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Syntaxin-1a and SNAP-25 expression level is increased in the blood samples of ischemic stroke patients

Cappelletti, Pamela, Filareti, Melania, Masuelli, Laura, Bei, Roberto, Hassanzadeh, Kambiz, Corbo, Massimo, Feligioni, Marco·2022·Scientific Reports
DOI 10.1038/s41598-022-18719-2PMC PMC9411545
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Abstract

The interest for the discovery of blood biomarkers for several neurological disorders, including Ischemic Stroke (IS), is growing and their identification in blood samples would be revolutionary allowing a fast and better pathology prediction or outcome and to collect information on patient recovery. The increased permeability of the blood–brain barrier, following a brain infarct, allows the detection of brain proteins in the blood flow. In this work, we analyzed the expression levels of two synaptic proteins Syntaxin (STX)-1a and Synaptosomal Associated Protein, 25 kDa (SNAP-25), in Peripheral Blood Mononuclear Cell (PBMC), serum and in Neuronal Derived Extracellular vesicles (NDEs) of IS patients, age and sex matched healthy control (HC) and younger HC (Y-HC). Interestingly, we identified STX-1a protein in the cytoplasm of PBMC and both STX-1a and SNAP-25 expression levels were significantly augmented in all IS patient’s blood fractions compared to control subjects. In addition, STX-1a blood levels correlated with the IS clinical scales National Institutes of Health Stroke Scale (NIH-SS) and the modified Barthel Index (BI). These results prompted us to speculate that STX-1a and SNAP-25 hematic fluctuations depict the brain damage after an ischemic attack and that their hematic detection could represent a novel and accessible IS biomarkers.

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Figure 1 Detection of STX-1a and SNAP-25 in peripheral blood of healthy donors. (a) WB analysis of the SNARE proteins in blood components. PBMCs, serum (SE), plasma (PL) and mouse brain cortex lysate (CTX, loaded as positive control) have been immunostained with STX-1a and SNAP-25 antibodies. STX-1a was detected in all blood components, but only in PBMCs at the predicted M.W., while SNAP-25 was observed in serum and plasma but not in blood cells. (b) Immunofluorescence analysis of STX-1a and SNAP-25 in PBMCs. Fluorescence analysis of PBMCs confirms the presence of STX-1a but not that of SNAP-25 (red channels). Nuclei have been labeled with the marker DAPI (blue channels). Original magnification: 60×. Bars correspond to 10 µm. (c) WB analysis of STX-1a and SNAP-25 in serum and plasma depleted from IgG and albumin. After the stripping of IgG and albumin from serum and plasma (as shown by Coomassie Blue staining) both STX-1a (at the shifted M.W) and SNAP-25 immune-recognition signals persist. For WB analysis 100 µg of blood components, 0.5 µg of mouse brain cortex lysate and 10 µL of flow through have been loaded in each lane. Uncropped WB and coomassie staining in (a) and (c) have been reported in Fig. S4 online.
Figure 1 Detection of STX-1a and SNAP-25 in peripheral blood of healthy donors. (a) WB analysis of the SNARE proteins in blood components. PBMCs, serum (SE), plasma (PL) and mouse brain cortex lysate (CTX, loaded as positive control) have been immunostained with STX-1a and SNAP-25 antibodies. STX-1a was detected in all blood components, but only in PBMCs at the predicted M.W., while SNAP-25 was observed in serum and plasma but not in blood cells. (b) Immunofluorescence analysis of STX-1a and SNAP-25 in PBMCs. Fluorescence analysis of PBMCs confirms the presence of STX-1a but not that of SNAP-25 (red channels). Nuclei have been labeled with the marker DAPI (blue channels). Original magnification: 60×. Bars correspond to 10 µm. (c) WB analysis of STX-1a and SNAP-25 in serum and plasma depleted from IgG and albumin. After the stripping of IgG and albumin from serum and plasma (as shown by Coomassie Blue staining) both STX-1a (at the shifted M.W) and SNAP-25 immune-recognition signals persist. For WB analysis 100 µg of blood components, 0.5 µg of mouse brain cortex lysate and 10 µL of flow through have been loaded in each lane. Uncropped WB and coomassie staining in (a) and (c) have been reported in Fig. S4 online.CC BY · PubMed Central
