Giuseppe Verdile & Ralph N Martins McCusker Foundation for Alzheimer’s Disease Research, Centre of Excellence for Alzheimer’s Disease Research and Care, School of Exercise, Biomedical and Health Sciences, Edith Cowan University, Joondalup and Hollywood Private Hospital, Nedlands, WA, Australia. School of Psychiatry and Clinical Neurosciences, University of Western Australia, Crawley, WA, Australia.

 Currently over 200,000 Australians have dementia and this number is estimated to increase to over 750,000 by 2050 with 175,000 new cases diagnosed each year (Access Economics Report, 2006). Worldwide, it has been estimated that one new case of dementia is diagnosed every 7 seconds (Ferri et al., 2005). The majority of these dementia cases are clinically diagnosed as Alzheimer’s disease (AD). Currently, acetylcholinesterase inhibitors (donepezil, rivastigmine and galantamine) and the N-methyl-D-aspartate receptor antagonist, memantine are the only available treatments for AD. However, these drugs only treat disease symptoms without targeting the underlying pathology and neurodegeneration. AD research has progressed rapidly in a short period of time to a point where researchers understand more about the underlying causes of AD resulting in the development and assessment of preventative or therapeutic strategies to combat the disease.

The disease was first described over 100 years ago by the German physician, Dr Alois Alzheimer who published a case report detailing the pathological changes in the brain of a 55-year-old woman with progressive dementia. The woman, known as Auguste, presented to Dr Alzheimer with behavioural and psychiatric symptoms including paranoia, delusions, hallucinations and impaired memory that progressively worsened over 5 years, until she died of a short illness in 1906. Dr Alzheimer examined her brain and described pathological hallmarks that today characterise AD. Observing these hallmarks on post-mortem examination is still today considered the only definite way to diagnose the disease.

For a number of decades following the first description by Dr Alzheimer, the mechanisms involved in the disease process remained elusive. In the mid 1980s a breakthrough was made at the University of Western Australia by Professor Colin Masters and his team (which included Professor Ralph Martins) in which the main protein constituent of amyloid plaques was isolated from an AD brain and identified as a small molecule that they called beta amyloid. Intensive research over the past 20 years has led to our current understanding of what causes AD, disease progression, what factors contribute to the disease process and how AD may be prevented, or treated effectively.

There have been a number of hypotheses that have been proposed to cause neurodegeneration that underlies AD. The most widely accepted and studied

Prof. Ralph Martins at the bench

 hypothesis is referred to as the “amyloid hypothesis”. Central to this hypothesis is accumulation of the beta amyloid (Aβ) protein. The exact mechanisms of how Aβ accumulates in the brain and leads to neurodegeneration are unclear. However it is thought that impaired clearance of Aβ from the brain or aberrant processing of its parent molecule, the amyloid precursor protein (APP), resulting in the accumulation of Aβ , initiates oxidative stress and inflammatory processes, culminating in neurodegeneration and dementia (reviewed in Verdile et al., 2004).

Aβ is a product of the enzymatic processing of a larger protein called APP (reviewed in Verdile et al., 2007). Intensive research has focused on the molecular mechanisms involved in this process. Particular focus has been on one of the enzymes critical for the generation of Aβ. The enzyme known as “gamma secretase” has been a major target for developing agents aimed at reducing Aβ production and thus inhibiting Aβ accumulation and aggregation. However, since gamma secretase is also involved in a number of normal cellular processes the majority of drugs aimed at inhibiting it have failed in pre-clinical animal trials due to severe side effects. Only one inhibitor of the gamma secretase enzyme is currently in the initial phases of human clinical trials. Our laboratory is currently trying to understand how this enzyme works and in particular the location specifically responsible for its activity on APP to generate Aβ. This will lead to the development of drugs that specifically target the enzyme’s role in generating Aβ  without affecting its other roles within the cell.

