Proteomic insights into MrgD receptor deficiency: motor hyperactive- and compulsive-like phenotype, and synaptic dysfunction in the nigrostriatal pathway

The renin-angiotensin system is composed by a proteolytic cascade of angiotensinogen leading to the formation of several bioactive peptides, including alamandine, which acts as an endogenous agonist of the MAS-related G protein-coupled receptor D (MrgD). Notably, this receptor is highly expressed in brain regions associated with the control of reward, cognition, and motor functions, including the nigrostriatal pathway. Behavioral studies done by our research group showed that MrgD receptor-deficient mice (KO) exhibit motor hyperactivity and a high compulsive-like phenotype, without anxiety-like phenotype or alterations in short or long-term memory. This study aimed to assess the proteome, phosphoproteome, and N-glycoproteome of the nigrostriatal pathway in C57Bl6/J (WT) and KO mice to uncover the molecular role of MrgD in the nigrostriatal pathway. Thus, substantia nigra (SN) and striatum (ST) regions were collected for proteomics (n=4). Proteins were extracted using the acid labile detergent SDC. Tryptic peptides were labeled with TMT-16plex and submitted to TiO₂ to allow the simultaneous enrichment of phosphorylated and sialic acid-containing glycopeptides. Non-modified and modified peptides samples were pre-fractionated into 20 and 12 fractions in high pH reverse-phase chromatography, respectively. Samples were analyzed using LC-MS/MS (Orbitrap Exploris 480). Raw data was deconvoluted using Maxquant against the SwissProt Mus musculus FASTA file were used. To validate proteome findings, digested peptides were loaded into the Evosep One coupled with timsTOF Pro and analyzed using a 30 SPD gradient (n=5). Data was deconvoluted in DIA-NN using the same FASTA file. An extensive functional validation was done. A total of 5,878 unique proteins, 19,209 class 1 phosphosites, and 5,301 N-glycosylated sites were identified, with 468 proteins, 190 phosphosites, and 12 N-glycosylated sites regulated (p<0.05, FC±1.2). KO mice showed alterations in biological processes related to dopaminergic synapses, motor activity control, and synaptic vesicle exocytosis. A remarkable down-regulation in phosphorylation and/or abundancy of proteins regulating Ca²⁺ dynamics such as Protein Kinase C (PKC), Calcium voltage-gated channel (Cav2), Ca²⁺/calmodulin-dependent protein kinase (Camk2α) was observed. In addition, key proteins in exocytosis such as Syntaxin (Stx1a), Syntaxin-binding protein (Stxbp1), and Complexin 2 (Cplx2), were down-regulated and glycosylated in KO group. In line with data, functional validation showed a reduction in exocytosis in the KO group, leading to an accumulation of neurotransmitters in the SN and decreased levels in the ST of KO mice. In contrast, the treatment of SN and ST with alamandine in WT increased exocytosis. In conclusion, the Alamandine/MrgD signaling has an important role in vesicle trafficking and release in the nigrostriatal system, highlighting its relevance to neuropsychiatric disorders involving motor control and impulsivity.