Figure 2 Analysis of the subcellular and cellular localization of STX-1a in PBMCs of healthy donors. (a) IF analysis of the cellular distribution of STX-1a in PBMCs. PBMCs have been immunostained with the cytoplasmic marker β-actin (upper panel) or the Golgi apparatus marker GM130 (lower panel) (green channels) and STX-1a (red channels). In the merge images, a clear colocalization of STX-1a with β-actin is evident thus indicating a cytoplasmic localization of the SNARE protein in PBMCs. A partial colocalization of STX-1a and GM130 is also shown. Original magnification: 60×. Bars correspond to 10 µm. (b) Colocalization analysis. Histogram showing the percentage of colocalized area compared to the total fluorescent area. (c) Pearson’s Coefficient. Histogram showing Pearson’s Coefficient. Green points and histograms represents the % of colocalization area (b) and the Pearson’s Coefficient (c) of STX-1a compared to β-actin, orange points and histograms represents the % of colocalization area (b) and the Pearson’s Coefficient (c) of STX-1a compared to GM130. (d) PBMC’s subcellular fractions analysis. WB analysis confirmed the cytoplasmic localization of STX-1a in PBMCs. The successful separation of the nuclear (Nc) and the cytoplasmic fractions (Cyto) of PBMCs is demonstrated by the presence of the nuclear marker lamin A/C only in the nuclear fraction and that of β-actin only in the cytoplasmic one. (e) STX-1a is expressed in both major PBMCs subtypes in cultures. WB analysis reveals that STX-1a is expressed in both monocytes (M) and lymphocytes (L) and that its expression is maintained over time in cultured cells. For IF analysis at least 15 cells for each subject (n = 5) have been analyzed. For WB analysis 4 µL of PBMCs subcellular fractions (i.e. Nc and Cyto), 100 µg of whole PBMCs, 0.5 µg of mouse brain cortex lysate and 2 × 105 of cultured PBMCs have been loaded in each lane. Bars represent the median value ± SD. Uncropped WB in (d) and (e) have been reported in Fig. S5 online.
Figure 2 Analysis of the subcellular and cellular localization of STX-1a in PBMCs of healthy donors. (a) IF analysis of the cellular distribution of STX-1a in PBMCs. PBMCs have been immunostained with the cytoplasmic marker β-actin (upper panel) or the Golgi apparatus marker GM130 (lower panel) (green channels) and STX-1a (red channels). In the merge images, a clear colocalization of STX-1a with β-actin is evident thus indicating a cytoplasmic localization of the SNARE protein in PBMCs. A partial colocalization of STX-1a and GM130 is also shown. Original magnification: 60×. Bars correspond to 10 µm. (b) Colocalization analysis. Histogram showing the percentage of colocalized area compared to the total fluorescent area. (c) Pearson’s Coefficient. Histogram showing Pearson’s Coefficient. Green points and histograms represents the % of colocalization area (b) and the Pearson’s Coefficient (c) of STX-1a compared to β-actin, orange points and histograms represents the % of colocalization area (b) and the Pearson’s Coefficient (c) of STX-1a compared to GM130. (d) PBMC’s subcellular fractions analysis. WB analysis confirmed the cytoplasmic localization of STX-1a in PBMCs. The successful separation of the nuclear (Nc) and the cytoplasmic fractions (Cyto) of PBMCs is demonstrated by the presence of the nuclear marker lamin A/C only in the nuclear fraction and that of β-actin only in the cytoplasmic one. (e) STX-1a is expressed in both major PBMCs subtypes in cultures. WB analysis reveals that STX-1a is expressed in both monocytes (M) and lymphocytes (L) and that its expression is maintained over time in cultured cells. For IF analysis at least 15 cells for each subject (n = 5) have been analyzed. For WB analysis 4 µL of PBMCs subcellular fractions (i.e. Nc and Cyto), 100 µg of whole PBMCs, 0.5 µg of mouse brain cortex lysate and 2 × 105 of cultured PBMCs have been loaded in each lane. Bars represent the median value ± SD. Uncropped WB in (d) and (e) have been reported in Fig. S5 online.CC BY · PubMed Central
Figure 3 WB analysis of a representative NDE purification from the serum of healthy donors. (a) Quality analysis of extracellular vesicle preparation. Extracellular vesicles samples are not contaminated by Golgi apparatus (GM130), endoplasmic reticulum (Erp57), lysosomes (CD107) and nucleus (lamin A/C). The presence of the HDL marker APO A1 is detectable in both total extracellular vesicles (T) and total extracellular vesicles depleted to NDE (T-N), but disappear in NDEs fractions (N). CD9 is a common exosome marker. (b) NDE characterization. WB analysis show an enrichment of neuronal markers (NSE and SV-2a) in NDEs with respect to the other two extracellular vesicles fractions. Moreover, NDE samples are negative to the astrocytes and the post synaptic markers GFAP and PSD95, respectively. (c) STX-1a and SNAP-25 are enriched in NDE fractions. The levels of both SNARE proteins are increased in NDE with respect to the two other extracellular vesicles fractions. (d) Representative EM analysis of NDE. The ultrastructural analysis of NDE shows the presence of rounded-shaped vesicles with a diameter range of 70–100 nm. Bars correspond to 50 nm. For WB analysis 100 µg of whole PBMCs, 50 µg of serum (SE), extracellular vesicles fractions (T, N, T-N) and 0.5 µg of mouse brain cortex lysate have been loaded in each lane. Uncropped WB in (a)–(c) have been reported in Fig. S6 online.
