Molecular cause and functional impact of altered synaptic lipid signaling due to a prg‐1 gene SNP
2015; Springer Nature; Volume: 8; Issue: 1 Linguagem: Inglês
10.15252/emmm.201505677
ISSN1757-4684
AutoresJohannes Vogt, Jenq‐Wei Yang, Arian Mobascher, Jin Cheng, Yunbo Li, Xingfeng Liu, Jan Baumgart, Carine Thalman, Sergei Kirischuk, Petr Unichenko, Guilherme Horta, Konstantin Radyushkin, Albrecht Stroh, Sebastian Richers, Nassim Sahragard, Ute Distler, Stefan Tenzer, Lianyong Qiao, Klaus Lieb, Oliver Tüscher, Harald Binder, Nerea Ferreirós, Irmgard Tegeder, Andrew J. Morris, Sergiu Gropa, Peter Nürnberg, Mohammad R. Toliat, Georg Winterer, Heiko J. Luhmann, Jisen Huai, Robert Nitsch,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoResearch Article15 December 2015Open Access Molecular cause and functional impact of altered synaptic lipid signaling due to a prg-1 gene SNP Johannes Vogt Corresponding Author Johannes Vogt Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Jenq-Wei Yang Jenq-Wei Yang Institute for Physiology and Pathophysiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Arian Mobascher Arian Mobascher Department of Psychiatry and Psychotherapy, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Jin Cheng Jin Cheng Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Yunbo Li Yunbo Li Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Xingfeng Liu Xingfeng Liu Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Jan Baumgart Jan Baumgart Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Carine Thalman Carine Thalman Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Sergei Kirischuk Sergei Kirischuk Institute for Physiology and Pathophysiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Petr Unichenko Petr Unichenko Institute for Physiology and Pathophysiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Guilherme Horta Guilherme Horta Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Konstantin Radyushkin Konstantin Radyushkin Focus Program Translational Neuroscience (FTN), University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Albrecht Stroh Albrecht Stroh Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Sebastian Richers Sebastian Richers Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Nassim Sahragard Nassim Sahragard Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Ute Distler Ute Distler Focus Program Translational Neuroscience (FTN), University Medical Center, Johannes Gutenberg-University, Mainz, Germany Institute for Immunology, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Stefan Tenzer Stefan Tenzer Institute for Immunology, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Lianyong Qiao Lianyong Qiao Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Klaus Lieb Klaus Lieb Department of Psychiatry and Psychotherapy, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Oliver Tüscher Oliver Tüscher Department of Psychiatry and Psychotherapy, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Harald Binder Harald Binder Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Nerea Ferreiros Nerea Ferreiros Institute of Clinical Pharmacology Goethe-University Hospital, Frankfurt am Main, Germany Search for more papers by this author Irmgard Tegeder Irmgard Tegeder Institute of Clinical Pharmacology Goethe-University Hospital, Frankfurt am Main, Germany Search for more papers by this author Andrew J Morris Andrew J Morris Division of Cardiovascular Medicine, Gill Heart Institute, University of Kentucky, Lexington, KY, USA Search for more papers by this author Sergiu Gropa Sergiu Gropa Department of Neurology, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Peter Nürnberg Peter Nürnberg Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany Search for more papers by this author Mohammad R Toliat Mohammad R Toliat Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany Search for more papers by this author Georg Winterer Georg Winterer Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany Search for more papers by this author Heiko J Luhmann Heiko J Luhmann Institute for Physiology and Pathophysiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Jisen Huai Jisen Huai Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Robert Nitsch Corresponding Author Robert Nitsch Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Johannes Vogt Corresponding Author Johannes Vogt Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Jenq-Wei Yang Jenq-Wei Yang Institute for Physiology and Pathophysiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Arian Mobascher Arian Mobascher Department of Psychiatry and Psychotherapy, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Jin Cheng Jin Cheng Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Yunbo Li Yunbo Li Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Xingfeng Liu Xingfeng Liu Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Jan Baumgart Jan Baumgart Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Carine Thalman Carine