Anne Martel, Large Scale Structures Group, D22 Instrument Scientist & Giovanna Fragneto, Large Scale Structures Group Leader, at the Institut Laue-Langevin (Grenoble, France)
As the world’s flagship centre for neutron science and technology, the Institut Laue-Langevin (ILL) provides scientists with a high flux of neutrons that feeds some 40 state-of-the-art instruments. These instruments are used in a wide variety of research, primarily focusing on fundamental science in biology, chemistry, condensed matter physics, nuclear physics and more. Having been established in 1967, this year marks ILL’s 50th anniversary of providing neutrons for science and society.
Health is a leading area in which the impact of neutron science and technology has high visibility, as neutrons provide a very effective analytical tool for investigating the properties of matter at both the atomic and molecular scales. ILL’s use of neutron scattering methods in medical research has significantly progressed our knowledge of the biological mechanisms underpinning various chronic conditions, from diabetes to Alzheimer’s Disease, and has contributed to the design of new chemicals including drugs and polymers. This research is incredibly important, as it ultimately has the potential to improve quality of life and life expectancy.
Fighting diabetes: a global epidemic on the rise
Chronic diseases are debilitating millions of people worldwide. Unlike infectious diseases, chronic conditions cannot be cured with medication nor prevented with vaccines. As such, these complex diseases warrant thorough research to improve treatment methods and patient outcome.
Diabetes is a chronic disease of global epidemic proportions, characterised by the body either not producing enough of, or ineffectively using insulin – a hormone that regulates blood sugar levels. According to the World Health Organisation (WHO), the number of people with diabetes worldwide reached 422 million in 2014, boosting prevalence to 8.5% among adults over 18 years of age. This figure is expected to rise to 642 million people by 2040, also quantifiable as one in ten adults. Type 2 Diabetes (T2D) comprises the majority of diabetes cases worldwide. It is a chronic lifelong condition that occurs due to the body’s ineffective use of the insulin it produces, causing blood glucose levels to become too high.
A key pathological consequence of T2D is the progressive failure and depletion of pancreatic β-cells, subsequently leading to defective glucose regulation. There has been increasing evidence suggesting this depletion is concomitant with the formation of amyloid aggregates in pancreatic islets of Langerhans. These fibrillar aggregates are primarily composed by Islet Amyloid PolyPeptide (IAPP), a hormone co-secreted with insulin by β-cells. Whilst in healthy conditions, a lower amount of IAPP is produced than insulin, the rate of IAPP production increases substantially during T2D development.
A significant amount of research has been carried out to understand the mechanism of IAPP-induced β-cell death. The concurrence of IAPP aggregation and cell depletion has developed the hypothesis that the aggregation is responsible for causing symptom onset, through disrupting ionic homeostasis and thus inducing cell death. However, the precise biological mechanisms underpinning IAPP’s involvement in cell depletion remain unclear. With the prediction that diabetes will continue to reach even greater epidemic proportions in the future, gaining a thorough understanding of the disease’s pathology is invaluable for improving current therapeutic approaches and developing more effective drugs.
At the ILL, we recently conducted a study in collaboration with researchers from the Institute for Molecular Engineering at the University of Chicago and Institut de Biologie Structurale in Grenoble, with the aim of enhancing our knowledge of the cytotoxic mechanisms of IAPP. We investigated the interaction between IAPP and model membranes – both membrane permeation and structural effects of IAPP – using a range of techniques including neutron scattering and reflectometry methods. This study was published on-line in the Journal of the American Chemical Society in December 2016(1).
The small-angle neutron scattering (SANS) experiments were conducted using ILL’s D22 diffractometer, which is used to study nanoscale materials. D22 is a particularly suitable instrument for studying structure in solution of biological molecules due to the high neutron flux and low background. Reflectometry experiments were carried out using ILL’s FIGARO, a horizontal neutron reflectometer. This high-flux, flexible resolution instrument has features suitable for a variety of studies in soft condensed matter, physics, biology and chemistry.
Using these neutron techniques together with other complementary characterisation tools available in the support laboraties of ILL, we were able to observe that amyloid aggregation and membrane permeation are two independent processes. These processes are in fact competitive, with aggregation inhibiting membrane permeation as a result.
Whilst it was previously hypothesised that IAPP aggregates cause membrane permeability, resulting in calcium leak, a signal triggering cell death, findings of our study favour a new hypothesis. We hypothesised that amyloid aggregation is a defense mechanism employed by the human body, whereby encapsulating IAPP into aggregates ‘silences’ the cytotoxic peptides and blocks them from interacting with cell membrane, thus lowering the toxicity of IAPP to pancreatic β-cells and delaying the onset of disease.
Furthermore, we observed that membrane permeation involves lipid depletion, the lipids being included in the aggregates as well. This mechanism, while compatible with the generally accepted hypothesis of the “toxic oligomer” does not involve the insertion of a pore-forming assembly of peptides.