Figure 3 WB analysis of a representative NDE purification from the serum of healthy donors. (a) Quality analysis of extracellular vesicle preparation. Extracellular vesicles samples are not contaminated by Golgi apparatus (GM130), endoplasmic reticulum (Erp57), lysosomes (CD107) and nucleus (lamin A/C). The presence of the HDL marker APO A1 is detectable in both total extracellular vesicles (T) and total extracellular vesicles depleted to NDE (T-N), but disappear in NDEs fractions (N). CD9 is a common exosome marker. (b) NDE characterization. WB analysis show an enrichment of neuronal markers (NSE and SV-2a) in NDEs with respect to the other two extracellular vesicles fractions. Moreover, NDE samples are negative to the astrocytes and the post synaptic markers GFAP and PSD95, respectively. (c) STX-1a and SNAP-25 are enriched in NDE fractions. The levels of both SNARE proteins are increased in NDE with respect to the two other extracellular vesicles fractions. (d) Representative EM analysis of NDE. The ultrastructural analysis of NDE shows the presence of rounded-shaped vesicles with a diameter range of 70–100 nm. Bars correspond to 50 nm. For WB analysis 100 µg of whole PBMCs, 50 µg of serum (SE), extracellular vesicles fractions (T, N, T-N) and 0.5 µg of mouse brain cortex lysate have been loaded in each lane. Uncropped WB in (a)–(c) have been reported in Fig. S6 online.CC BY · PubMed Central
Figure 4 Quantitative analysis of STX-1a in PBMCs and of SNAP-25 in sera in all population analyzed. (a, e) Representative WB analysis of STX-1a in PBMCs (a) and of SNAP-25 in serum (e) in IS and HC subjects. PBMCs are immunostained for STX-1a (a) and serum for SNAP-25 (e). For each sample, the STX-1a densitometric value is normalized to that of β-actin, while the SNAP-25 densitometric values is normalized to that of total blood proteins content revealed by Coomassie Blue staining. (b) Densitometric analysis STX-1a expression levels in PBMCs. STX-1a expression levels have been analyzed by using One-way ANOVA with post-hoc Tukey test: STX-1a significantly increase in PBMCs of IS patients with respect to HC and Y-HC subjects (p ≤ 0.0001), while no differences are observed between control groups (p = 0.97). (c, d) Pearson’s correlation analysis between expression of STX-1a in IS PBMC and the clinical stroke scales. STX-1a levels in PBMCs of IS patients present a moderate negative correlation with NIH-SS (p = 0.03) (c) and a moderate positive correlation with BI (d) (p = 0.03). (f) Densitometric analysis SNAP-25 expression levels in sera. SNAP-25 expression levels have been analyzed by using One-way ANOVA with post-hoc Tukey test: SNAP-25 levels are significantly increased in serum of IS patients with respect to HC and Y-HC subjects (p ≤ 0.0001). A significant increase of SNAP-25 expression is also observed in HC with respect to Y-HC subjects (p ≤ 0.0001). (g) WB analysis of SNAP-25 in sera depleted of IgG and albumin. The increased SNAP-25 levels in IS patient are not influenced by the presence of serum proteins. For WB analysis 100 µg of whole PBMCs and of serum (SE) have been loaded in each lane. In quantitative graphs (panels b and f) each point in a frame depicts the value for a single subject (n = 30 IS patients, 30 HC subjects and 15 young HC subjects) while bars represent the median value ± SD. ***p ≤ 0.001. In panels (b) and (f) light blue points and histograms correspond to Y-HC subjects, blue points and histograms correspond to HC subjects, red points and histograms correspond to IS patients. Uncropped WB and coomassie staining in (a), (e) and (g) have been reported in Fig. S7 online.
