Supplementation with γ-glutamylcysteine (γ-GC) lessens oxidative stress, brain inflammation and amyloid pathology and improves spatial memory in a murine model of AD.

INTRODUCTION: The accumulation of oxidative stress, neuroinflammation and abnormal aggregation of amyloid ß-peptide (Aß) have been shown to induce synaptic dysfunction and memory deficits in Alzheimer's disease (AD). Cellular depletion of the major endogenous antioxidant Glutathione (GSH) has been linked to cognitive decline and the development of AD pathology. Supplementation with γ-glutamylcysteine (γ-GC), the immediate precursor and the limiting substrate for GSH biosynthesis, can transiently augment cellular GSH levels by bypassing the regulation of GSH homeostasis. METHODS: In the present study, we investigated the effect of dietary supplementation of γ-GC on oxidative stress and Aß pathology in the brains of APP/PS1 mice. The APP/PS1 mice were fed γ-GC from 3 months of age with biomarkers of apoptosis and cell death, oxidative stress, neuroinflammation and Aß load being assessed at 6 months of age. RESULTS: Our data showed that supplementation with γ-GC lowered the levels of brain lipid peroxidation, protein carbonyls and apoptosis, increased both total GSH and the glutathione/glutathione disulphide (GSH/GSSG) ratio and replenished ATP and the activities of the antioxidant enzymes (superoxide dismutase (SOD), catalase, glutamine synthetase and glutathione peroxidase (GPX)), the latter being a key regulator of ferroptosis. Brain Aß load was lower and acetylcholinesterase (AChE) activity was markedly improved compared to APP/PS1 mice fed a standard chow diet. Alteration in brain cytokine levels and matrix metalloproteinase enzymes MMP-2 and MMP-9 suggested that γ-GC may lower inflammation and enhance Aß plaque clearance in vivo. Spatial memory was also improved by γ-GC as determined using the Morris water maze. CONCLUSION: Our data collectively suggested that supplementation with γ-GC may represent a novel strategy for the treatment and/or prevention of cognitive impairment and neurodegeneration.

The Precursor to Glutathione (GSH), γ-Glutamylcysteine (GGC), Can Ameliorate Oxidative Damage and Neuroinflammation Induced by Aß40 Oligomers in Human Astrocytes.

Glutathione (GSH) is one of the most abundant thiol antioxidants in cells. Many chronic and age-related diseases are associated with a decline in cellular GSH levels or impairment in the catalytic activity of the GSH biosynthetic enzyme glutamate cysteine ligase (GCL). γ-glutamylcysteine (GGC), a precursor to glutathione (GSH), can replenish depleted GSH levels under oxidative stress conditions, by circumventing the regulation of GSH biosynthesis and providing the limiting substrate. Soluble amyloid-ß (Aß) oligomers have been shown to induce oxidative stress, synaptic dysfunction and memory deficits which have been reported in Alzheimer's disease (AD). Calcium ions, which are increased with age and in AD, have been previously reported to enhance the formation of Aß40 oligomers, which have been casually associated with the pathogenesis of the underlying neurodegenerative condition. In this study, we examined the potential beneficial effects of GGC against exogenous Aß40 oligomers on biomarkers of apoptosis and cell death, oxidative stress, and neuroinflammation, in human astrocytes. Treatment with Aß40 oligomers significantly reduced the cell viability and apoptosis of astrocyte brain cultures and increased oxidative modifications of DNA, lipids, and protein, enhanced pro-inflammatory cytokine release and increased the activity of the proteolytic matrix metalloproteinase enzyme, matric metalloproteinase (MMP)-2 and reduced the activity of MMP-9 after 24 h. Co-treatment of Aß40 oligomers with GGC at 200 µM increased the activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) and led to significant increases in the levels of the total antioxidant capacity (TAC) and GSH and reduced the GSSG/GSH ratio. GGC also upregulated the level of the anti-inflammatory cytokine IL-10 and reduced the levels of the pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and attenuated the changes in metalloproteinase activity in oligomeric Aß40-treated astrocytes. Our data provides renewed insight on the beneficial effects of increased GSH levels by GGC in human astrocytes, and identifies yet another potential therapeutic strategy to attenuate the cytotoxic effects of Aß oligomers in AD.

Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein).

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni-Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

An analysis of the changes in soluble hydrogenase and global gene expression in Cupriavidus necator (Ralstonia eutropha) H16 grown in heterotrophic diauxic batch culture.

BACKGROUND: Soluble hydrogenases (SH) are enzymes that catalyse the oxidation of molecular hydrogen. The SH enzyme from Cupriavidus necator H16 is relatively oxygen tolerant and makes an attractive target for potential application in biochemical hydrogen fuel cells. Expression of the enzyme can be mediated by derepression of the hox promoter system under heterotrophic conditions. However, the overall impact of hox derepression, from a transcriptomic perspective, has never been previously reported. RESULTS: Derepression of hydrogenase gene expression upon fructose depletion was confirmed in replicate experiments. Using qRT-PCR, hoxF was 4.6-fold up-regulated, hypF2 was up-regulated in the cells grown 2.2-fold and the regulatory gene hoxA was up-regulated by a mean factor of 4.5. A full transcriptomic evaluation revealed a substantial shift in the global pattern of gene expression. In addition to up-regulation of genes associated with hydrogenase expression, significant changes were observed in genes associated with energy transduction, amino acid metabolism, transcription and translation (and regulation thereof), genes associated with cell stress, lipid and cell wall biogenesis and other functions, including cell motility. CONCLUSIONS: We report the first full transcriptome analysis of C. necator H16 grown heterotrophically on fructose and glycerol in diauxic batch culture, which permits expression of soluble hydrogenase under heterotrophic conditions. The data presented deepens our understanding of the changes in global gene expression patterns that occur during the switch to growth on glycerol and suggests that energy deficit is a key driver for induction of hydrogenase expression in this organism.

Global cellular responses to ß-methyl-amino-L-alanine (BMAA) by olfactory ensheathing glial cells (OEC).

This study utilised a proteomics approach to identify any differential protein expression in a glial cell line, rat olfactory ensheathing cells (OECs), treated with the cyanotoxin ß-methylamino-l-alanine (BMAA). Five proteins of interest were identified, namely Rho GDP-dissociation inhibitor 1 (RhoGDP1), Nck-associated protein 1 (NCKAP1), voltage-dependent anion-selective channel protein 1 (VDAC1), 3-hydroxyacyl-CoA dehydrogenase type-2 (3hCoAdh2), and ubiquilin-4 (UBQLN4). Four of these candidates, nuclear receptor subfamily 4 group A member 1 (Nur77), cyclophilin A (CyPA), RhoGDP1 and VDAC1, have been reported to be involved in cell growth. A microarray identified UBQLN4, palladin and CyPA, which have been implicated to have roles in excitotoxicity. Moreover, the NCKAP1, UBQLN4, CyPA and 3hCoAdh2 genes have been associated with abnormal protein aggregation. Differential expression of genes involved in mitochondrial activity, Nur77, 3hCoAdh2, VDAC1 and UBQLN4, were also identified. Confirmatory reverse transcription quantitative PCR (RT-qPCR) analysis of transcripts generated from the genes of interest corroborated the differential expression trends identified in the global protein analysis. BMAA induced cell cycle arrest in the G2/M phase of OEC and apoptosis after 48 h at concentrations of 250 µM and 500 µM. Collectively, this work advances our understanding of the mechanism of BMAA-mediated glial-toxicity in vitro.

Therapeutic approaches to modulating glutathione levels as a pharmacological strategy in Alzheimer's disease.