Thalman Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Sergei Kirischuk Sergei Kirischuk Institute for Physiology and Pathophysiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Petr Unichenko Petr Unichenko Institute for Physiology and Pathophysiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Guilherme Horta Guilherme Horta Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Konstantin Radyushkin Konstantin Radyushkin Focus Program Translational Neuroscience (FTN), University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Albrecht Stroh Albrecht Stroh Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Sebastian Richers Sebastian Richers Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Nassim Sahragard Nassim Sahragard Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Ute Distler Ute Distler Focus Program Translational Neuroscience (FTN), University Medical Center, Johannes Gutenberg-University, Mainz, Germany Institute for Immunology, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Stefan Tenzer Stefan Tenzer Institute for Immunology, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Lianyong Qiao Lianyong Qiao Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Klaus Lieb Klaus Lieb Department of Psychiatry and Psychotherapy, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Oliver Tüscher Oliver Tüscher Department of Psychiatry and Psychotherapy, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Harald Binder Harald Binder Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Nerea Ferreiros Nerea Ferreiros Institute of Clinical Pharmacology Goethe-University Hospital, Frankfurt am Main, Germany Search for more papers by this author Irmgard Tegeder Irmgard Tegeder Institute of Clinical Pharmacology Goethe-University Hospital, Frankfurt am Main, Germany Search for more papers by this author Andrew J Morris Andrew J Morris Division of Cardiovascular Medicine, Gill Heart Institute, University of Kentucky, Lexington, KY, USA Search for more papers by this author Sergiu Gropa Sergiu Gropa Department of Neurology, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Peter Nürnberg Peter Nürnberg Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany Search for more papers by this author Mohammad R Toliat Mohammad R Toliat Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany Search for more papers by this author Georg Winterer Georg Winterer Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany Search for more papers by this author Heiko J Luhmann Heiko J Luhmann Institute for Physiology and Pathophysiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Jisen Huai Jisen Huai Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Robert Nitsch Corresponding Author Robert Nitsch Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Search for more papers by this author Author Information Johannes Vogt 1,‡, Jenq-Wei Yang2,‡, Arian Mobascher3,‡, Jin Cheng1, Yunbo Li1, Xingfeng Liu1, Jan Baumgart1, Carine Thalman1, Sergei Kirischuk2, Petr Unichenko2, Guilherme Horta1, Konstantin Radyushkin4, Albrecht Stroh1, Sebastian Richers1, Nassim Sahragard1, Ute Distler4,5, Stefan Tenzer5, Lianyong Qiao1, Klaus Lieb3, Oliver Tüscher3, Harald Binder6, Nerea Ferreiros7, Irmgard Tegeder7, Andrew J Morris8, Sergiu Gropa9, Peter Nürnberg10, Mohammad R Toliat10, Georg Winterer10,‡, Heiko J Luhmann2,‡, Jisen Huai1,‡ and Robert Nitsch 1,‡ 1Institute for Microscopic Anatomy and Neurobiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany 2Institute for Physiology and Pathophysiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany 3Department of Psychiatry and Psychotherapy, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany 4Focus Program Translational Neuroscience (FTN), University Medical Center, Johannes Gutenberg-University, Mainz, Germany 5Institute for Immunology, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany 6Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany 7Institute of Clinical Pharmacology Goethe-University Hospital, Frankfurt am Main, Germany 8Division of Cardiovascular Medicine, Gill Heart Institute, University of Kentucky, Lexington, KY, USA 9Department of Neurology, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany 10Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 6131 17 8091/8072; E-mail: [email protected] *Corresponding author. Tel: +49 6131 17 8091/8072; E-mail: [email protected] EMBO Mol Med (2016)8:25-38https://doi.org/10.15252/emmm.201505677 See also: B Stutz & TL Horvath (January 2016) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Loss of plasticity-related gene 1 (PRG-1), which regulates synaptic phospholipid signaling, leads to hyperexcitability via increased glutamate release altering excitation/inhibition (E/I) balance in cortical networks. A recently reported SNP in prg-1 (R345T/mutPRG-1) affects ~5 million European and US citizens in a monoallelic variant. Our studies show that this mutation leads to a loss-of-PRG-1 function at the synapse due to its inability to control lysophosphatidic acid (LPA) levels via a cellular