Targeting ‘misfolded proteins’ to combat neurodegenerative diseases

As in other European countries, the incidence of degenerative brain diseases in Malta is increasing at a fast pace. The number of people suffering from Alzheimer's Disease, for instance, is expected to double in the next 20 years.

There are currently no cures for either Parkinson's or Alzheimer's, but a team of Maltese researchers are making headway in delaying the disease process with their innovation, which targets what is referred to in scientific terms as 'protein misfolding'.

The team, based at the Department of Physiology and Biochemistry of the University of Malta and led by Dr Neville Vassallo, has in fact drawn the attention of the Centre for Neuropathology of the renowned Ludwig-Maximilians University of Munich - which in fact sent two German students to Malta to base their Master's degrees on the subject and who also got hands-on experience during the first phase of the study.

The team's findings have recently been published in three important scientific journals, including 'Biochim. Biophys. Acta.' which is considered to be one of the 100 most influential journals in Biology and Medicine.

On meeting Dr Vassallo on campus, he ushered me in to the laboratory where he was conducting, alongside his students, an experiment connected to his ongoing research.

Vassallo says that "with increased life expectancy, age-related conditions such as dementia are on the rise and something needs to be done to attenuate the massive burden on society at large, including our national healthcare system".

"Although we are looking at encouraging results from our research on the subject, one naturally cannot promise pie-in-the-sky and that such diseases will be eradicated once and for all. However, if with an effective disease-modifying treatment you give a 70-year-old with Alzheimer's or Parkinson's the opportunity of an extra 10 years of quality-life, by inhibiting the degenerative brain process associated with these conditions, it would be a significant improvement - a breath of fresh air, especially for the patient."

So I ask Vassallo to let go of the scientific jargon for a minute and explain to me in layman's terms what his research on both diseases entails.

"We are dealing with conditions - Alzheimer's and Parkinson's - both of which involve severe loss of neurons (core components of the nervous system) in specific areas of the brain. Typically, this occurs in elderly people but can also occur in much younger individuals, especially when associated with inheritance of particular genetic traits.

"With Alzheimer's, one experiences loss of memory (dementia) whereas in Parkinson's the main symptoms are motor, namely tremor and immobility. However, although these two neurodegenerative diseases are highly distinct and manifest themselves in different ways, today we know that at a molecular level, they are actually very similar."

Vassallo further explains: "Brain neurons, like other cells within the body, contain normal proteins but disease occurs when specific proteins fold abnormally, creating 'knots' (scientifically known as 'aggregates') which start to clump together and  deposit as 'amyloid plaques' in the brain - the ultimate cause behind the destruction of the neurons (nerve cells).

"If one looks at brain tissue under a microscope, one will witness these 'plaques' that litter specific regions of the brain, causing degeneration."

What was the primary focus of the team of experts?

"During our research we focused on two particular aspects - firstly, the formation of amyloid aggregates and secondly, the damage caused by amyloid to the outer shell (membrane) of neurons in the brain."

The team hasn't worked directly on humans or animals as yet, but plans are in hand to make use of established disease models in the fruit fly Drosophila melanogaster during the next major phase of the study.

"We replicated the disease process in the laboratory in order to be able to test and screen several compounds and extracts efficiently; we thus haven't worked on humans or animals as yet, but intend to do so in the next phase of our study.

In vivo is the experimentation using a whole, living organism as opposed to a partial or dead organism.

"This was done by inducing specific proteins - which we either purchased or obtained from our colleagues in Munich - to misfold in the laboratory. In this manner, we induced the formation of the amyloid aggregates, thereby mimicking what takes place in the brain over a decade or so, in a matter of hours in the lab."

I ask Vassallo about the central idea behind the project.

"The central push was to identify how diverse compounds or extracts can prevent the formation of the clumps and/or their ability to protect neuronal membranes from damage. The result: a massive decrease in damage to the brain," Vassallo says.

So what compounds did Vassallo and his team turn to?

"Our approach was to identify potent compounds typically enriched in the Mediterranean diet - wine, tomatoes, olive oil, fish, and so on. We also used extracts from pomegranates, prickly pears, and black tea (the latter proving to be extremely promising in terms of results), and last but not least an extract from a marine alga - Padina pavonica.

