ultimately leads to the anabolism of the essential pyridine nucleotide, nicotinamide adenine dinucleotide. TRP catabolism results in the production of several important metabolites, including the major immune tolerance-inducing metabolite KYN, and the neurotoxin and excitotoxin quinolinic acid. Dendritic cells (DCs) have been shown to mediate immunoregulatory roles that mediated by TRP catabolism. However, characterization of the KP in human DCs has so far only been partly delineated. It is critical to understand which KP enzymes are expressed and which KP metabolites are produced to be able to understand their regulatory effects on the immune response. In this study, we characterized the KP in human monocyte-derived DCs (MDDCs) in comparison with the human primary macrophages using RT-PCR, high-pressure gas chromatography, mass spectrometry, and immunocytochemistry. Our results show that the KP is entirely expressed in human MDDC. Following activation of the KP using interferon gamma, MDDCs can mediate apoptosis of T h cells in vitro. Understanding the molecular mechanisms regulating KP metabolism in MDDCs may provide renewed insight for the development of novel therapeutics aimed at modulating immunological effects and peripheral tolerance.

Increasing evidence suggests an important role of alpha-synuclein (α-Syn) in the pathogenesis of Parkinson's disease (PD). The inter-neuronal spread of α-Syn via exocytosis and endocytosis has been proposed as an explanation for the neuropathological findings of PD in sub-clinical and clinical phases. Therefore, interfering the uptake of α-Syn by neurons may be an important step in slowing or modifying the propagation of the disease. The purposes of our study were to investigate if the uptake of α-Syn fibrils can be specifically interfered with monomeric β-Amyloid1-40 (Aβ40) and to characterise the core acting site of interference. Using a radioisotope-labelled uptake assay, we found an 80 % uptake reduction of α-Syn fibrils in neurons interfered with monomeric Aβ40, but not β-Amyloid1-42 (Aβ42) as compared to controls. This finding was further confirmed by enzyme-linked immunosorbent assay (ELISA) with α-Syn uptake reduced from about 80 % (Aβ42) to about 20 % (Aβ40) relative to controls. To define the region of Aβ40 peptide capable of the interference, we explored shorter peptides with less amino acid residues from both the C-terminus and N-terminus. We found that the interference effect was preserved if amino acid residue was trimmed to position 11 (from N-terminus) and 36 (from C-terminus), but dropped off significantly if residues were trimmed beyond these positions. We therefore deduced that the "core acting site" lies between amino acid residue positions 12-36. These findings suggest α-Syn uptake can be interfered with monomeric Aβ40 and that the core acting site of interference might lie between amino acid residue positions 12-36.

Quinolinic acid (QUIN) is an excitotoxin that has been implicated in the pathogenesis of several neurodegenerative diseases including Alzheimer's disease (AD). While QUIN has been shown to induce neuronal and astrocytic apoptosis as well as excitotoxic cell death, other mechanisms such as autophagy remain unexplored. We investigated the role of Cathepsin D (CatD) and Beclin-1 (Bc1) in QUIN-treated primary human astrocytes and neurons. We demonstrated that the expression patterns of CatD, a lysosomal aspartic protease associated with autophagy, are increased at 24 h after QUIN treatment. However, unlike CatD, the expression patterns of Bc1, a tumour suppressor protein, are significantly reduced at 24 h after QUIN treatment in both brain cell types. Furthermore, we showed that the NMDA ion channel blockers, MK801, can attenuate QUIN-induced changes CatD and Bc1 expression in both astrocytes and neurons. Taken together, these results suggest that induction of deficits in CatD and Bc1 is a significant mechanism for QUIN toxicity in glial and neuronal cells. Maintenance of autophagy may play a crucial role in neuroprotection in the setting of AD.

Ciguatoxin (CTX), is a toxic compound produced by microalgae (dinoflagellate) Gambierdiscus spp., and is bio-accumulated and bio-transformed through the marine food chain causing neurological deficits. To determine the mechanism of CTX-mediated cytotoxicity in human neurons, we measured extracellular lactate dehydrogenase (LDH) activity, intracellular levels of nicotinamide adenine dinucleotide (NAD(+)) and H2AX phosphorylation at serine 139 as a measure for DNA damage in primary cultures of human neurons treated with Pacific (P)-CTX-1B and P-CTX-3C. We found these marine toxins can induce a time and dose-dependent increase in extracellular LDH activity, with a concomitant decline in intracellular NAD(+) levels and increased DNA damage at the concentration range of 5-200 nM. We also showed that pre- and post-treatment with rosmarinic acid (RA), the active constituent of the Heliotropium foertherianum (Boraginaceae) can attenuate CTX-mediated neurotoxicity. These results further highlight the potential of RA in the treatment of CTX-induced neurological deficits.