uptake mechanism which appears to depend on proper glycosylation altered by this SNP. PRG-1+/− mice, which are animal correlates of human PRG-1+/mut carriers, showed an altered cortical network function and stress-related behavioral changes indicating altered resilience against psychiatric disorders. These could be reversed by modulation of phospholipid signaling via pharmacological inhibition of the LPA-synthesizing molecule autotaxin. In line, EEG recordings in a human population-based cohort revealed an E/I balance shift in monoallelic mutPRG-1 carriers and an impaired sensory gating, which is regarded as an endophenotype of stress-related mental disorders. Intervention into bioactive lipid signaling is thus a promising strategy to interfere with glutamate-dependent symptoms in psychiatric diseases. Synopsis Synaptic phospholipids are potent bioactive factors known to increase glutamatergic transmission in excitatory neurons, and they are normally cleared from the synaptic cleft by PRG-1. A common loss-of-function SNP in PRG-1 affects the pathophysiology and behavior in a way reminiscent of psychiatric disorders. The human PRG-1 SNP (R345T), present in a monoallelic variant, abolished PRG-1 function by impeding its ability for LPA internalization due to altered glycosylation. Monoallelic PRG-1 deficiency affected cortical information processing, leading to decreased somatosensory filter function in rodents and humans, and impaired resilience during stress-related behaviors, an endophenotype of psychiatric disorders. Pharmacological intervention specifically targeting phospholipid signaling rescued cortical somatosensory filter function to wild-type levels, opening a new therapeutic perspective for stress-related mental dysfunctions. Introduction Accurate synaptic transmission is a fundamental requirement for normal brain function (Turrigiano, 2011), and signaling alterations at the excitatory synapse have been related to psychiatric disorders such as schizophrenia (Harrison & Weinberger, 2005; Coyle, 2006; Belforte et al, 2010). Recent research shows that bioactive lipid signaling and protein–lipid interaction play major roles in all steps of endo- and exocytosis processes, including synaptic vesicle cycling (Di Paolo et al, 2004). Plasticity-related gene 1 (PRG-1, also assigned as LPPR4), a neuron-specific molecule in the brain, is an important postsynaptic control element of this pathway (Trimbuch et al, 2009). Previously, we demonstrated that the absence of PRG-1 (Brauer et al, 2003), which is involved in synaptic phospholipid signaling, leads to hippocampal hyperexcitability via increased glutamate release at the synapse (Trimbuch et al, 2009). Recently, a single nucleotide polymorphism (SNP, rs138327459, NHLBI Exome Sequencing Project https://esp.gs.washington.edu/drupal/) was detected in humans resulting in an arginine (R) to threonine (T) exchange at position 345 of the amino acid chain (mouse homolog located at position 346). Our electrophysiological studies using re-expression of PRG-1R346T in PRG-1−/− mice by in utero electroporation revealed that this mutation results in a loss-of-PRG-1 function at the synapse. On a molecular level, we could show that PRG-1R346T lacked the ability to support uptake of lysophosphatidic acid (LPA) into intracellular compartments due to altered O-glycosylation of S347 next to the SNP site, while in-depth quantitative analysis revealed no role for serine phosphorylation at this position. PRG-1 heterozygous mice which are an animal correlate for human monoallelic PRG-1+/mut carriers showed altered cortical information processing and stress-related behavioral deficits indicative for mental disorders. Using specific inhibitors of phospholipid synthesis, we could show that modulation of bioactive phospholipid levels upstream of PRG-1 reverted cortical network function and behavior toward wild-type (wt) levels. In line with experimental data, electrophysiological assessment using the P50 sensory gating auditory paradigm (which corresponds to the pre-pulse inhibition (PPI) tested in mice) revealed an altered sensory gating in monoallelic R345T PRG-1 carriers which were identified among 1,811 human volunteers in a population-based cohort. Since similar alterations of cortical excitability and sensory gating have been described as an endophenotype of schizophrenia and stress-related disorders (Turetsky et al, 2007; Javitt et al, 2008), our results indicate a novel therapeutic strategy targeting synaptic bioactive lipid signaling in altered cortical information processing related to mental disorders. Results PRG-1R346T is a loss-of-function mutation PRG-1 was shown to be involved in internalization of lysophosphatidic acid (LPA) to intracellular postsynaptic compartments (Trimbuch et al, 2009), thereby controlling LPA levels in the synaptic cleft. To understand the molecular consequences of the human SNP resulting in arginine (R) to threonine (T) exchange at position 345 in the amino acid chain of PRG-1, we established HEK cell