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Prosaposin and GPR37 in synucleinopathies

Periodic Reporting for period 4 - PROGSY (Prosaposin and GPR37 in synucleinopathies)

Reporting period: 2020-02-01 to 2022-01-31

Parkinson´s disease (PD) is the most common movement disorder and it 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. 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 was to examine the roles of prosaposin and GPR37 in relation to parkinsonism. GPR37 is a G protein coupled receptor which easily misfolds and is found in Lewy bodies. 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. Taking these data into account, prosaposin and GPR37 stands out as prominent targets for modulation of PD progression.
This research programme used advanced imaging technology and elucidated mechanisms whereby GPR37 aggregates and form protein complexes with relevance for PD. It also used animal models to study whether GPR37 and/or prosaposin can protect against toxic effects of alpha-synuclein and other parkinsonian toxins. It was found that both prosaposin and GPR37 have protective actions. Using brain tissue and cerebrospinal fluid from patients with PD, our studies have demonstrated the potentials of N-terminal GPR37 fragments and prosaposin to serve as diagnostic markers.
GPR37 is exceptional among GPCRs having a high propensity for intracellular receptor accumulation and aggregation leading to neurotoxicity (see Figure). However, unexpectedly, our results suggest that GPR37 is neuroprotective in dopaminergic when located at the plasma membrane. Prosaposin has been claimed to be a agonist at GPR37 and GPR37L1. The programme therefore aimed at generating 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. During this first period of the programme, I spent time summarizing the literature and posing key research questions related to the topic. This review has been published in Trends of Pharmacological Sciences. The research has been conducted around four specific objectives related to prosaposin, GPR37 and GPR37L1. The first objective studies the structural basis for the high propensity of GPR37 to misfold and aggregate. In collaboration, in silico models of GPR37 (see figure) and GPR37L1 have been made. These models have contributed to identify residues relevant for function and ligand binding. In particular, we found that GPR37 contains an amino acid in a TM region which is uncommon among class A GPCR’s. We have mutated this amino acid residue and expressed mutated constructs in cell lines and found that it governs GPR37 trafficking to the cell surface. Using these homology models of GPR37 and GPR37L1, we have also perform a molecular docking experiments with a large chemical library and identified compounds which we are now evaluating in cellular assays. In the second objective we investigated GPR37 and GPR37L1 dimerization and multimerization in live cells using three quantitative imaging methods: Fluorescence Cross-Correlation Spectroscopy (FCCS), Förster Resonance Energy Transfer, and Fluorescence Lifetime Imaging Microscopy. Our data show that GPR37 and GPR37L1 form homo- and heterodimers in live cells. Importantly, aggregation of GPR37, but not GPR37L1, was identified in the cytoplasm, which could be counteracted by Parkin overexpression. These data provide further evidence that GPR37 participate in cytosolic aggregation processes implicated in PD pathology. By using FCCS, we have found that GPR37 and GPR37L1 differentially interacts with the two splice forms of dopamine D2 receptors. The third objective concerns animal models with altered levels of prosaposin, GPR37 and GPR37L1. GPR37 KO mice have a dopamine neuron deficit, enhanced striatal GABA levels and deficient corticostriatal LTP. They also respond stronger to 6-OHDA-induced neurotoxicity. The data indicates that properly functional GPR37 may counteract aging processes and parkinsonism. We have generated conditional prosaposin, GPR37 KO and GPR37L1 KO mice and crossed them with DAT-CreER mice. Deficits in locomotion and emotionality are seen in prosaposin KO mice. These deficits are reversed by viral constructs overexpressing prosaposin. In fact, such viral constructs also counteract toxicity by AAV-alpha-synuclein overexpression. In a fourth aim, we proposed to use prosaposin/GPR37 as targets for biomarker development. We have reported that N-terminal fragments of GPR37 and prosaposin can aid as diagnostic cerebrospinal fluid biomarkers in PD. Several scientific publications have been reported and there will be additional publications. The data has been presented at several international conferences.
Central to this proposal was a detailed exploration of the recently described bidirectional role of GPR37 in PD. GPR37 is a G protein coupled receptor which easily misfolds and is found in Lewy bodies in PD. In contrast, my laboratory has found that properly folded GPR37, located at the plasma membrane, exerts neuroprotection against parkinsonian toxins in cell lines.
Several of the methodological approaches used in the proposal were also unconventional. In particular, fluorescence correlation spectroscopy (FCS) and fluorescence cross correlation spectroscopy (FCCS) studies 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 (temporal autocorrelation) or molecular brightness distribution. 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. Using these methods, our data show that GPR37 and GPR37L1 form homo- and heterodimers with dopamine D2 receptors in live cells. We also used a novel FCS multidetector and could demonstrate that aggregation of GPR37, but not GPR37L1, was identified in the cytoplasm.
Our data also support that prosaposin and GPR37 promotes dopamine function and counteracts dopamine neurodegeneration opening up therapeutic possibilities. Finally, we have shown that the N-terminal part of GPR37 can be demonstrated in cerebrospinal fluid, which is very unusual for a GPCR. Interestingly, we have reported that N-terminal fragments of GPR37 can aid as diagnostic cerebrospinal fluid biomarkers in PD.
GRP37 folding and trafficking
In silco structure of GPR37
GRP37 Schematic figure