Accumulating evidence has suggested the involvement of oxidative stress in the pathogenesis of Alzheimer's disease (AD). The main endogenous antioxidant, glutathione (GSH), has been shown to decline with ageing and in several age-related degenerative diseases, including AD. Potential options for replenishing GSH levels as a therapeutic target to treat these conditions include the administration of GSH itself, and low toxicity forms of the limiting amino acid for GSH synthesis; cysteine. However, passive GSH uptake is limited due to an unfavourable concentration gradient between the plasma and cytosol. Similarly, cysteine prodrugs have demonstrated limited efficacy to elevate depleted GSH levels in several in vivo and in vitro models of disease. It has been suggested that the decline in GSH levels in AD, may be associated with down regulation of GSH homeostasis rather than substrate limitation. Cellular GSH homeostasis is regulated by non-allosteric feedback inhibition exerted by GSH on glutamate cysteine ligase (GCL), which is responsible for the synthesis of the GSH precursor γ-glutamylcysteine (GGC). In conditions involving down regulated GSH homeostasis, GGC serves as a crucialrate-limiting substrate for GSH synthetase, the main enzyme responsible for condensing glycine with GGC to form the final thiol tripeptide, GSH. In this review, we focus on the therapeutic potential of GGC to elevate cellular GSH levels. We also discuss the efficacy of GGC prodrugs which would be taken up and converted by the unregulated GS to GSH, and the administration of modified GSH compounds, such as GSH esters that could potentially overcome the concentration gradient that prohibits passive GSH uptake, in AD.

Gliotoxicity of the cyanotoxin, ß-methyl-amino-L-alanine (BMAA).

The amino acid variant ß-methyl-amino-L-alanine (BMAA) has long been associated with the increased incidence and progression of the amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC). Previous studies have indicated that BMAA damages neurons via excitotoxic mechanisms. We have challenged rat olfactory ensheathing cells (OECs) with exogenous BMAA and found it to be cytotoxic. BMAA also induces a significant increase in Ca2+ influx, enhanced production of reactive oxygen species (ROS), and disrupts mitochondrial activity in OECs. This is the first study investigating BMAA toxicity using pure glial cells. These findings align BMAA with the three proposed mechanisms of degeneration in ALS, those being non-cell autonomous death, excitotoxicity and mitochondrial dysfunction.

Lipid raft-dependent endocytosis of close homolog of adhesion molecule L1 (CHL1) promotes neuritogenesis.

CHL1 plays a dual role by either promoting or inhibiting neuritogenesis. We report here that neuritogenesis-promoting ligand-dependent cell surface clustering of CHL1 induces palmitoylation and lipid raft-dependent endocytosis of CHL1. We identify ßII spectrin as a binding partner of CHL1, and we show that partial disruption of the complex between CHL1 and ßII spectrin accompanies CHL1 endocytosis. Inhibition of the association of CHL1 with lipid rafts by pharmacological disruption of lipid rafts or by mutation of cysteine 1102 within the intracellular domain of CHL1 reduces endocytosis of CHL1. Endocytosis of CHL1 is also reduced by nifedipine, an inhibitor of the L-type voltage-dependent Ca(2+) channels. CHL1-dependent neurite outgrowth is reduced by inhibitors of lipid raft assembly, inhibitors of voltage-dependent Ca(2+) channels, and overexpression of CHL1 with mutated cysteine Cys-1102. Our results suggest that ligand-induced and lipid raft-dependent regulation of CHL1 adhesion via Ca(2+)-dependent remodeling of the CHL1-ßII spectrin complex and CHL1 endocytosis are required for CHL1-dependent neurite outgrowth.

Excitotoxic potential of the cyanotoxin ß-methyl-amino-L-alanine (BMAA) in primary human neurons.