lines with stable expression of the mouse homolog of this SNP, PRG-1R346T, and found similar membrane localization when compared to wild-type PRG-1 (wtPRG-1, Fig 1A). After application of fluorescence labeled LPA (TopFluor (TF)-LPA) and removal of surface bound TF-LPA using 0.001% SDS, PRG-1R346T-expressing cells displayed a lower fluorescence signal in intracellular compartments (Fig 1B). This finding was quantified by FACS analysis (example shown in Fig 1C) revealing a significantly reduced capacity of PRG-1R346T-expressing cells to internalize TF-LPA when compared to wtPRG-1 (Fig 1D). To unambiguously prove the internalization of LPA, we applied a chemically modified, unnatural LPA (C17-LPA) on wtPRG-1-expressing cells which showed a statistically significant internalization of C17-LPA (Fig 1E) and the intracellular presence of its metabolite C17-MAG (Fig 1F) when compared to HEK cells which lack any PRG-1 expression. In line with previous results, PRG-1R346T-expressing cells did not show a significant increase of intracellular C17 LPA nor of its metabolite C17-MAG providing further evidence that PRG-1R346T lacks the capacity to mediate internalization of LPA into cells (Fig 1E and F). Figure 1. PRG-1R346T: loss-of-function mutation reduced LPA internalization due to altered O-glycosylation A. PRG-1 and PRG-1R346T are expressed on HEK293 cell membranes. B. Conventional fluorescence images of transfected and TopFluor (TF)-LPA-stimulated cells after washing with 0.001% SDS. Scale bar: 10 μm. C, D. FACS analysis of internalized TF-LPA of PRG-1- and PRG-1R346T-transfected cells (n = 7 each, **P = 0.0015; t-test). E, F. Incubation with C-17 LPA and subsequent washing as described above, led to a significant increase of TF-LPA in PRG-1-expressing cells but not in PRG-1R346T-expressing cells. In line, C-17 MG, a degradation product of C-17 LPA, was significantly increased in PRG-1-expressing cells but not in control or PRG-1R346T-expressing cells (n = 4 each; Kruskal–Wallis test (for C-17 LPA P = 0.0066; for C-17 MAG P = 0.0048) with Dunn's multiple comparison test, *P < 0.05 for C-17 LPA and C-17 MAG). G. Quantitative assessment of S347 phosphorylation using isotopically labeled peptides and mass spectrometry of purified wild-type PRG-1 and PRG-1R346T from stable transfected HEK293 cells revealed low total phosphorylation levels for both protein variants (n = 3 measurements). H. TF-LPA uptake as analyzed by FACS was significantly decreased in S347A and S347D mutant PRG-1 (n = 12 per condition, ***P < 0.0001; one-way ANOVA with Bonferroni's multiple comparison test). I. Western blot (WB) of IP using a specific PRG-1 antibody revealed decreased levels of O-GlcNAc in PRG-1R346T when compared to wild-type PRG-1. J. Densitometric analysis of n = 7 WB from IP of PRG-1- and PRG-1R346T-expressing cells revealed a significantly decreased O-GlcNAc amount in PRG-1R346T (unpaired t-test, *P = 0.0107). Data information: All experiments were done in parallel. Bar diagrams represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint PRG-1R346T alters O-glycosylation, but not phosphorylation of neighboring S347 Since PRG-1R346T was properly expressed at the membrane, we hypothesized a posttranslational modification being involved in the loss of function in TF-LPA internalization. Interestingly, the SNP changing R345 (arginine) to 345T (threonine) is located in close proximity to a frequently reported phosphorylation site (Munton et al, 2007; Trinidad et al, 2008; Huttlin et al, 2010; Wisniewski et al, 2010; Goswami et al, 2012). Therefore, we addressed the question to what extent the neighboring S (serine) at position 346 in humans and at position 347 in mouse is phosphorylated under wild-type conditions, and whether this phosphorylation is eventually altered due to the mutation of R to T, which alters typical phosphorylation motifs (Pearson & Kemp, 1991). Using quantitative mass spectrometry analysis and isotopically labeled peptides corresponding to the peptide containing S347, we detected that, although described as a potential activity-related phosphorylation site (Munton et al, 2007), S347 neighboring the SNP site showed only minor phosphorylation (4.73% of total PRG-1, Fig 1G). In addition, in PRG-1R346T, we could neither detect any relevant change in phosphorylation of S347 (3.33% of total PRG-1, Fig 1G), nor any phosphorylation of the T at position 346. To further assess whether S347 phosphorylation is a potential relevant cause for the loss of LPA uptake of PRG-1R346T, we established HEK cell lines with a stable expression of PRG-1S347A and PRG-1S347D, which either mimic the non-phosphorylated (S347A) status or the phosphorylated status (S347D) of S347. Both cell lines lacked the capacity to significantly internalize TF-LPA above baseline levels (Fig 1H), strongly arguing against an important role for phosphorylation explaining the loss of function of PRG-1R346T in TF-LPA internalization, however, pointing to the need of S347 at this position. Another intracellular posttranslational