"The Mediterranean diet has a high content of what we call 'polyphenols' -

a structural class of mainly natural organic chemicals characterised by the presence of large multiples of phenol structural units," Vassallo said.

The team selected around 20 purified polyphenols which are found in the Mediterranean diet.

Several epidemiological (population-based) studies have shown that the Mediterranean diet helps stave off many age-related conditions.

Unlike the typical American diet, the Mediterranean diet is very low in red meat and poultry; high in fruits, nuts, legumes, vegetables, and cereals; is high in fish; and permits low-to-moderate amounts of wine while also uses olive oil as the main source of fat.

Vassallo continues: "Therefore, natural products from the Med. diet and marine plants, including the readily available supplies, have assisted our team in identifying the top extracts, significantly proving to be a potential method in the fight against such diseases."

Vassallo's team has gone a step further by using technology such as fluorescence correlation spectroscopy which helps detect molecules, highlighting any significant leaks that could take place in the same way in the brain. This is an ideal way for identifying how effective a drug is.

"Other promising results through the experiments performed by our team and to ultimately be able to treat such diseases, is the use of lipid vesicle permeabilisation  - a method that assesses how sticky amyloid clumps turn out to be and whether the above-mentioned compounds prevent destruction of membranes of nerve cells."

So what's so innovative about the team's approach?

"Clumping occurs early in the disease so there's a potential for disease-modified therapy with the promising results emerging from our research. "A major problem in treatment of neurogenerative diseases is that the treatment currently available only alleviates the symptoms, while leaving the underlying pathology in the brain to progress unhindered.

"In simple terms, our team is targeting a primary cause of these disorders through the use of natural products," Vassallo said.

Results from the study have not only revealed that patients who have early-stage formation of clumps may benefit from this treatment which would eventually be administered to patients with such conditions, but would also benefit patients with already-formed clumps (the disease in more advanced stages).

I grow curious as to what the next step of this ambitious project would be.

"First and foremost, our research work so far has been duly acknowledged by the scientific community through publications in prominent scientific journals - a necessary and positive step in displaying our efforts and being innovative on the subject - and which has helped highlight the study's initial results.

"Secondly, in the next stage of our research we will be selecting the best four or five compounds we have found to be most effective in all our assays thus far, and thoroughly examine their ability to modify disease progression in an in vivo model which will therefore bring us a step closer to the patient."

The preferred in vivo model to be used, according to Vassallo, is the fruit fly Drosophila melanogaster, as discussed earlier. Vassallo will be collaborating with a colleague from his same department at the University, Dr Ruben Cauchi, who is an expert on fruit fly genetics and research.

The joint team's project has just been accepted and shortlisted among the Research and Innovation (R&I) programmes to be funded by the Malta Council of Science and Technology (MCST).

When asked if his team was the only one working on neurogenerative diseases, Vassallo said that as far as he knew, there were no other local experts working on the same subject.

When asked how close his team was to approaching the end of the study and eventually transform this precious information to treatment and have it administered to patients, Vassallo said that is still several years away. Nevertheless, there are already epidemiological studies which associate the Mediterranean diet with reduced incidence of dementia and even Parkinson's disease.

"Dr Charles Scerri of the Department of Pathology (who also formed part of our research team), and myself just published a review paper entitled 'The Mediterranean diet and dementia of the Alzheimer type', to give an overview of all the studies that exist and show this relationship. In our own research, however, we took this relationship a step further.

"Wine, for instance, includes a wide variety of polyphenols, but through our research we have determined which are the most promising polyphenols to treat age-related neurodegeneration. And as a result we may have paved the way to developing nutraceutical supplements in the near future. The polyphenolic content of one supplement would, for instance, be equivalent to that of several glasses of wine."

When asked about the participation of students who were sent from the University of Munich to participate in his study, Vassallo replied that such initiatives bring recognition to the University of Malta as an institution at the forefront of scientific research and innovation, and which is integrated into a European-wide network of excellence. Moreover, these scholarships help create the opportunity and environment for foreign students to visit and live in Malta for appreciable lengths of time.

The project, co-funded by the Malta Council for Science and Technology and the University of Malta, was carried out in collaboration with a local SME, the Institute of Cellular Pharmacology Ltd. The next phase of the study is expected to commence in October. Dr Cauchi will join the team in the next, and critical, phase of the study.