Search of brain-enriched proteins in salivary extracellular vesicles for their use as mental disease biomarkers: A pilot study of the neuronal glycoprotein M6a

Background: Mental disorders affect millions of people worldwide. Their etiology is complex and the fact that the main effects occur in the brain hampers biochemical diagnosis. Therefore, biomarker finding in peripheral fluids such as serum or saliva is desirable. Here, we searched for biomarkers in salivary extracellular vesicles (EVs). Then, we focused on the protein M6a, related to neuronal connectivity and associated with several mood disorders to study its usefulness in saliva for the diagnosis of depression and anxiety. Methods: Biomarker candidates were searched by proteomic analysis of human salivary EVs. M6a presence in salivary EVs was validated by transmission electron microscopy and Western blot. M6a levels were measured by ELISA in saliva samples of 88 individuals classified as control, depressed or anxious. Results: We identified ten protein candidates in salivary EVs: OLIG2, PMP2, CNP, CAMK2A, SLC25A22, MLLT11, HTR2A, MAPT, ATP2B2 and M6a, all associated with emotional disorders. Salivary M6a levels positively correlated with the scores for the perceived stress scale in individuals diagnosed with depression. Furthermore, saliva samples of depressed patients treated with serotonin reuptake inhibitors (SSRI) or benzodiazepines differed in their M6a levels with respect to untreated patients. Limitations: The main limitation of this study lies in the low number of patients involved, which warrants replication. Conclusions: Salivary EVs contain promising biomarker candidates for mental disorders. Further studies will help validate them for their potential use in diagnosis. Our results lead us to propose M6a as a workable molecule to take into account as a possible stress biomarker.Fil: Monteleone, Melisa Carolina. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Billi, Silvia Cristina. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Viale, Luciano. Gobierno de la Provincia de Buenos Aires. Hospital Interzonal Especializado de Agudos y Cronicos San Juan de Dios.; Argentina. Universidad Nacional de San Martín; ArgentinaFil: Catoira, Natalia P.. Gobierno de la Provincia de Buenos Aires. Hospital Interzonal Especializado de Agudos y Cronicos San Juan de Dios.; ArgentinaFil: Frasch, Alberto Carlos C.. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Brocco, Marcela Adriana. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

ER-Bound Protein Tyrosine Phosphatase PTP1B Interacts with Src at the Plasma Membrane/Substrate Interface

PTP1B is an endoplasmic reticulum (ER) anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC) in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM) associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC). Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research

Neural glycoprotein M6a is released in extracellular vesicles and modulated by chronic stressors in blood

Abstract Membrane neuronal glycoprotein M6a is highly expressed in the brain and contributes to neural plasticity promoting neurite growth and spine and synapse formation. We have previously showed that chronic stressors alter hippocampal M6a mRNA levels in rodents and tree shrews. We now show that M6a glycoprotein can be detected in mouse blood. M6a is a transmembrane glycoprotein and, as such, unlikely to be free in blood. Here we demonstrate that, in blood, M6a is transported in extracellular vesicles (EVs). It is also shown that M6a-containing EVs are delivered from cultured primary neurons as well as from M6a-transfected COS-7 cells. Released EVs containing M6a can be incorporated into COS-7 cells changing its phenotype through formation of membrane protrusions. Thus, M6a-containing EVs might contribute to maintain cellular plasticity. M6a presence in blood was used to monitor stress effects. Chronic restraint stress modulated M6a protein level in a sex dependent manner. Analysis of individual animals indicated that M6a level variations depend on the stressor applied. The response to stressors in blood makes M6a amenable to further studies in the stress disorder field

Prenatal stress changes the glycoprotein GPM6A gene expression and induces epigenetic changes in rat offspring brain