The normal biological function of Aβ is not yet determined. Some studies have reported that at low levels, Aβ can be neuroprotective, with antioxidant properties. However the main emphasis of researchers is to determine the pathological functions of Aβ. Where once the amyloid plaques were considered the toxic form of Aβ, it is now generally accepted that they only represent the “tombstone” (i.e. a marker of neuronal death). Instead many studies have now provided evidence that the intermediates between small aggregates (or oligomers) result in neuronal dysfunction and death. Many studies have shown that metals ions such as copper (Cu⁺²) potentiate the toxic properties of Aβ . Although the mechanisms of this metal ion-Aβ  interaction are not fully understood, it is apparent that Aβ is involved in the chemistry of how toxic bi-products of cellular metabolism (free radicals/pro-oxidants) such as hydrogen peroxide (H₂O₂) are produced resulting in oxidative stress and ultimately cell death. Drugs aimed at inhibiting Aβ aggregation or associated neurotoxicity are being developed. Two Australian companies have been developing agents aimed at inhibiting Aβ -associated toxicity. Prana Biotechnology has had some success with its drug PBT2 which is currently in human clinical trials. This drug is based on removing copper ions to reduce Aβ aggregation and its associated neurotoxicity. Our West Australian company, Alzhyme, has developed small molecules that specifically target Aβ to reduce its aggregation and neurotoxicity.

Prof. Ralph Martins group (Giuseppe Verdile is at the back left)

It is now becoming apparent that a number of factors including genetic mutations/variations, dietary, hormonal and lifestyle factors impact on the risk of developing AD. Furthermore, many studies have shown that these factors contribute to the accumulation of Aβ within the brain (reviewed in Verdile et al., 2004; Barron et al., 2006; Martins et al., 2006). Many of these risk factors are now targeted for developing effective therapeutic and preventative strategies for combating AD. For example age-related changes in reproductive hormones (i.e. sex hormones and the gonadotropins) are known to increase the risk of developing AD and contribute to Aβ  accumulation and neuronal death. Hormone replacement therapies have been successful in animal trials; but have been rather controversial in human trials. This is particularly true for oestrogen replacement trials where larger longitudinal clinical studies are needed to determine whether oestrogen is beneficial in AD (reviewed in Barron et al., 2006). Another sex hormone that is receiving attention is testosterone in men. Research from our group and others have shown that decline in testosterone levels in men is associated with cognitive decline and increased blood levels of Aβ contributing to the risk of developing AD (reviewed in Fuller et al., 2007). Cell culture and animal studies have also shown that testosterone is beneficial in reducing Aβ accumulation and neuronal death (reviewed in Pike et al., 2006). We are currently assessing, in a small cohort of men, the benefits of testosterone therapy at improving cognition and lowering blood Aβ levels. However, large-scale randomised, placebo-controlled studies that assess cognitive, biochemical and brain imaging parameters are required to determine if testosterone has benefits for AD.

Another hormone referred to as luteinizing hormone (LH), which stimulates sex hormone production, also contributes to AD risk and pathology. Our laboratory and that of A/Professor Craig Atwood (University of Wisconsin) has done some of the pioneering work that has lead to this hormone being a target for developing treatments for AD. Voyager Pharmaceuticals has had some success with a gonadotropin lowering agent, leuprolide, in human clinical trials. We are currently assessing another agent in animal trials which has been shown to lower LH levels and has the added benefit of attenuating neurotoxicity associated with Aβ.

It is becoming increasingly apparent that dietary and lifestyle factors such as physical inactivity, obesity, type II diabetes, decreases in levels of HDL (good cholesterol) and increases in LDL (bad cholesterol) and lower intake of antioxidants are contributing factors to the increased risk of developing AD. Together with an aging population these factors may explain the increasing prevalence of AD in developed and developing nations. Nutritional supplements such as green tea, fish oil (omega-3 essential fatty acids) and the curry spice curcumin have shown some benefits in reducing the risk of AD and attenuating Aβ  brain accumulation in animal studies. We are currently assessing whether combining these supplements has added benefits in preventing AD. Another potential preventative strategy is undertaking physical activity. There is some epidemiological evidence that exercise has benefits in improving cognition and memory. Evidence has also shown that mice models of AD undertaking voluntary exercise (e.g. running on a wheel) show improved memory and reduced Aβ deposition in their brains. Although the benefits of exercise are evident, studies are still required to determine what type (walking, cycling or weight resistance) and how much is required to show potential as a preventative strategy for AD.

It has been estimated that preventing only a small proportion of AD cases will result in massive economic savings. By delaying onset by 5 months, it is estimated that the number of new cases would reduce by 5% annually, creating a saving of $1.3 billion by 2020. If onset was delayed by 5 years, there would be an incredible 50% reduction in new cases each year, and a $13.5 billion saving by 2020. In order to achieve these aims, improved diagnostic and predictive techniques are sorely needed. Preventative strategies must go together with improved diagnostics and risk assessment.

