The Epidemiology of Parkinson's Disease in Croatia and the Influence of Genetic Factors and Microbiota on the Progression and Treatment Outcomes of the Disease

Parkinson's disease (PD) is the second most common neurodegenerative disease, which affects 2-3% of the general population above 65 years. There are significant differences in incidence depending on geographical location, race, and ethnicity. The exact cause of the disease is still unknown, but the role of genetic and environmental factors has already been established. Certain genetic forms of the disease make up for a small percentage, so it is thought that environmental factors have a more significant impact on the development of the disease. The incidence of PD is higher in people exposed to significant quantities of pesticides and traumatic brain injury, while there is a smaller incidence in smokers and people consuming more significant quantities of caffeine.

Moreover, intestinal and oral microbiota is regarded as a significant factor in the pathogenesis of the disease, due to the discovered floral imbalance in people suffering from PD. At this point, it is unknown whether the dysbiosis of the intestinal microbiota precedes the disease or it is the result of the disturbed neuronal communication of the liver-brain axis. Since there has not been a proper epidemiological study conducted in Croatia so far, there is no knowledge of the number of cases of PD linked with genetic and environmental factors, doing this project that more scientifically and socially impactful.

A specific pathophysiological event in PD is the loss of neurons in the substantia nigra, along with the accumulation of the intracellular protein α-synuclein in the entire central nervous system. It is important to note that PD is asymptomatic for an extended period since the symptoms start manifesting only when there is a loss of more than 70-80% of dopaminergic neurons. Dysfunction of glial cells, cells that are usually in charge of keeping homeostatic conditions in the central nervous system accompanies PD. The dysfunction leads to neuroinflammation and microglial activation, phagocytosis and the destruction of neurons. On the other hand, there is a lack of a proper nutritive and reparative reaction from astrocytes, glial cells involved in neuroprotection and injury repair in the CNS. Unfortunately, there is still no specific biomarker known for PD, however alterations of neurodegenerative, nutritive, and immunological markers in these disorders enable experimental monitoring of disease progression. Recent research proposed a link between intestinal microflora and glial cells in physiological conditions, but also in the pathogenesis of various neurodegenerative disorders. It is currently thought that the first pathophysiological changes, occur inside the intestinal nervous system, which is in direct contact with the intestinal microflora. This way the intestinal microbiota could modulate the pathogenesis of PD and contribute to the interindividual variability of the clinical presentation. Even though a small number of studies did focus on this part of the pathogenesis of the disease, the real effect of the intestinal and oral microflora dysbiosis on the pathogenesis of PD is not established. Also, there have been no studies which explore the relationship between genetic and environmental factors, making prospective studies with de novo PD patients necessary in shedding light on the mean effects of the dysbiosis on the disease progression and treatment outcome.

The project will finish in four years, with the first 20 months dedicated to the first phase (genetic-epidemiological research), and the entirety of the 48 months for the second phase of the project (prospective clinical research) Participants: The research group in the first phase of the research includes patients with PD throughout the Republic of Croatia. The population would include all the available afflicted currently residing in Croatia, which are members or work with the Croatian PD patient's association. The estimated response is 10% of the PD population (500-750 patients) The patients will fill the epidemiologic questionnaires and patients with familial PD will be selected. The expected emergence of the hereditary form of the disease in the PD population is around 10-15% (50-75). The second phase of the research (prospective clinical trial) includes two subject groups. The first will be made up from patients from the Clinical Hospital Centers in Rijeka, which have not been actively treated yet (drug-naive), while the second will consist of healthy controls in the same age range. The target number of subjects in both of these two groups is 50. Inclusion criteria for participation of drug-naïve patients is a confirmed PD diagnosis according to the Movement Disorder Society Clinical Diagnostic criteria. The healthy control group will consist of participants from the same age group with the same nutritional status (body mass index), as well as living in the same geographical area as the test group. Exclusion criteria for participation include unique diet plans, chronic bowel disease, any autoimmune disorder, acute infectious diseases, acute active inflammation, patient history positive for significant gastrointestinal surgical procedures and an active antibiotic, probiotic, nutritive supplement, corticosteroid, and immunosuppressive drugs.

The first phase - genetic-epidemiological study (20 months): In the first nine months, throughout Croatia, participants who are members or work with the Croatian PD patient's association will be recruited. At the end of the first year of the research, the participants will have their whole exomes sequenced with the help of next-generation sequencing (NGS), to find genetic variants in Croatian patients. Through the second year of research, result analysis and dissemination are planned, as well as counseling in participants with a confirmed familial variant of the disease. For this purpose, the participants will give blood samples for genetic analysis. The target number of subjects in this phase of the research is 50. The participants will also be asked to fill out several structured study forms, which should provide an estimation of the genetic background of the participant, along with information on their biorhythm, sleep patterns, diet, life goals, and general stress.