Report
Teaser, summary, work performed and final results
Periodic Reporting for period 3 - PROGSY (Prosaposin and GPR37 in synucleinopathies)
Teaser
Parkinson´s disease (PD) is the most common movement disorder and affects 1 % of the population above 60 years of age. The related condition, Dementia with Lewy Bodies (DLB) is the second most common form of dementia. Since people are living longer, the prevalence of PD and...
Summary
Parkinson´s disease (PD) is the most common movement disorder and affects 1 % of the population above 60 years of age. The related condition, Dementia with Lewy Bodies (DLB) is the second most common form of dementia. Since people are living longer, the prevalence of PD and DLB is set to rise dramatically in the future. In PD, progressive loss of dopaminergic neurons results in a number of symptoms including tremor, bradykinesia and rigidity. Today, there are several clinically used therapies which enhance dopamine function and thereby alleviate the main symptoms of PD, at least at the early stages. However, none of them can halt progression of the disease. At a subcellular level, PD and DLB are characterized by accumulation of intracellular protein aggregates called Lewy bodies. The main constituent of Lewy bodies is alpha-synuclein and, consequently, PD and DLB are referred to as synucleopathies. The next breakthrough in the treatment of PD will be aimed at slowing down disease progression based on insights into the underlying pathogenic process. The overall objective of this proposal is to examine the role of GPR37in relation to parkinsonism. GPR37 is a G protein coupled receptor which easily misfolds and is found in Lewy bodies. GPR37 has actually been reported to be a core component of Lewy bodies, suggesting that it may be involved in early pathological events of PD. Intriguingly, my laboratory has found that properly folded GPR37, located at the plasma membrane, exerts neuroprotection against parkinsonian toxins in cell lines. GPR37 has also been reported to be activated by the neuroprotective factor prosaposin. I have recently summarized the literature and posed key research questions on these topics (Leinartaité L, Svenningsson P. Folding Underlies Bidirectional Role of GPR37/Pael-R in Parkinson Disease. Trends Pharmacol Sci. 2017 Aug;38(8):749-760.)
The overall aim of this programme is to generate mechanistic insight into the role of prosaposin, GPR37 and GPR37L1 as novel diagnostics and targets for the development of neuroprotective pharmacological therapies against synucleopathies, particularly PD.
Work performed
The research is conducted around four specific objectives. The first objective studies the structural basis for the high propensity of GPR37 to misfold and aggregate. In collaborative efforts, structural models of GPR37 and GPR37L1 are being made. They are used for the identification of residues relevant for function and ligand binding as well as for molecular docking experiments with large chemical libraries. The second objectives uses fluorescence correlation spectroscopy for single molecule trafficking studies of GPR37 and GPR37L1 in living cells. By using fluorescence cross correlation spectroscopy (FCCS), studies are being made on the aggregation of GPR37 and on how this receptor and GPR37L1 interacts with the two splice forms of dopamine D2 receptors, called D2R-long (D2RL) and D2R-short (D2RS). The third objective concerns animal models with altered levels of prosaposin, GPR37 and GPR37L1 in defined neuronal circuitries, particularly dopaminergic. Detailed behavioral studies are performed under baseline conditions or after experimental parkinsonism induced by intranigral AAV6-alpha-synuclein overexpression or intracerebral 6-OHDA injections. Postmortem, histological and neurochemical analyses are being made to monitor neurodegeneration. In the fourth aim, we are using prosaptides/GPR37 as potential targets for radioligand development towards PET imaging of Lewy bodies. This aim has been extended to wet biomarker studies of GPR37 and prosaposin.
Final results
\"Several of the methodological approaches in this programme are unconventional. In particular, fluorescence correlation spectroscopy (FCS) and fluorescence cross correlation spectroscopy are not commonly used to examine ligand-GPCR interactions. This is somewhat surprising as these methodologies are quantitative with single molecule-sensitivity and reveal detailed information about molecular numbers and their transporting properties. FCS technology is characterized by very high \"\"single-molecule\"\" sensitivity and very high time resolution. Dual-color FCCS relies on the co-variance of signals from two spectrally distinct fluorescent markers to establish complex formation. If the two fluorescent markers constitute the same complex, the fluorescence fluctuations of the two fluorophores will correlate, thus giving rise to a positive cross-correlation in the statistical analysis. The use of FCCS in the study of complex formation does not produce false negatives arising from unbound molecules residing in the near vicinity of each other, as do many other methods used for this purpose. s (PLA). Within this programme, we use FCS/FCCS to study membrane insertion and kinetics along with multimerization and aggregation of GPR37 and GPR37L1.
By a combination of unconventional and more conventional approaches described above, we expect that this programme shall clarify several aspects of the nature of prosaposin, GPR37 and GPR37L1. Hopefully, these insights can be developed into novel diagnostics and/or neuroprotective pharmacological therapies against PD and DLB. In a wider perspective, the project will also generate technical advancements that could be of general interest for dynamic studies of ligand-GPCR interactions.\"