Richard Dargusch

The Regulation of Glucose Metabolism by HIF-1 Mediates a Neuroprotective Response to Amyloid Beta Peptide

It is frequently argued that both amyloid beta (Abeta) and oxidative stress are involved in the pathogenesis of Alzheimers disease (AD). We show here that clonal nerve cell lines and primary cortical neurons that are resistant to Abeta toxicity have an enhanced flux of glucose through both the glycolytic pathway and the hexose monophosphate shunt. AD brain also has increased enzymatic activities in both pathways relative to age-matched controls. The Abeta-induced changes in glucose metabolism are due to the activation of the transcription factor hypoxia inducible factor 1 (HIF-1). As a result of Abeta-induced changes in glucose metabolism, Abeta-resistant cells are more readily killed by glucose starvation and by classes of antipsychotic drugs that inhibit glucose uptake.

Cellular Mechanisms of Resistance to Chronic Oxidative Stress

Oxidative stress is implicated in several pathologies such as AIDS, Alzheimers disease, and Parkinsons disease, as well as in normal aging. As a model system to study the response of cells to oxidative insults, glutamate toxicity on a mouse nerve cell line, HT-22, was examined. Glutamate exposure kills HT-22 via a nonreceptor-mediated oxidative pathway by blocking cystine uptake and causing depletion of intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species and, ultimately, apoptotic cell death. Several HT-22 subclones that are 10-fold resistant to exogenous glutamate were isolated and the mechanisms involved in resistance characterized. The expression levels of neither heat shock proteins nor apoptosis-related proteins are changed in the resistant cells. In contrast, the antioxidant enzyme catalase, but not glutathione peroxidase nor superoxide dismutase, is more highly expressed in the resistant than in the parental cells. In addition, the resistant cells have enhanced rates of GSH regeneration due to higher activities of the GSH metabolic enzymes gamma-glutamylcysteine synthetase and GSH reductase, and GSH S-transferases activities are also elevated. As a consequence of these alterations, the glutamate resistant cells are also more resistant to organic hydroperoxides and anticancer drugs that affect these GSH enzymes. These results indicate that resistance to apoptotic oxidative stress may be acquired by coordinated changes in multiple antioxidant pathways.

ERK Activation by the Polyphenols Fisetin and Resveratrol Provides Neuroprotection in Multiple Models of Huntington's Disease

Huntingtons disease (HD) is an inherited, progressive and ultimately fatal neurodegenerative disorder that is characterized by psychiatric, cognitive and motor symptoms. Among the pathways implicated in HD are those involving mitogen-activated protein kinase signaling and particularly the Ras-extracellular signal-regulated kinase (ERK) cascade. Studies in both cells and animal models suggest that ERK activation might provide a novel therapeutic target for the treatment of HD but compounds that specifically activate ERK are few. To test the hypothesis that pharmaceutical activation of ERK might be protective for HD, a polyphenol, fisetin, which was previously shown to activate the Ras-ERK cascade, was tested in three different models of HD: PC12 cells expressing mutant Httex1 under the control of an inducible promoter, Drosophila expressing mutant Httex1 and the R6/2 mouse model of HD. The results indicate that fisetin can reduce the impact of mutant huntingtin in each of these disease models. Prompted by this observation, we determined that the related polyphenol, resveratrol, also activates ERK and is protective in HD models. Notably, although more than a dozen small molecule inhibitors of ERK activation are in clinical trials, very few small molecule activators of ERK signaling are reported. Thus, fisetin, resveratrol and related compounds might be useful for the treatment of HD by virtue of their unique ability to activate ERK.

Distribution of the Cystine/Glutamate Antiporter System x−c in the Brain, Kidney, and Duodenum

System x− c, one of the main transporters responsible for central nervous system cystine transport, is comprised of two subunits, xCT and 4F2hc. The transport of cystine into cells is rate limiting for glutathione synthesis, the major antioxidant and redox cofactor in the brain. Alterations in glutathione status are prevalent in numerous neurodegenerative diseases, emphasizing the importance of proper cystine homeostasis. However, the distribution of xCT and 4F2hc within the brain and other areas has not been described. Using specific antibodies, both xCT and 4F2hc were localized predominantly to neurons in the mouse and human brain, but some glial cells were labeled as well. Border areas between the brain proper and periphery including the vascular endothelial cells, ependymal cells, choroid plexus, and leptomeninges were also highly positive for the system x− c components. xCT and 4F2hc are also present at the brush border membranes in the kidney and duodenum. These results indicate that system x− c is likely to play a role in cellular health throughout many areas of the brain as well as other organs by maintaining intracellular cystine levels, thereby resulting in low levels of oxidative stress. (J Histochem Cytochem 54: 549–557, 2006)

Fisetin Lowers Methylglyoxal Dependent Protein Glycation and Limits the Complications of Diabetes

The elevated glycation of macromolecules by the reactive dicarbonyl and α-oxoaldehyde methylglyoxal (MG) has been associated with diabetes and its complications. We have identified a rare flavone, fisetin, which increases the level and activity of glyoxalase 1, the enzyme required for the removal of MG, as well as the synthesis of its essential co-factor, glutathione. It is shown that fisetin reduces two major complications of diabetes in Akita mice, a model of type 1 diabetes. Although fisetin had no effect on the elevation of blood sugar, it reduced kidney hypertrophy and albuminuria and maintained normal levels of locomotion in the open field test. This correlated with a reduction in proteins glycated by MG in the blood, kidney and brain of fisetin-treated animals along with an increase in glyoxalase 1 enzyme activity and an elevation in the expression of the rate-limiting enzyme for the synthesis of glutathione, a co-factor for glyoxalase 1. The expression of the receptor for advanced glycation end products (RAGE), serum amyloid A and serum C-reactive protein, markers of protein oxidation, glycation and inflammation, were also increased in diabetic Akita mice and reduced by fisetin. It is concluded that fisetin lowers the elevation of MG-protein glycation that is associated with diabetes and ameliorates multiple complications of the disease. Therefore, fisetin or a synthetic derivative may have potential therapeutic use for the treatment of diabetic complications.

