Establishment of mass spectrometric determination of the biomarkers cAMP and cGMP and investigation of potential pathogenic processes (GPR6, telomerase, microglial activation) in animal models of Parkinson´s disease

Abstract

Parkinson´s disease (PD) is a neurodegenerative disorder with a characteristic loss of nigrostriatal dopaminergic neurons and disturbances of the motoric system. There is an unmet medical need for the discovery of disease modifying treatment strategies and identification of reliable biomarkers. The aim of my PhD thesis was to establish an analytical method for the determination of the free radical biomarkers o-, m-, p-tyrosine, nitrophenylalanine and nitrotyrosine by HPLC as well as the biomarkers cyclic adenosine-3´,5´-monophosphate (cAMP) and cyclic guanosine-3´,5´-monophosphate (cGMP) by liquid chromatography/tandem mass spectrometry (LC-MS/MS). In order to improve the knowledge about PD pathogenesis, I investigated the impact of astrocyte-specific IKK2 activation and telomerase deficiency in the MPTP model of PD using neurochemical and behavioural readouts as well as immunohistochemistry. I established the neuroinflammatory-based LPS (lipopolysaccharide) mouse model of PD and investigated new treatment strategies for PD including GPR6 deficiency, angiotensin II receptor 1 (AT1) antagonism by telmisartan, NADPH oxidase inhibition by apocynin and the increase of single, O-glycosidically linked -N-acetylglucosamine (O-GlcNAc) on proteins by Thiamet G. The HPLC method for the determination of the free radical biomarkers showed good sensitivity (up to 5 nM) and linearity (up to 100 µM). The increased formation of the free radical biomarkers after in vitro incubation of D-phenylalanine with peroxyinitrite and in vivo injection of 6-hydroxydopamine (6-OHDA) in mice verified the use of this biomarkers for free radical determination. The LC-MS/MS method for cAMP and cGMP showed high sensitivity (limit of quantification 0.5 nM). It was suitable to measure cAMP and cGMP in plasma, cerebrospinal fluid (CSF) and brain tissue. ...