The goal of the Neuromedicine group is to bring together the various expertise in the Institute for Biomedicine to focus on existing and emerging neurological problems facing society. Through the use of molecular genotyping and phenotyping platforms (i.e. 'omics' platforms such as genomics, transcriptomics, metabolomics and proteomics), and experimental model systems, the group conducts projects aimed at supporting the population and case oriented research into some of the pressing neurological and neuropsychiatric issues facing society.
Increasing life expectancy makes neurodegenerative diseases the most pressing health problem for the coming generation, with associated health care costs amounting to many billions of Euros each year, and an immeasurable suffering to society. According to estimates by the World Health Organisation (WHO: World Health Report 2001) more than one billion people suffer from disorders of the central nervous system (CNS). Diseases caused by degeneration of the CNS (neurodegeneration) are currently the fourth most significant healthcare issue facing the world. By 2040, the World Health Organisation predicts that neurodegenerative disorders such as dementia and Parkinson's disease will have overtaken cancer to become the second leading cause of death.
Similarly, chronic pain is a major challenge in terms of human health and well-being. Approximately 20% of the population has an on-going pain problem and many are poorly treated with current medications. Because pain affects the elderly disproportionally, it becomes an increasingly important and expensive problem as our population continues to age. The growing use of neurotoxic drugs to treat a variety of diseases in the population, coupled with the recognition of gender differences in both pain mechanisms and the clinical presentation of pain syndromes further emphasize the need to better understand the underlying biological mechanisms of pain, so that improved therapies can be offered to patients.
According to a systematic review of data and statistics from community studies in European Union (EU) countries, Iceland, Norway and Switzerland: 27% of the adult population (aged 18–65) had experienced at least one of a series of mental disorders in the past year (this included problems arising from substance use, psychoses, depression, anxiety, and eating disorders). Neuropsychiatric disorders are currently the third leading cause of disability-adjusted life years (DALYs) in Europe, with approximately 4 out of every 15 people affected by anxiety or any form of depression. With an estimated >83 million people affected, mental disorders are primed to become one of the largest causes of disability by 2030. Closely overlapping with this, problems with falling asleep or daytime sleepiness are estimated to affect approximately 35 to 40% of the adult population in some countries annually and are a significant cause of morbidity and mortality. Although chronic sleep loss is common in today's society, many people are unaware of the potential adverse health effects of habitual sleep restriction. Under strict experimental conditions, short-term restriction of sleep results in a variety of adverse physiologic effects, including hypertension, activation of the sympathetic nervous system, impairment of glucose control, and increased inflammation. Ultimately poor sleep can contribute to poor mental and physical health, leading to increased levels of obesity, diabetes, neuropsycuiatric disorders and altered immune system functioning that can contribute to neurodegenerative disease amongst other manifestations.
The European Brain Council have estimated the cost of brain disorders to Europe alone at €800 billion in 2010. Taken together, one can easily see why the study of the molecular and genetic mechanisms of ageing neurological problems such as Parkinson's disease and neurodegeneration, pain, sleep and neuropsychiatric disorders in the population represent important areas of research for the population.
Overall, our research strategy is to use deep molecular phenotyping alongside suitable cellular and animal model systems to better understand the fundamental mechanisms of disease processes and through this process derive knowledge to drive translational programs that will return benefit to patients through improved diagnostic capacity, better disease management and the development of rational treatments.
For our Parkinson's disease research, using a combination of these approaches and resources we hope to identify important molecular pathways of pathogenesis and extend these findings to other neurological diseases. The potential of this approach is already apparent from some of the rarer monogenic PD genes identified, where Parkin, PINK1 and DJ-1 have important roles in the molecular pathways involved in the clearance of damaged mitochondria. As part of the focus in the Neuromedicine group we are currently identifying and studying Parkin interacting proteins in the mitochondria and their roles in the maintenance of mitochondrial integrity.
Pathways involved in the maintenance of healthy mitochondria are, however, important for many diseases including other forms of neurodegeneration, cancer and cardiovascular disease, therefore insights gained from studying one disease (PD) may have more wider application to other diseases. We hope such genetic data and any experimental models arising from it can be used to both improve diagnostic and therapeutic options for patients, and screen for novel ways to slow disease progression and extend the quality of life.