A broadly neuroprotective derivative of curcumin.

The plant polyphenolic curcumin alters the response of nerve cells to some forms of toxic stress. The steroid‐like compound, cyclohexyl bisphenol A, has broad neuroprotective properties that are very distinct from those of curcumin. To incorporate both families of biological activities into a single molecule, a pyrazole derivative of curcumin, called CNB‐001, was synthesized. CNB‐001 acquires a new activity and is far superior in neuroprotection assays to either parental molecule, but retains some of the properties of both. It is neuroprotective in cell culture assays for trophic factor withdrawal, oxidative stress, excitotoxicity, and glucose starvation, as well as toxicity from both intracellular and extracellular amyloid. While the creation of CNB‐001 was based upon an uncommon approach to drug design, it has the potential of a lead drug candidate for treating multiple conditions involving nerve cell death.

Modulation of p25 and inflammatory pathways by fisetin maintains cognitive function in Alzheimer's disease transgenic mice.

Alzheimers disease (AD) is the most common type of dementia. It is the only one of the top ten causes of death in the USA for which prevention strategies have not been developed. Although AD has traditionally been associated with the deposition of amyloid β plaques and tau tangles, it is becoming increasingly clear that it involves disruptions in multiple cellular systems. Therefore, it is unlikely that hitting a single target will result in significant benefits to patients with AD. An alternative approach is to identify molecules that have multiple biological activities that are relevant to the disease. Fisetin is a small, orally active molecule which can act on many of the target pathways implicated in AD. We show here that oral administration of fisetin to APPswe/PS1dE9 double transgenic AD mice from 3 to 12 months of age prevents the development of learning and memory deficits. This correlates with an increase in ERK phosphorylation along with a decrease in protein carbonylation, a marker of oxidative stress. Importantly, fisetin also reduces the levels of the cyclin‐dependent kinase 5 (Cdk5) activator p35 cleavage product, p25, in both control and AD brains. Elevated levels of p25 relative to p35 cause dysregulation of Cdk5 activity leading to neuroinflammation and neurodegeneration. These fisetin‐dependent changes correlate with additional anti‐inflammatory effects, including alterations in global eicosanoid synthesis, and the maintenance of markers of synaptic function in the AD mice. Together, these results suggest that fisetin may provide a new approach to the treatment of AD.

Isolation and Characterization of Novel Presenilin Binding Protein

Approximately 50% of familial Alzheimers disease (AD) cases are linked to the presenilin (PS) gene. This suggests that an altered function of mutated PSs accounts for a fundamental process leading to AD. Here we identify a new PS binding protein, PBP, which is highly expressed in cerebral cortex and hippocampus. immunohistochemical studies and cell fractionation analysis show that PBP redistributes from cytoplasm to membranes in the presence of PS. In addition, PBP is deficient in the soluble fraction of sporadic AD brains.

A Novel Neurotrophic Drug for Cognitive Enhancement and Alzheimer's Disease

Currently, the major drug discovery paradigm for neurodegenerative diseases is based upon high affinity ligands for single disease-specific targets. For Alzheimers disease (AD), the focus is the amyloid beta peptide (Aß) that mediates familial Alzheimers disease pathology. However, given that age is the greatest risk factor for AD, we explored an alternative drug discovery scheme that is based upon efficacy in multiple cell culture models of age-associated pathologies rather than exclusively amyloid metabolism. Using this approach, we identified an exceptionally potent, orally active, neurotrophic molecule that facilitates memory in normal rodents, and prevents the loss of synaptic proteins and cognitive decline in a transgenic AD mouse model.

Increase in Expression Levels and Resistance to Sulfhydryl Oxidation of Peroxiredoxin Isoforms in Amyloid β-Resistant Nerve Cells

Peroxiredoxins (Prxs) are a ubiquitously expressed family of thiol peroxidases that reduce hydrogen peroxide, peroxynitrite, and hydroperoxides using a highly conserved cysteine. There is substantial evidence that oxidative stress elicited by amyloid β (Aβ) accumulation is a causative factor in the pathogenesis of Alzheimer disease (AD). Here we show that Aβ-resistant PC12 cell lines exhibit increased expression of multiple Prx isoforms with reduced cysteine oxidation. Aβ-resistant PC12 cells also display higher levels of thioredoxin and thioredoxin reductase, two enzymes critical for maintaining Prx activity. PC12 cells and rat primary hippocampal neurons transfected with wild type Prx1 exhibit increased Aβ resistance, whereas mutant Prx1, lacking a catalytic cysteine, confers no protection. Using an antibody that specifically recognizes sulfinylated and sulfonylated Prxs, it is demonstrated that primary rat cortical nerve cells exposed to Aβ display a time-dependent increase in cysteine oxidation of the catalytic site of Prxs that can be blocked by the addition of the thiol-antioxidant N-acetylcysteine. In support of previous findings, expression of Prx1 is higher in post-mortem human AD cortex tissues than in age-matched controls. In addition, two-dimensional gel electrophoresis and mass spectrometry analysis revealed that Prx2 exists in a more oxidized state in AD brains than in control brains. These findings suggest that increased Prx expression and resistance to sulfhydryl oxidation in Aβ-resistant nerve cells is a compensatory response to the oxidative stress initiated by chronic pro-oxidant Aβ exposure.