Grant Stuchbury

Alzheimer’s associated inflammation, potential drug targets and future therapies

Summary.Alzheimer’s disease is the most common cause of dementia in the elderly population. The most widely used treatment for Alzheimer’s disease at present is acetylcholinesterase inhibitors, which aim to prolong cognitive function through increased synaptic activity, without providing neuroprotection. This treatment is only symptomatic and provides modest outcomes for patients. The recent elucidation of the inflammatory pathways involved in Alzheimer’s disease however, has opened doors for better treatment and prevention by identification of areas of therapeutic intervention that target the cause of the disease rather than the symptoms. This review describes the inflammatory pathways that are thought to be present in Alzheimer’s disease and some of the new therapies that have shown promise, via alteration or inhibition of these pathways. Some of the therapies included in this review, which have already demonstrated beneficial effects in the treatment of Alzheimer’s disease, or have the potential to do so, are nonsteroidal anti-inflammatory drugs, statins, RAGE antagonists and antioxidants.

Methylglyoxal impairs glucose metabolism and leads to energy depletion in neuronal cells—protection by carbonyl scavengers

Advanced glycation end products (AGEs) are found in various intraneuronal protein deposits such as neurofibrillary tangles in Alzheimers disease and Lewy bodies in Parkinsons disease. Among the many reactive carbonyl compounds and AGE precursors, methylglyoxal is most likely to contribute to intracellular AGE formation, since it is extremely reactive and constantly produced by degradation of triosephosphates. Furthermore, methylglyoxal levels increase under pathophysiological conditions, for example, when trisosephosphate levels are elevated, the expression or activity of glyoxalase I is decreased, as is the case when the concentration of reduced glutathione, the rate-determining co-factor of glyoxalase I, is low. However, the effects of methylglyoxal on mitochondrial function and energy levels have not been studied in detail. In this study, we show that methylglyoxal increases the formation of intracellular reactive oxygen species and lactate in SH-SY5Y neuroblastoma cells. Methylglyoxal also decreases mitochondrial membrane potential and intracellular ATP levels, suggesting that carbonyl stress-induced loss of mitochondrial integrity could contribute to the cytotoxicity of methylglyoxal. The methylglyoxal-induced effects such as ATP depletion and mitochondrial dysfunction can be prevented by pre-incubation of the cells with the carbonyl scavengers aminoguanidine and tenilsetam. In a clinical context, these compounds could not only offer a promising therapeutic strategy to reduce intracellular AGE-accumulation, but also to decrease the dicarbonyl-induced impairment of energy production in aging and neurodegeneration.

The molecular basis of the prevention of Alzheimer's disease through healthy nutrition

The Alzheimers disease (AD) brain shows numerous pathological phenomena, including amyloid plaques, neurofibrillary tangles, elevated levels of advanced glycation endproducts and their receptor, oxidative damage and inflammation, all of which contribute to neurodegeneration. In this review, we consider these neuropathologies associated with AD and propose that inflammation and oxidative stress play major pathogenic roles throughout disease progression. It is believed that oxidative stress and inflammation not only play major roles early in the disease, but that they act in a reinforcing cycle, amplifying their damaging effects. Therefore, epidemiological studies indicate that anti-inflammatory, antioxidant and neuroprotective agents including those from medicinal plants and health promoting foods may protect against AD, possibly through scavenging of reactive oxygen species, cytokine downregulation and strengthening the neurons antioxidant defense. This concept is further supported by evidence that certain diets (such as a Mediterranean diet) have been associated with a lower incidence of AD. This review highlights specific foods and diets thought to lower the risk of developing AD and discusses the potential of healthy nutrition in disease prevention.

Hydrogen peroxide is a true first messenger.

Hydrogen peroxide has been shown to act as a second messenger mediating intracellular redox-sensitive signal transduction. Here we show that hydrogen peroxide is also able to transmit pro-inflammatory signals from one cell to the other and that this action can be inhibited by extracellularly added catalase. If these data can be further substantiated, hydrogen peroxide might become as important as nitric oxide as a small molecule intercellular (first) messenger.

Optimizing the generation of stable neuronal cell lines via pre-transfection restriction enzyme digestion of plasmid DNA.

Transfection of mammalian cell lines is a widely used technique that requires significant optimization, including transfection method or product used, DNA vector, cell density, media composition and incubation time. Generation and isolation of stable transfectants from the large pool of untransfected or only transiently transfected cells can be laborious and time-consuming. Transfection of DNA is usually performed with a non-linearized plasmid, since it is assumed that cutting the plasmid beforehand leads to a lower efficiency of transfection or the degradation of linearized DNA by cytosolic nucleases. However, the transfected circular plasmid will be linearized by a random cut within the cell and it might be possible that sensitive parts of the plasmid such as the resistance gene or the gene of interest are destroyed upon linearization. On the other hand, linearizing a plasmid before transfection by a single, defined cut with a selected restriction enzyme in a non-coding area of the gene has the advantage of ensuring the integrity of all necessary gene elements of the plasmid. In this study, we have compared these different methods in order to increase both transient and stable transfection efficiency in mammalian cells. We report that linearization of plasmid DNA prior to transfection can increase both the efficiency of stable clone generation and target gene expression, but is dependant on the site of linearization within the vector.

The Thiamine Analogue and Advanced Glycation Endproducts Crosslink Breaker ALT-711 does not Interfere with Transketolase Activity

The enzyme transketolase (sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) is involved in the pentose phosphate pathway (PPP) and catalyses the transfer of a 2-carbon fragment from a 5-carbon keto sugar (xylulose-5-P) to a 5-carbon aldo sugar (ribose-5-P) to form a 7-carbon keto sugar (sedoheptulose- 7-P) and a 3-carbon aldo sugar (glyceraldehyde-3-P). Transketolase requires thiamine pyrophosphate as a co-factor. Advanced glycation endproducts (AGEs) are implicated in the complications of diabetes and aging, primarily via adventitious and crosslinking of tissue proteins. ALT-711 is an AGE crosslink breaker and has been tested as an intervention therapy in established complications of diabetes. It has been noticed that it has a similar structure to that of thiamine and it was hypothesized that it might inhibit transketolase by replacing the active co-factor rendering the enzyme inactive. In this study, we have established a novel microtiter plate format transketolase assay which determines the concentration of NADH by measuring its fluorescence. Using this assay, it was found that ALT-711 does not inhibit the activity of transketolase up to concentration of 5 mM. We conclude that ALT-711 does not interfere with transketolase activity at clinically relevant concentrations.