Some of the main research programs of the Neuromedicine group at the Institute for Biomedicine are summarized below.
Research focus and highlights
Genetics of Parkinson's disease
The largest pedigree of Parkin-mutation carriers known to date was identified in South-Tyrol. In collaboration with the group of Christine Klein at the Medical University of Lübeck (Germany), precious genetic and tissue resources have been collected from family members. These are being used to study important molecular pathways in Parkinson's disease, especially in relation to designing possible interventions that can be used to rescue genetic defects. Fibroblasts from patients are being reprogrammed into functional dopaminergic cells to create novel cellular models. Alpha-synuclein (aSyn) and LRRK2 represents both strong etiological and associated factors, while MAPT greatly increases risk of PD in the general population. As these proteins play fundamental roles in neuronal function and maintenance, being involved in neurotransmission, vesicle dynamics, cytoskeletal function, endosome-lysosome system, we believe that deeper understanding of the cellular properties, roles and dysfunctions of these proteins and the systems they are involved in systems is needed.
Identification of interaction partners of the PARK2 gene product Parkin
Recent work has shown that Parkin selectively translocates from the cytosol to depolarized mitochondria and subsequently induces their autophagic removal. A comprehensive set of novel Parkin-interacting proteins has been identified by affinity pull-down interaction screens. Such proteins will contribute to gain further biological insights into the mechanisms of the ubiquitin-proteasome system, the fission/fusion dynamics and the mitophagy pathway.
Mitochondria are dynamic organelles known to be the key energy producers in the cell, which are responsible for generating the bulk of cellular ATP. It has become apparent that mitochondria are important in a number of pathologies, including neurodegenerative, muscular, cardiovascular, and metabolic diseases and cancer. A large body of evidence has been accumulated confirming that mitochondria play an important role in the development of Parkinson's disease (PD). Dopaminergic neurons are particularly vulnerable to the functional deterioration of mitochondria due to their high energy demands. Damaged mitochondria can cause overproduction of free radicals that can damage the cells, they can affect calcium regulation within the cells and the intracellular spaces, which directly affects neuronal health, they can affect axonal transport, and they can cause cell death.
Our work currently focuses on several aspects of mitochondrial function in PD, which include vulnerability to chemical stressors, mitochondrial reactive oxygen species (mROS), mitochondrial respiration, mitochondrial branching, and levels of mitochondrial and cytoplasmic Ca2+ in neuroblastoma cell lines, as well as fibroblast lines and iPSC-derived dopaminegic neurons from PD patients and healthy controls. Moreover, we are defining the metabolic profile of selected metabolites obtained from media of cultured cells and plasma samples of PD patients vs. controls.
In addition to the specific mitochondria in PD work, we also have projects examining the role of mitochondrial DNA copy number, and accumulations mitochondrial mutation in ageing and disease.
Functional screening and validation in Parkinson's disease (PD)
Over recent years several genome-wide association studies (GWAS) have identified close to 20 loci for PD at genome-wide significance levels. At many of these loci the gene responsible has not been identified. Our functional work involves screening genes at these loci, and also candidate genes arising from sequencing in patients, in order to determine whether novel pathways or genes can be identified that offer the possibilities of innovative therapy in the future.
Dyskinesia, Impulse control and Sleep disorders in Parkinson's disease (PD)
Some of the major clinical problems arising from PD or its chronic treatment involve onset of motor fluctuations and dyskinesias, behaviour problems due to impulse control or poor quality sleep. By collecting a patient cohort and suitable controls characterised for these endophenotypes in PD (DISP study), we hope to use various molecular, metabolomic and genetic profiling techniques in an attempt to discover the mechanisms behind some of these symptoms. Any interesting candidate genes identified through this process can be followed up in suitable functional validation projects.
Genes involved in sleep disorders
Sleep disorders currently affect up to 30% of the population and negatively influence health. In the population-based studies of the Institute for Biomedicine we are gathering information on various sleep parameters in order to better investigate genetic causes of sleep disorders. We have identified components of an ATP-sensitive potassium channel that associate with sleep duration raising the hope that novel treatments can be discovered in the future.
