Abstract:
Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease and affects 150 000 – 250 000 people in Germany. Although the disease is not curable yet, great progress has been made in the past few years to treat the symptoms and improve the quality of life of these patients. However, reliable biomarkers to detect the disease in an early stage before the first symptoms occur are so far unavailable. Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage could be detected in many PD patients, but their analysis requires either post-mortem brain sections, a brain biopsy, or CSF. Extracellular ves-icles (EVs) are a potential new source of biomarkers. They are nanoparticles made of a lipid bilayer and can be found in serum and CSF and can also pass the blood-brain barrier. EVs carry mtDNA making them suitable for genetic analysis.
In this work, different EV isolation methods from serum and CSF were tested and corresponding protocols were established (ultracentrifugation, size-exclusion chro-matography, precipitation techniques). A commercial kit (ExoQuick™) was chosen for the downstream analysis as the most promising method. EVs were characterized by their appearance, their size, and their protein composition. Extravesicular DNA was digested, and DNA was extracted from the EV preparation and characterized by PCR showing enrichment of mtDNA in these samples. Furthermore, an immunoprecipitation technique was modified to use the transmembrane protein L1CAM as a potential marker for neuronal-derived EVs in serum, but this method turned out to be unsuitable for the downstream analysis.
A cohort with 35 PD patients (serum and CSF samples) and 35 healthy controls (only serum samples) was built to assess the suitability of mtDNA in EVs as a biomarker for PD. The primary endpoint was the ratio of two mitochondrial genes (ND4 and ND1) as an established parameter to detect deletion in the mtDNA. EVs from the serum and CSF samples were isolated using the ExoQuick™ kit, their DNA was extracted, and gene expression was measured by using quantitative real-time PCR. The ND4/ND1 ratio showed no difference between serum EVs from PD patients and controls, but a significant difference could be observed when comparing serum and CSF EVs from the PD patients. mtDNA deletion measured with the ND4/ND1 ratio in serum EVs did not turn out to be a reliable biomarker for PD. However, CSF EVs might serve as better biomarkers and further research is required to explore their promising potential.
ExoQuick