Fig 1.
The Repeated Interrupted Stress (RIS) protocol induces changes in body weight, behavior and hippocampal M6a levels.
One day after the end of RIS, stressed (RIS, n = 10) and non-stressed (C, n = 10) mice were tested. A. RIS induced a significant reduction in body weight gain, *p<0.01. B. RIS significantly increased the time spent immobile in the forced swim test (FST), *p<0.05. C. RIS also caused a significant reduction in the expression levels of the hippocampal glycoprotein M6a (p<0.01). Each lane represents one individual. Equal amounts of proteins were loaded per lane. Dashed line indicates where the image was cropped. An empty naïvee (E) was left between control and RIS animals. Full scans can be seen in S1 Fig. Prior to antibody hybridization membranes were cut at 40kDa using the Molecular Weight marker as reference. Membranes were treated with antibodies anti-M6a and with anti- tubulin for normalization purposes. Data are presented as mean ± SD, for statistical analysis a two-tailed t-Student test was used.
Fig 2.
RIS leads to divergent individual stress responses.
Body weight gain, FST and M6a levels were evaluated in a new cohort of animals. A. RIS induced a general reduction in body weight gain (****: p<0.0001). B. RIS increased the time spent immobile in the FST (****: p<0.0001). C. RIS also reduced M6a hippocampal levels (*: p<0.05). However, a great interindividual variability was observed. D. The M6a/Tubulin level vs. time spent immobile was plotted to classify animals. Dotted lines indicate cut-off values and were calculated as the mean value plus two standard deviations (time spent immobile) or minus (M6a/Tubulin) two standard deviations. All control animals that exhibited low time spent immobile and high M6a levels hence are located in the Q2 quadrant. Animals in the Q4 quadrant displayed low levels of M6a and high time spent immobile, and were considered stressed. Animals that were subjected to RIS but displayed high levels of M6a (Q3) were classified as less sensitive to stress.
Fig 3.
Qualitative analysis Venn diagram showing proteins detected in EVs-C, EVs-RIS or EVs-Q3.
Before A. and after B. exclusion of keratins and immunoglobulin chains. Here, the following 10 proteins were exclusively detected in EVs-C: DESP: Desmoplak in, F13B: Coagulation factor XIII B chain, H2A2A/2B/2C: histone 2A type A, B and C, PLAK: Junction plakoglobin, 1433Z: 14-3-3 zeta/delta protein, FBLN3: EGF-containing fibulin-like extracellular matrix protein 1, UBB: Ubiquitin B, G3P Glyceraldehyde-3-phosphate dehydrogenase. Seven proteins were exclusively detected in EVs-RIS: ACTBL: Beta-actin-like protein 2, FA5 and FA10: coagulation factors V and X, PSA6: Proteasome subunit alpha type-6, B3AT: Band 3 anion transport protein also known as Solute carrier family 4 (anion exchanger) member 1 (SLC4A1), APOC3: Apolipoprotein C-III, RET4: Retinol-binding protein 4 and two proteins were exclusively detected in EVs-Q3: CES1D: Carboxylesterase 1D and PSA7 proteasome subunit alpha type-7.C. Proteomic analysis of extracellular vesicles (EVs) shows that many (21/36) of the candidate proteins are expressed in the brain. Venn analysis of the differentially expressed proteins between the experimental conditions (Candidates, identified after quantitative proteomic approach (LFQ-analysis) detailed in supplementary file sheet 8), the proteome of mouse brain extracellular vesicles (Brain EVs) and mouse brain proteins (Brain, http://www.mousebrainproteome.com/).
Fig 4.
M6a selectively expressed in neurons can be detected in serum.
A. Representative image of brain (cerebral cortex) slices from postnatal day 5 pups electroporated in-utero Anti-GFAP and anti-NeuN antibodies were used as astrocyte and neuronal markers respectively. M6a tagged with green fluorescent protein (GFP) under the T-alpha promoter was electroporated in-utero to directly target neurons. Immunofluorescence detection of glial fibrillary acid protein (GFAP, red), GFP (green) and the neuronal marker NeuN (gray) in coronal brain slices indicated that cells positive for M6a-GFP signal did not overlap with GFAP positive cells and had neuronal morphology (see magnifications). B. Image shown in A rotated to show the molecular cortical layer to dorsal. C. Representative image of brain (cerebral cortex) slices from postnatal day 5 non electroporated pups. Immunofluorescence detection of glial fibrillary acid protein (GFAP, red), neuronal marker NeuN (gray) and GFP (green). No GFP signal could be detected D. Western blot analysis of EVs isolated from serum of a non-electroporated control rat and 5 adult rats electroporated in-utero with the construct. Anti-GFP antibody was used to detect both GFP (~ 27 kDa) and M6a-GFP (~ 70 kDa). As seen, the recombinant protein was detected in the EVs isolated from the serum of all electroporated animals but not in the control. Full scan can be seen in S5 Fig.
Fig 5.
Intranasal administration of bulk EVs isolated from the serum of RIS mice induces behavioral and molecular changes like those observed in stressed animals.
Twenty-four hours after EV administration, mice were evaluated with the forced swim test (FST). A. Time spent immobile increased in animals that received EVs derived from RIS animals (EVs-RIS) compared with animals that received PBS, *p<0.05.n = 5/group, ANOVA and by Holm-Sidak’s multiple comparisons test. One week after EV administration body weight gain B, and C FST performance were evaluated. No differences were found among groups. D. Western blot and the corresponding densitometric quantification of changes in the content of M6a in hippocampal homogenates one week after EV administration. Each lane represents one individual, n = 5/group. Dashed line indicates where the image was cropped. Prior to antibody hybridization membranes were cut at 40kDa using the Molecular Weight marker as reference. Full scan can be seen in S6 Fig. Equal amounts of proteins were loaded per lane. Tubulin levels were used to normalize protein load. M6a levels were significantly reduced in animals that received EVs-RIS animals compared to those who received EVs-C or PBS (n = 4-5/group, ANOVA followed by Holm-Sidak’s multiple comparisons test *p < 0.05).