The toxicity of the cyanobacterial modified amino acid, BMAA, has been described in rat, mouse and leech neurons. Particular emphasis has been placed on the potential ability of BMAA to induce neuronal damage via excitotoxic mechanisms. Here we present data indicating that the effects observed on lower organisms are also evident in a human model. Our data indicates that BMAA induces increased intracellular Ca²âº influx, DNA damage, mitochondrial activity, lactate dehydrogenase (LDH) release and generation of reactive oxygen species (ROS). The amelioration of LDH release in the presence of the N-methyl-D-aspartate (NMDA) receptor antagonist MK801 indicates that the neurotoxic effects of BMAA are mediated via NMDA receptor activation. Additionally, we have shown that BMAA induces the expression of neuronal nitric oxide synthase (nNOS) and caspase-3 indicating that it can stimulate apoptosis in human neurons, presumably via activation of NMDA receptors.

Does α-amino-ß-methylaminopropionic acid (BMAA) play a role in neurodegeneration?

The association of α-amino-ß-methylaminopropionic acid (BMAA) with elevated incidence of amyotrophic lateral sclerosis/Parkinson's disease complex (ALS/PDC) was first identified on the island of Guam. BMAA has been shown to be produced across the cyanobacterial order and its detection has been reported in a variety of aquatic and terrestrial environments worldwide, suggesting that it is ubiquitous. Various in vivo studies on rats, mice, chicks and monkeys have shown that it can cause neurodegenerative symptoms such as ataxia and convulsions. Zebrafish research has also shown disruption to neural development after BMAA exposure. In vitro studies on mice, rats and leeches have shown that BMAA acts predominantly on motor neurons. Observed increases in the generation of reactive oxygen species (ROS) and Ca(2+) influx, coupled with disruption to mitochondrial activity and general neuronal death, indicate that the main mode of activity is via excitotoxic mechanisms. The current review pertaining to the neurotoxicity of BMAA clearly demonstrates its ability to adversely affect neural tissues, and implicates it as a potentially significant compound in the aetiology of neurodegenerative disease. When considering the potential adverse health effects upon exposure to this compound, further research to better understand the modes of toxicity of BMAA and the environmental exposure limits is essential.

Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice.

Obsessive-compulsive disorder (OCD) is an anxiety-spectrum disorder characterized by persistent intrusive thoughts (obsessions) and repetitive actions (compulsions). Dysfunction of cortico-striato-thalamo-cortical circuitry is implicated in OCD, although the underlying pathogenic mechanisms are unknown. SAP90/PSD95-associated protein 3 (SAPAP3; also known as DLGAP3) is a postsynaptic scaffolding protein at excitatory synapses that is highly expressed in the striatum. Here we show that mice with genetic deletion of Sapap3 exhibit increased anxiety and compulsive grooming behaviour leading to facial hair loss and skin lesions; both behaviours are alleviated by a selective serotonin reuptake inhibitor. Electrophysiological, structural and biochemical studies of Sapap3-mutant mice reveal defects in cortico-striatal synapses. Furthermore, lentiviral-mediated selective expression of Sapap3 in the striatum rescues the synaptic and behavioural defects of Sapap3-mutant mice. These findings demonstrate a critical role for SAPAP3 at cortico-striatal synapses and emphasize the importance of cortico-striatal circuitry in OCD-like behaviours.

Expression of the spermatogenic cell-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) in rat testis.

The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1,417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS (94% identity) and human GAPD2 (83% identity) isozymes. Northern blotting of rat tissue RNAs detected the 1.5 kb Gapds transcript in the testis and not in RNA from liver, spleen, epididymis, heart, skeletal muscle, brain, seminal vesicle, and kidney. The rat Gapds mRNA was first detected at day 29 of postnatal testis development, an age which coincides with the initial post-meiotic differentiation of round spermatids. When isolated rat spermatogenic cell RNA was probed for Gapds expression, transcripts were detected only in round spermatids and condensing spermatids, but not in pachytene spermatocytes, demonstrating haploid expression of the Gapds gene. However, immunohistochemical staining of rat testis sections with anti-GAPDS antisera did not detect GAPDS in round spermatids, but localized the protein only to stage XIII and later condensing spermatids as well as testicular spermatozoa, indicating that Gapds expression is translationally regulated. The current results are similar to those previously obtained for mouse GAPDS and human GAPD2, suggesting that reliable comparisons can be made between these species in toxicant screening and contraceptive development.