With our observations resulting in a new hypothesis about the interaction between IAPP and membranes, further investigations into the role of IAPP in the pathology of T2D are clearly necessary. This can help with the development of drugs that are better tailored to tackle β-cell depletion in the pancreatic islets of Langerhans. Additionally, with our hypothesis suggesting that IAPP aggregates are a safety mechanism to reduce the spread of toxins, drug innovation could be re-directed towards drugs that promote rather than inhibit amyloid aggregation.
Understanding chronic diseases through model biological membranes
At the ILL, we specialise in using model biological membranes to help us in the fight against chronic diseases such as amyloidoses - a group of diseases characterised by the aggregation and deposition of insoluble protein fibres in key organs of the body.
The main constituent of the fibrillar aggregates across all amyloidoses is a specific amyloid peptide: in Alzheimer’s disease, it is the Aβ peptide, in Parkinson’s disease, it is the α-synuclein peptide and in T2D, as explained above, it is the Islet Amyloid PolyPeptide (IAPP). As a common point between all amyloidoses, abnormal cell death tends to occur in the regions where these aggregates are localised. The final aggregates are not cytotoxic, leading to the hypothesis that intermediary species occurring along the aggregation process, are the cytotoxic species that cause the onset of disease symptoms.
ILL’s commitment to medical research is further demonstrated by our involvement in an integrated infrastructure initiative for neutron scattering and muon spectroscopy, called the SINE2020 Joint Research Activity (JRA) on Chemical Deuteriation. The aim of the initiative is to integrate all the research infrastructures within the European Research Area that are in the neutron scattering and muon spectroscopy fields, to facilitate the Pan-European coordination of research activities in these areas.
As is highlighted by the diabetes study above, at the ILL, the SINE2020 JRA involves the production of model biological membranes from natural lipids, and the investigation of their characteristics using neutron techniques, which provide high performance tools for the analysis of biological membranes.
These artificial membranes are made to mimic real membranes in the human body as closely as possible, allowing scientists to use them to accurately study the biological mechanisms underpinning various human diseases. Our challenge is to accommodate the complexity of natural membrane while simplifying the system to a point where we can understand the role of each element. Besides T2D, ILL instruments have also provided unique information related to the mechanism of cell loss occurring in Alzheimer’s and Parkinson’s diseases.
The advancement of medicine and increase in global living standards means that the human race is living longer than ever before. In 2013, the United Nations published a report estimating that by 2050, over 20% of the world’s population will be 65 or older. Our ageing population means that more of us are being diagnosed with age-related illnesses.
This includes Alzheimer’s Disease, a chronic condition and leading cause of dementia, characterised by a progressive decline in cognitive function. In 2015, there were an estimated 46.8 million people living with dementia, and there are over 9.9 million new cases appearing each year worldwide, costing the world economy over $800 billion. With this figure predicted to rise to an estimated 131.5 million cases by 2050, understanding Alzheimer’s is incredibly important.
The mechanisms underlying Alzheimer’s Disease are yet to be entirely understood, but genetic, pathological and biochemical observations indicate that the progressive production and accumulation of β-amyloid (Aβ) peptides play a pivotal role. Neurons release Aβ peptides in a soluble form that progressively generate different molecular assemblies, from oligomeric to multimeric structures, ending up as fibrillar aggregates. Therefore, the role of first-stage Aβ peptide aggregation in the development of Alzheimer’s Disease and damage to the cells in the brain is widely accepted, yet the precise interactions remain unclear.
A collaboration between ILL, the department of Medical Biotechnologies and Translations Medicine at the University of Milano, and the department of Molecular Biochemistry and Pharmacology at the Institut for Pharmacological Research Mario Negri carried out neutron reflectometry experiments to understand the biology underpinning Alzheimer’s Disease. This study was published in Nature Scientific Reports in 2016(2).
Image - D17 is a neutron reflectometer with horizontal scattering geometry suitable for the study of surface structures in solids and solid/liquid interfaces over a wide range of length scales. Credit: ILL, B. Lehn
The existence and extent of the interaction between Aβ peptides and a lone customised biomimetic membrane was investigated, together with their dependence on the aggregation state of the peptide. The model biological membrane, containing phospholipids, GM1 and cholesterol in biosimilar proportions, was designed to imitate the outside layer of neurons found in the brain.
The D17 instrument at the ILL was used to investigate the interactions between different forms of Aβ peptides with the synthetic membrane. D17 is a neutron reflectometer designed to be as flexible as possible in modes of operation and resolution, and is suitable for the analysis of surface structures in both solids and solid/liquid interfaces.
Neutrons are particularly adapted to measuring lighter elements such as those found in biological materials, and are highly penetrating without causing damage in the way the equivalent X-ray beam would. In a neutron reflectivity experiment involving stratified samples, such as with this model biological membrane, neutron reflectometers are best-placed to give detailed insight into its structure and properties.