Prenatal stress (PS) exerts strong impact on fetal brain development and on adult offspring brain functions. Previous work demonstrated that chronic stress alters the mRNA expression of GPM6A, a neuronal glycoprotein involved in filopodium extension. In this work, we analyzed the effect of PS on gpm6a expression and the epigenetic mechanisms involved. Pregnant Wistar rats received restraint stress during the last week of gestation. Male offspring were sacrificed on postnatal days 28 and 60. Hippocampus and prefrontal cortex samples were analyzed for gene expression (qPCR for mRNAs and microRNAs), methylation status (bisulfite conversion) and protein levels. Hippocampal neurons in culture were used to analyze microRNA overexpression effects. Prenatal stress induced changes in gpm6a levels in both tissues and at both ages analyzed, indicating a persistent effect. Two CpG islands in the gpm6a gene were identified. Variations in the methylation pattern at three specific CpGs were found in hippocampus, but not in PFC samples from PS offspring. microRNAs predicted to target gpm6a were identified in silico. qPCR measurements showed that PS modified the expression of several microRNAs in both tissues, being microRNA-133b the most significantly altered. Further studies overexpressing this microRNA in neuronal cultures showed a reduction in gmp6a mRNA and protein level. Moreover filopodium density was also reduced, suggesting that GPM6A function was affected. Gestational stress affected gpm6a gene expression in offspring likely through changes in methylation status and in posttranscriptional regulation by microRNAs. Thus, our findings propose gpm6a as a novel target for epigenetic regulation during prenatal stress.Fil: Monteleone, Melisa Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Adrover, Ezequiela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Fisico-Química Biológicas; ArgentinaFil: Pallares, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Fisico-Química Biológicas; ArgentinaFil: Antonelli, Marta Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Fisico-Química Biológicas; ArgentinaFil: Frasch, Alberto Carlos C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Brocco, Marcela Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentin

Expression and distribution of BiFC constructs.

<p>(A) Diagram of fusion proteins. YN (residues 1–154) and YC (residues 155–238) fragments of enhanced YFP were fused to the N-terminus of wild type (WT) and substrate trap mutant D181A (DA) PTP1B. The same fragments were fused at the C-terminus of mouse Src and Fyn. The amino acids of the linker region are indicated in italics. (B) Constructs were transiently expressed in CHO-K1 cells and probed in Western blots with anti-PTP1B (left panel), anti-Src (middle panel), and anti-Fyn (right panel) antibodies. YC and YN fragments of YFP add ∼10 and 18 kDa, respectively, to the partner fused proteins (PTP1B, Src and Fyn). Arrowheads indicate the migration of the endogenous proteins. Anti-PTP1B does not recognize the endogenous CHO-K1 protein; thus, a cell extract from PTP1B knockout cells reconstituted with human PTP1B was probed with this antibody and shown in the lane marked as PTP1BWT. Subcellular distribution of constructs used for BiFC was assessed by fluorescence microscopy. CHO-K1 cells expressing YC-PTP1BWT (C, C′, D), YC-PTP1BDA (E, E′, F), and SYF cells expressing Fyn-YN (G, H), and Src-YN (I, J) were immunolabeled with anti-PTP1B (C, Ć, E, É), anti-Fyn (G) and anti-Src (I) followed by secondary antibodies conjugated with Alexa Fluor 568 nm. Images on the red channel show that YC-PTP1BWT (C, Ć) and YC-PTP1BDA (E, É) localize in the ER, as expected (Ćand É are magnifications of regions within boxes in C and E, respectively). In addition, YCPTP1BDA accumulates in small puncta (arrows in É). Fyn-YN (G) and Src-YN (I) are enriched at the cell margin and in a perinuclear compartment. All constructs display background fluorescence at the green channel in which BiFC is analyzed (D, F, H, J). (K-N) Starved SYF cells expressing Src-YN were plated for 30 min in the absence (K, L) and in the presence (M, N) of serum. Note that Src-YN localizes in a perinuclear compartment in the absence of serum (L) and redistributes to peripheral, radial focal adhesions in the presence of serum (N), as expected. (K, M) Surface reflectance interference contrast images showing the membrane in contact with the substrate. Dashed lines indicate the perimeter of cells. Scale bar, 40 µm.</p

Distribution of the BiFC signal.