Parkinson's disease (PD) is a multicentred neurodegenerative disorder characterised by the accumulation and aggregation of alpha-synuclein (α-syn) in several parts of the central nervous system. However, it is well established that PD can generate symptoms of constipation and other gastrointestinal problems and α-syn containing lesions have been identified in intestinal nerve cells. In this study, we show that α-syn can be taken up and accumulate in primary human foetal enteric neurons from the gastrointestinal tract and can be transferred between foetal enteric neurons. Impaired proteosomal/lysosomal degradation can promote the uptake and accumulation of α-syn in enteric neurons. Enteric neurons exposed to α-syn can also lead to impaired mitochondrial complex I activity, reduced mitochondrial function, and NAD(+) depletion culminating in cell death via energy restriction. These findings demonstrate neuron-to-neuron transmission of α-syn in enteric neurons, providing renewed evidence for Braak's hypothesis and the aetiology of PD.

Autism is a debilitating neurodevelopment disorder characterised by stereotyped interests and behaviours, and abnormalities in verbal and non-verbal communication. It is a multifactorial disorder resulting from interactions between genetic, environmental and immunological factors. Excitotoxicity and oxidative stress are potential mechanisms, which are likely to serve as a converging point to these risk factors. Substantial evidence suggests that excitotoxicity, oxidative stress and impaired mitochondrial function are the leading cause of neuronal dysfunction in autistic patients. Glutamate is the primary excitatory neurotransmitter produced in the CNS, and overactivity of glutamate and its receptors leads to excitotoxicity. The over excitatory action of glutamate, and the glutamatergic receptors NMDA and AMPA, leads to activation of enzymes that damage cellular structure, membrane permeability and electrochemical gradients. The role of excitotoxicity and the mechanism behind its action in autistic subjects is delineated in this review.

Increasing evidence suggests that an inflammatory microenvironment promotes invasion by glioblastoma (GBM) cells. Together with p38 mitogen-activated protein kinase (MAPK) activation being regarded as promoting inflammation, we hypothesized that elevated inflammatory cytokine secretion and p38 MAPK activity contribute to expansion of GBMs. Here we report that IL-1β, IL-6, and IL-8 levels and p38 MAPK activity are elevated in human glioblastoma specimens and that p38 MAPK inhibitors attenuate the secretion of pro-inflammatory cytokines by microglia and glioblastoma cells. RNAi knockdown and immunoprecipitation experiments suggest that the p38α MAPK isoform drives inflammation in GBM cells. Importantly, p38 MAPK inhibition strongly reduced invasion of U251 glioblastoma cells in an inflammatory microenvironment, providing evidence for a p38 MAPK-regulated link between inflammation and invasiveness in GBM pathophysiology.

There is growing evidence implicating the kynurenine pathway (KP) and particularly one of its metabolites, quinolinic acid (QUIN), as important contributors to neuroinflammation in several brain diseases. While QUIN has been shown to induce neuronal and astrocytic apoptosis, the exact mechanisms leading to cell death remain unclear. To determine the mechanism of QUIN-mediated excitotoxicity in human brain cells, we measured intracellular levels of nicotinamide adenine dinucleotide (NAD(+)) and poly(ADP-ribose) polymerase (PARP) and extracellular lactate dehydrogenase (LDH) activities in primary cultures of human neurons and astrocytes treated with QUIN. We found that QUIN acts as a substrate for NAD(+) synthesis at very low concentrations (<50 nM) in both neurons and astrocytes, but is cytotoxic at sub-physiological concentrations (>150 nM) in both the cell types. We have shown that the NMDA ion channel blockers, MK801 and memantine, and the nitric oxide synthase (NOS) inhibitor, L-NAME, significantly attenuate QUIN-mediated PARP activation, NAD(+) depletion, and LDH release in both neurons and astrocytes. An increased mRNA and protein expression of the inducible (iNOS) and neuronal (nNOS) forms of nitric oxide synthase was also observed following exposure of both cell types to QUIN. Taken together these results suggests that QUIN-induced cytotoxic effects on neurons and astrocytes are likely to be mediated by an over activation of an NMDA-like receptor with subsequent induction of NOS and excessive nitric oxide (NO(*))-mediated free radical damage. These results contribute significantly to our understanding of the pathophysiological mechanisms involved in QUIN neuro- and gliotoxicity and are relevant for the development of therapies for neuroinflammatory diseases.

Oxidative imbalance is a prominent feature in Alzheimer's disease and ageing. Increased levels of reactive oxygen species (ROS) can result in disordered cellular metabolism due to lipid peroxdation, protein-cross linking, DNA damage and the depletion of nicotinamide adenine dinucleotide (NAD(+)). NAD(+) is a ubiquitous pyridine nucleotide that plays an essential role in important biological reactions., from ATP production and secondary messenger signaling, to transcriptional regulation and DNA repair. Chronic oxidative stress may be associated with NAD(+) depletion and a subsequent decrease in metabolic regulation and cell viability. Hence, therapies targeted toward maintaining intracellular NAD(+) pools may prove efficacious in the protection of age-dependent cellular damage, in general, and neurodegeneration in chronic central nervous system inflammatory diseases such as Alzheimer's disease, in particular.