modification, critically important for brain function is O-GlcNAC glycosylation (Rexach et al, 2008), which occurs at S and T residues and has a complex interplay with phosphorylation (Tarrant et al, 2012). Using immunoprecipitation studies and specific antibodies, we analyzed O-GlcNAC glycosylation in wtPRG-1 in comparison with PRG-1R346T and detected significantly reduced glycosylation levels in PRG-1R346T-expressing cells (Fig 1I and J), indicating an interference of the R346T mutation with its neighboring S347 O-glycosylation site. Since neither A nor D is a target for O-GlcNAC glycosylation and the cell lines expressing PRG-1S347A and PRG-1S347D lacked the capacity of internalizing TF-LPA (Fig 1H), these data point to the necessity of an O-glycosylation at S for proper PRG-1 function, which is altered in PRG-1R346T. In sum, these data on posttranslational modifications provide evidence for the fact that not changes in phosphorylation, but proper O-glycosylation of S at position 346 in humans interfered by the R345T SNP is crucial for PRG-1 function. PRG-1R346T is a loss-of-function mutation at the synapse To analyze potential functional consequences of the human SNP in neurons, we moved on in assessing PRG-1R346T function at the synapse and performed whole-cell patch-clamp recordings from layer IV spiny stellate neurons in the S1BF of PRG-1−/− animals that re-expressed PRG-1R346T delivered by in utero electroporation (Fig 2A). This re-expression resulted in proper localization of PRG-1R346T protein at the spine head membrane, excluding an impact of this mutation in protein targeting (Fig 2B). While re-expression of wtPRG-1 in PRG-1-deficient neurons was reported to rescue the frequency of miniature excitatory postsynaptic currents (mEPSC) to wild-type levels in hippocampal CA1 pyramidal cells (Trimbuch et al, 2009), PRG-1R346T, albeit present at the proper dendritic localization, failed to achieve this functional rescue. This is consistent with a loss of function of PRG-1R346T, presumably due to its inability to support LPA internalization (Fig 2A and C). Figure 2. PRG-1R346T fails to rescue PRG-1−/− phenotype and to induce significant TF-LPA uptake A, B. In utero electroporation (IUE) of PRG-1R346T lead to membrane expression of this construct. C, D. In vitro whole-cell patch-clamp recordings from PRG-1−/− layer IV spiny stellate neurons (S1BF) and of spiny stellate neurons reconstituted with PRG-1R346T by IUE. Original traces are shown in (A) (n = 11 PRG-1−/− neurons and 11 PRG-1R346T-reconstituted neurons from 4 mice; t-test). E. Bright field image and regions of interest (ROIs, yellow) of primary neuronal cultures containing PRG-1−/− neurons. F. Detection of transfected neurons via a co-transfected IRES-GFP cassette by fluorescent illumination. Scale bar: 50 μm. G. Baseline fluorescence (calculated as Δf/f) of transfected and non-transfected neurons prior to TF-LPA stimulation. H. PRG-1wt-transfected neurons displayed a clear increase in fluorescence intensity after TF-LPA application (left), while PRG-1R346T-transfected neurons were not different from non-transfected PRG-1−/− neurons (right). I. Fluorescence intensity analysis of the ROIs revealed significant TF-LPA uptake in PRG-1wt-transfected neurons but not in PRG-1R346T-transfected neurons (n = 25 neurons per condition; repeated-measures ANOVA with Bonferroni post hoc correction, ***P < 0.0001). J. Expression of GFP or of PRG-1R346T in wild-type neurons did not alter TF-LPA uptake when compared to non-transfected neurons (n = 57 wild-type, 10 GFP-transfected and 10 PRG-1R346T-transfected neurons; Kruskal–Wallis test with Dunn's multiple comparison test). K. Bright field image and fluorescence image overview showing TF-LPA in neurons with ROIs (yellow) of a primary neuronal culture. L. Fluorescence analysis revealed a significantly lower TF-LPA uptake in PRG-1+/− neurons when compared to wild-type neurons (n = 15 neurons per group; unpaired t-test, ***P < 0.0001). Data information: Bar diagrams represent mean ± SEM. Download figure Download PowerPoint To directly assess the functional consequences of PRG-1R346T in neurons in terms of their capacity to internalize LPA (Trimbuch et al, 2009), we used primary cortical neurons obtained from PRG-1−/− animals either transfected with a prg-1wt or with a prg-1R346T expressing construct (Fig 2E and F) and measured internalization of LPA which was tagged by a fluorescence label (TopFluor, TF-LPA) in a live imaging mode. TF-LPA internalization capacity of either PRG-1wt or PRG-1R346T was compared to PRG-1-deficient neurons in the same experiment (see also Fig 1G for baseline levels). To exclude bias by the transfection procedure, we assessed the TF-LPA uptake capacity of GFP-transfected PRG-1−/− neurons finding no difference to the non-transfected neurons (Fig EV1). While expression of PRG-1wt in neurons induced a significant increase in TF-LPA internalization, PRG-1R346T expression failed to do so (Fig 2H and I). To rule out a dominant-negat
Referência(s)