Advances have been made in developing diagnostic criteria for greater accuracy in AD diagnosis and moreover, developing diagnostic tools for early detection of the disease. The current diagnostic criteria can only give a possible or probable diagnosis of AD when symptoms arise. Only on post-mortem examination of the brain for the presence of hallmarks such as amyloid plaques can a definite diagnosis be given. Current efforts on improving diagnosis are aimed at identifying genetic and biological markers for the disease. Combined with neuropsychological and brain imaging assessment, it is envisaged that these markers will not only give a more accurate diagnosis than current methods but will also allow early diagnosis of the disease allowing early intervention studies. Imaging beta amyloid deposition in the brain is a recent technique that has great potential as an early diagnostic tool. This technique uses a radiolabelled tag, referred to as the Pittsburgh Compound-B (PIB) (named after the USA city where it was discovered) which binds amyloid allowing plaque deposition to be visualised using positron emission tomography (PET) (Price et al., 2005). For the first time this technique allows the identification of amyloid deposition within the brain while the individual is alive.

It is widely believed that for a treatment/preventative strategy to be effective, it would have to be administered at the early stages of the disease process prior to symptoms developing. This is the basis for a large, longitudinal study that we are currently undertaking in individuals with subjective memory complaints (i.e. those people that think they have a memory problem). A variety of neuropsychological, clinical and biochemical analyses are performed to generate a “high risk” profile. We are also investigating potential early predictors of Alzheimer’s disease in another large study which is recruiting 1000 volunteers from Perth and Melbourne to investigate early predictive markers of AD. This collaborative study between Edith Cowan University, University of Melbourne and CSIRO is the largest of its kind and will investigate neuropsychological, biomarkers and brain imaging parameters to improve diagnostic and predictive techniques. Following along these lines we are also part of a Dementia Collaborative Research centre with Professor Marc Budge from ANU, Professor Colin Masters and Professor David Ames from the University of Melbourne and Alzheimer’s Association of Victoria, to investigate the potential of assessing blood levels of beta amyloid as a diagnostic test for AD.

Although further research is required, it is clear that leaps and bounds have been made in AD research. Where 10 or 20 years ago little hope was given for AD sufferers, today we are optimistic that effective diagnostic tools and appropriate therapeutic or preventative strategies will be developed to combat this devastating disease.


References

1. Access Economics Report: Dementia Estimates and Projections: Australian States and Territories, Feb 2005

2. Barron A, Verdile G and Martins RN. (2006) The role of gonadotropins in Alzheimer’s disease: potential neurodegenerative mechanisms. Endocrine 29: 257-69.

3. Ferri CP, Prince M, Brayne C, Brodaty H, Fratiglioni L, Ganguli M, Hall K, Hasegawa K, Hendrie H, Huang Y, Jorm A, Mathers C, Menezes PR, Rimmer E, Scazufca M (2005) Global Prevalence of dementia: a Delphi consensus study, Lancet, 366: 2112-17.

4. Fuller SJ, Tan RS, Martins RN. Androgens in the etiology of Alzheimer’s disease in aging men and possible therapeutic interventions (2007). J Alzheimers Dis. 12(2):129-42.

5. Martins IJ, Hone E, Foster JK, Sünram-Lea SI, Gnjec A, Fuller SJ, Nolan D, Gandy SE, Martins RN. Apolipoprotein E, cholesterol metabolism, diabetes, and the convergence of risk factors for Alzheimer’s disease and cardiovascular disease. Mol Psychiatry. 2006 Aug; 11(8):721-36.

6. Pike CJ, Rosario ER, Nguyen TV. (2006) Androgens, aging, and Alzheimer’s disease. Endocrine. 29(2):233-41.

7. Price JC, Klunk WE, Lopresti BJ, Lu X, Hoge JA, Ziolko SK, Holt DP, Meltzer CC, DeKosky ST, Mathis CA (2005). Kinetic modeling of amyloid binding in humans using PET imaging and Pittsburgh Compound-B. J Cereb Blood Flow Metab. 25: 1528-47.

8. Verdile, G., Fuller, S., Atwood, C.S., Laws, S.M., Gandy, S.E. and Martins, R.N. (2004). The role of beta amyloid in Alzheimer’s disease: still a cause of everything or the only one who got caught? Pharmacological Research, 50: 397-409.

9. Verdile G, Gandy SE and Martins RN. (2007) The role of presenilin and it interacting proteins in the biogenesis of Alzheimer’s beta amyloid. Neurochemical Research 32: 609-623