Genetics of Restless Legs Syndrome (RLS)
Genetic contributions to RLS have been consistently recognized from population and family studies. The prevalence of RLS in population microisolates of the Val Venosta (MICROS) was 8.9%. A genomewide linkage scan identified a locus on chromosome 2q33 (RLS4), which spans a candidate region of 8.2 cM. In our ongoing CHRIS study, aimed at surveying 10,000 or more inhabitants of the Val Venosta, we see similar frequencies, giving us several hundred RLS sufferers to work with in trying to elucidate genes contributing to this painful and disruptive syndrome.
Familial clustering of migraine and patent foramen ovale (PFO)
An increased prevalence of PFO in migraine with aura (MA) patients is widely documented in the litrature. In the population of the Val Venosta in South Tyrol, we also observe a high prevalence of PFO among migraine patients in some of the studied pedigrees. This suggests that PFO might be associated with familial migraine, strengthening the hypothesis of a common genetic mechanism underlying the two conditions. We are attempting to use the structure of the population here to dissect out genetic contributions to migraine.
As part of the first round of the CHRIS study, we are assessing various pain phenotypes in addition to migraine and headache in the population, including chronic pain, perceived pain sensitivity through questionnaire and pain sensitivity through use of algometry. In future follow up of the population we hope to expand pain phenotyping to include more quantitative testing. The data obtained so far will be a launching point for determining genetic factors contributing to pain problems in the society, which when combined with data from suitable screening in model systems, will let us build an active research program in this important area.
As part of the CHRIS study we are ascertaining by questionnaire various psychiatric phenotypes including depression, anxiety, addiction, affective disorder, traumatic events, ADHD, life orientation, personality, and satisfaction.
The Neuromedicine group is responsible for setting up and running the cutting-edge confocal system of the Institute for Biomedicine (Leica SP8-X). The system employs 3 laser sources (UV, Argon and White Light), 4 detectors for fluorescence (two hybrid, two photomultipliers) plus one for brightfield/phase contrast, and uses Acousto-Optical Beam Splitter technology. This combination of features provides complete spectral freedom in both the excitation and the detection of fluorescence, and makes the system "future-proof" as it allows tuning to the characteristics of any fluorophore. The system is also equipped with an environmental chamber with controlled temperature, CO2 and moisture supply for live cell imaging, and a resonant scanner (8KHz) for the visualization of very fast cellular events (for example, for calcium imaging).
A collaboration with MicroPhoton Devices, the Politechnico Milano and the University of Cambridge will soon result in the addition of multi-channel spectral FLIM capabilities to the system.
iPS cell generation and reprogramming
In order to study the molecular mechanisms underlying neurological disorders in a biologically relevant cell model, we are generating induced pluripotent stem (iPS) cells by reprogramming of patient- skin fibroblasts and PBMNCs from human frozen buffy coats. These iPS cells are then differentiated into DA neurons, which represent a very important resource for the in vitro generation of a cell model that can be used to investigate the pathophysiology of disorders and to identify novel therapeutic approaches.
High resolution respirometry
Mitochondrial dysfunction has been a longstanding theme implicated in the etiopathogenesis of Parkinson's disease (PD). Evidence for a direct relationship between mitochondrial dysfunction and PD came from reports of moderate decrease of the mitochondrial respiratory chain complex I activity in the substantia nigra of patients with sporadic PD.
High-resolution respirometry is one of the modern trends in mitochondrial physiology; it enables simultaneous measurement of oxygen flux, ROS production, mt-membrane potential, Ca2+ and ATP production, many parameters characterizing mitochondrial function in routine assays using small samples of biological material that can be isolated mitochondria, cultured cells, tissue preparations and human biopsies. We are currently using the Oxygraph from Oroboros to perform our high-resolution respirometry.
The CRISPR/Cas9 technology is a recent development in gene editing approaches and is currently having a major impact across the life sciences as well as for biotechmology. It's main tool is the Caspase 9 of Streptococcus pyogenes which functions like a pair of molecular scissors and can be easily brought to any desired locus by a short guiding RNA fragment (sgRNA). Having targeted the enzyme to the locus of interest it can either destroy a gene, create mutations or even bring a new piece of DNA into the genome. The technique has facilitated genome editing in many ways and is becoming a standard technology for biomedical research. We are using the technology to tag and/or edit genes specific for Parkinson's disease in order to allow us to create in vitro isogenic disease models in an easy and straightforward way and to monitor the impact of Parkinson-related mutations during neuronal development.