<p>CHO-K1 cells were co-transfected with BiFC pairs and analyzed by fluorescence microscopy. (A-C) Representative BiFC distribution of YC-PTP1BWT/Src-YN is shown. Most cells show the BiFC signal as bright fluorescence puncta associated with a network pattern of lower fluorescence intensity (A, magnifications in B and C). (D–G) Representative BiFC distribution of YC-PTP1BDA/Src-YN is shown. Note that BiFC is exclusively seen as bright puncta (magnifications in F, G), sometimes more dense in the perinuclear region (arrow in D). Scale bar in A: 25 µm. Magnifications in B, C, F, and G are at 200% of the original images (E) Image taken under surface reflection interference contrast. (H–J) Representative CHO-K1 cell co-transfected with the YC-PTP1BWT/Src-YN pair and then fixed and processed for immunofluorescence detection of calnexin, using Alexa Fluor 568-conjugated secondary antibodies. (H) Calnexin labeling, (I) BiFC, (J) merge of both channels. (K) Cytofluorogram showing the high correlation between red/green pixels corresponding to the calnexin and BiFC images, respectively. Arbitrary units (a.u.) represent grey level values from 12-bit images. Pearson’s correlation coefficient close to 1 reveals positive correlation. Manders’ coefficients M1 and M2 estimate the amount of co-localizing signal from the calnexin image to the BiFC image and viceversa, respectively. Both M1 and M2 coefficients are close to 100% indicating an almost perfect co-localization. Dashed lines indicate the perimeter of cells.</p

BiFC in ventral membranes.

<p>CHO-K1 cells were co-transfected with YC-PTP1B and either Src-YN (A–D) or SrcT-YN (E–H). Membrane preparations were obtained by sonication of the cells previously exposed to hypotonic conditions. After fixation with paraformaldehyde, Src-YN (A) and YC-PTP1B (E) were detected by specific primary antibodies and Alexa Fluor 568-conjugated secondary antibodies. The BiFC signal was displayed in puncta that tightly colocalized with the Src-YN staining (C). In contrast, BiFC signal was undetectable when using SrcT-YN (F, G). SRIC analysis showed dark/light patterns of the ventral membrane in contact with the substrate (D, H). Dashed lines indicate the perimeter of cells. Scale bar: 25 µm.</p

Schematic view of BiFC results.

<p>ER membranes (green) are positioned close to the plasma membrane (brown) by microtubules (black). PTP1B anchored to the cytosolic surface of the ER membrane (green pins) can interact with Src (red pins) associated to the cytosolic side of the plasma membrane. The substrate trap mutant PTP1BDA enhances the interaction leading to the visualization of large puncta (inset, and green beads on the ER in the upper/lower view). TIRF microscopy reveals BiFC puncta at the ventral membrane and SRIC shows that some of BiFC puncta occur in close contact with the substrate (SRIC dark spots).</p

BiFC analysis by TIRFM.

<p>CHO-K1 cells were co-transfected with YC-PTP1BWT/Src-YN and then fixed and processed for immunofluorescence detection of Src (A) and PTP1B (D) by wide field (wf). The BiFC signal of the same cells was observed by wide field (B, E) and under TIRF illumination (C, F). Yellow boxes in D and F were magnified in G and H. Note that BiFC puncta visualized under TIRF illumination (H) overlap with the ER network seen with anti-PTP1B (G). Also note that some puncta (white arrows in H and insets) display a comet-like appearance with a grading intensity of fluorescence brightest at the tip, suggesting a “dipping down” toward the substrate and into the region of exponentially increasing excitation of the evanescent field. Several BiFC puncta (yellow arrowheads) coincide with dark spots visualized by SRIC (I). Scale bar in A, 25 µm; scale bar in I, 2.5 µm.</p