Differential mRNA expression and protein localization of the SAP90/PSD-95-associated proteins (SAPAPs) in the nervous system of the mouse.

The supramolecular anchoring/signaling complex at the postsynaptic density of glutamatergic synapses has been proposed to play a key role in regulating synaptic function and plasticity. One class of proteins present in the complex is the SAP90/PSD-95-associated protein family (SAPAPs). The SAPAPs, identified by their direct interaction with PSD-95 family proteins, were initially proposed to function in the anchoring/signaling complex as linker proteins between glutamate receptor binding proteins and the cytoskeleton. However, recent studies have indicated that the SAPAPs also bind to signaling molecules and may thus have multiple roles at synapses. Four homologous genes encoding SAPAP proteins have been previously identified. As a first step toward understanding the physiological function of the SAPAPs, we have investigated in detail, at both the mRNA and protein levels, the localization of the individual SAPAP genes in the adult murine nervous system. We find that the SAPAP mRNAs are highly, yet differentially, expressed in many regions of the brain, including the hippocampus and cerebellum. Furthermore, SAPAP3 mRNA is targeted to dendrites, whereas SAPAP1, -2, and -4 mRNAs are detected mainly in cell bodies. The SAPAP proteins are localized at synapses in a manner consistent with mRNA expression. Surprisingly, in addition to glutamatergic synapse localization, antibody staining also reveals that the SAPAP proteins are localized at cholinergic synapses, including neuronal cholinergic synapses and the neuromuscular junction. Together, these results indicate that the SAPAPs are general components of excitatory synapses and that each of these proteins may perform a distinct function.

Localization of the sperm protein SP22 and inhibition of fertility in vivo and in vitro.

We previously established that levels of the sperm membrane protein, SP22, are highly correlated with the fertility of sperm from the cauda epididymidis of rats exposed to both epididymal and testicular toxicants, and that a testis-specific SP22 transcript is expressed in postmeiotic germ cells. In this study, polyclonal and monoclonal antibodies were generated to study the expression of SP22 in the testis and epididymis, and to determine whether SP22 plays a coincidental or causal role in fertility. Polyclonal antiserum was raised in sheep against full-length recombinant rat SP22 (rSP22). Hybridoma clones were generated from mice immunized with rSP22 and boosted with native SP22; positive clones were used for ascites production. Immunoblots indicated that affinity-purified anti-rSP22 immunoglobulin (Ig) and ascites Ig recognized denatured and native SP22, respectively. Linear epitope mapping of the 189-amino acid SP22 sequence revealed 3 distinct peptide sequences recognized by anti-rSP22 Ig, and 1 sequence recognized by ascites Ig. Cytoplasm of round spermatids and heads of elongating/elongated spermatids immunostained with both anti-rSP22 and ascites antibodies. Isolated rete testis sperm revealed discrete staining over the cytoplasmic droplet, whereas staining was apparent over the equatorial segment of the head by the time sperm reached the caput epididymidis. Clear cells were, interestingly, immunostained along the length of the epididymis. Ascites Ig and anti-SP22 Ig each recognized the equatorial segment of sperm heads from rat, hamster, bull, rabbit, and human. Ascites Ig and affinity-purified anti-rSP22 Ig each significantly inhibited the fertility of cauda epididymal sperm from the rat in vivo, as well as the fertilization rates of cauda epididymal sperm in vitro. Moreover, affinity-purified anti-rSP22 significantly inhibited in vitro fertilization of both zona-intact and zona-free hamster oocytes, suggesting that SP22 may play a role in both the zona penetration and membrane fusion steps of fertilization.