The study focused on two particular conditions: 1) when the Aβ peptide reaches the raft-mime surface already in the membrane-active structured-oligomer state, and 2) when early, unstructured Aβ peptide forms are administered and peptide oligomerisation possibly takes place at the membrane. Both showed membrane-active species of Aβ peptide, namely early-labile and structure oligomers although differences exist in the extent and depth of interaction. Interestingly, these differences point at unexpected relative impacts on the membrane.
The neutron reflectometry experiments allowed to observe that structured oligomers are embedded in the outer leaflet of the membrane where they constitute a seed for further Aβ addition and elongation. Conversely, it was found that early labile oligomers, easily dissolving to monomers, are captured by the membrane and deeply dig in towards the opposite side – this observation that there is an eventual deeper impact of monomers as compared to oligomers is surprising based on current concepts.
Another new concept as a result of this study hypothesises a role for a short N-terminal sequence of Aβ in destabilising the cellular membrane. Together with the known ability of seeds, formed by small peptide aggregates, in driving Aβ recruitment towards fiber formation, this new vision potentially enables us to identify a new method to stop these Aβ sections from weakening the outer membrane layer. This could bring us one step closer to identifying new drug targets and effective mechanisms that can stop the progression of Alzheimer’s Disease.
A similar study was also conducted on the interaction between model biological membranes and α-synuclein, the peptide involved in Parkinson’s Disease, in collaboration with the European Spallation Source, Department of Chemistry at Lund University, Wallenberg Neuroscience Centre at Lund University, Oak Ridge National Laboratory, Centre for Neurodegenerative Science at Van Andel Research Institute and Keele University. This study, which was published in ACS Chemical Neuroscience in 20133, aimed to understand the molecular determinants for adsorption of monomeric α-synuclein to model biological membranes.
Parkinson’s Disease is a chronic neurological condition characterised by neurons perishing and several regions of the brain becoming progressively damaged over many years, often resulting in involuntary tremors, slow movement and inflexible muscles. Similarly to Alzheimer’s Disease, Parkinson’s is an age-related illness, with older age being a key risk factor of its development.
The neuropathological marker of Parkinson’s Disease is the presence of Lewy bodies, which are intraneuronal aggregates primarily composed of an amyloid form of the protein α-synuclein. It is thought that interactions between α-synuclein oligomers and cellular membranes underpin the pathology of Parkinson’s, but the precise biological mechanisms of this interaction remain uncertain. With more than 10 million people worldwide living with Parkinson’s, further research on the biological mechanisms underpinning the condition is of great importance.
The D17 neutron reflectometer at the ILL was also used in this study to investigate the interaction between α-synuclein and model membranes. Neutron reflectometry measurements revealed that α-synuclein adsorbs to bilayers that contain anionic lipids; an association governed by electrostatic interactions and the polarised nature of the amyloid protein.
In terms of structural changes, the interaction between the protein and membrane results in mutually disruptive structural perturbations. Whilst membrane surfaces can alter the rate at which amyloid-forming proteins convert into aggregates and amyloidogenic proteins, these aggregates can, in turn, compromise the structural integrity of the membranes.
As with the other two studies, the findings of this study have demonstrated how neutrons can provide an effective analytical tool when studying biomedical problems and indicate the need for continued research into the biological processes and cytotoxic mechanisms associated with Parkinson’s Disease and other chronic diseases.
Although all three studies have aimed to enrich our knowledge on the precise interactions between specific amyloid peptides and membranes – two factors so small in scale that they cannot be seen by the human eye – the ultimate goal of this research has much wider implications. At the ILL, we carry out research in many health fields for society; enabling the populations who face chronic conditions, including the above T2D, Alzheimer’s Disease and Parkinson’s Disease examples, to one day have a longer life expectancy and better quality of life.
Our intense neutron beams, sophisticated instrumentation, optimised sample preparation tools and application of neutron scattering methods contribute to the design and development of new drugs that are better tailored to combat the cytotoxic mechanisms associated with disease progression and improving the lives of its sufferers.
1. Membrane permeation versus Amyloidogenicity: a multi-technique study of Islet Amyloid PolyPeptide interaction with model membranes, Martel et al., Journal of the American Chemical Society, 139 (1), 2016, DOI: 10.1021/jacs.6b06985
2. Amyloid‑β Peptides in interaction with raft-mime model membranes: a neutron reflectivity insight, Rondelli et al., Nature Scientific Reports 6, 20997, 2016, DOI: 10.1038/srep20997
3. Adsorption of α-Synuclein to supported lipid bilayers: positioning and role of electrostatics, Hellstrand et al., ACS Chemical Neuroscience, 4 (10), 2013, DOI: 10.1021/cn400066t