Figure 4 Quantitative analysis of STX-1a in PBMCs and of SNAP-25 in sera in all population analyzed. (a, e) Representative WB analysis of STX-1a in PBMCs (a) and of SNAP-25 in serum (e) in IS and HC subjects. PBMCs are immunostained for STX-1a (a) and serum for SNAP-25 (e). For each sample, the STX-1a densitometric value is normalized to that of β-actin, while the SNAP-25 densitometric values is normalized to that of total blood proteins content revealed by Coomassie Blue staining. (b) Densitometric analysis STX-1a expression levels in PBMCs. STX-1a expression levels have been analyzed by using One-way ANOVA with post-hoc Tukey test: STX-1a significantly increase in PBMCs of IS patients with respect to HC and Y-HC subjects (p ≤ 0.0001), while no differences are observed between control groups (p = 0.97). (c, d) Pearson’s correlation analysis between expression of STX-1a in IS PBMC and the clinical stroke scales. STX-1a levels in PBMCs of IS patients present a moderate negative correlation with NIH-SS (p = 0.03) (c) and a moderate positive correlation with BI (d) (p = 0.03). (f) Densitometric analysis SNAP-25 expression levels in sera. SNAP-25 expression levels have been analyzed by using One-way ANOVA with post-hoc Tukey test: SNAP-25 levels are significantly increased in serum of IS patients with respect to HC and Y-HC subjects (p ≤ 0.0001). A significant increase of SNAP-25 expression is also observed in HC with respect to Y-HC subjects (p ≤ 0.0001). (g) WB analysis of SNAP-25 in sera depleted of IgG and albumin. The increased SNAP-25 levels in IS patient are not influenced by the presence of serum proteins. For WB analysis 100 µg of whole PBMCs and of serum (SE) have been loaded in each lane. In quantitative graphs (panels b and f) each point in a frame depicts the value for a single subject (n = 30 IS patients, 30 HC subjects and 15 young HC subjects) while bars represent the median value ± SD. ***p ≤ 0.001. In panels (b) and (f) light blue points and histograms correspond to Y-HC subjects, blue points and histograms correspond to HC subjects, red points and histograms correspond to IS patients. Uncropped WB and coomassie staining in (a), (e) and (g) have been reported in Fig. S7 online.CC BY · PubMed Central
Figure 5 Confirmation of increased STX-1a levels in PBMCs of IS patients with respect to HC subjects. (a) Representative WB of STX-1a expression levels in PBMC cytoplasmic fractions. Representative WB analysis of cytoplasmic and nuclear fractions of PBMCs. For each sample, the STX-1a densitometric values have been normalized to that of β-actin. The successful separation of the nuclear (Nc) and the cytoplasmic fractions (Cyto) of PBMCs is demonstrated by the presence of the nuclear marker lamin A/C in the nuclear fraction and that of β-actin in the cytoplasmic one. (b) Quantitative analysis of cytoplasmic STX-1a. STX-1a expression levels have been analyzed by using One-way ANOVA with post-hoc Tukey test: STX-1a significantly increase in PBMCs of IS patients with respect to HC (p ≤ 0.0001) and Y-HC (p ≤ 0.003) subjects, while no difference is observed between the two control groups (p = 0.7). (c) Representative Immunofluorescence analysis of STX-1a in PBMCs of IS patients and HC subjects. PBMCs have been labeled with β-actin (green) and STX-1a (red). The STX-1a fluorescence intensity has been normalized by dividing the measure of its intensity by that of the β-actin subtracted from that measured for the nucleus (DAPI immunostaining). (d) Quantitative analysis STX-1a levels in PBMCs. The normalized STX-1a expression levels have been analyzed by using One-way ANOVA with post-hoc Tukey test: STX-1a levels are significantly increased in PBMCs of IS patients with respect to HC subjects (p = 0.005). Original magnification: 60X. Bars correspond to 10 µm. For WB analysis 4 µL of each fraction (Nc and Cyto) and 100 µg of whole PBMCs have been loaded in each lane. For IF analysis at least 15 cells for each subject have been analyzed. In quantitative graphs (panels b and d) each point in a frame depicts the value for a single subject (n = 5–10 IS patients, 5–10 HC subjects and 0–5 young HC subjects, respectively in (b) and (d) while bars represent the median value ± SD. **p ≤ 0.005; ***p ≤ 0.001. Uncropped WB in a) have been reported in Fig. S8 online.
Figure 5 Confirmation of increased STX-1a levels in PBMCs of IS patients with respect to HC subjects. (a) Representative WB of STX-1a expression levels in PBMC cytoplasmic fractions. Representative WB analysis of cytoplasmic and nuclear fractions of PBMCs. For each sample, the STX-1a densitometric values have been normalized to that of β-actin. The successful separation of the nuclear (Nc) and the cytoplasmic fractions (Cyto) of PBMCs is demonstrated by the presence of the nuclear marker lamin A/C in the nuclear fraction and that of β-actin in the cytoplasmic one. (b) Quantitative analysis of cytoplasmic STX-1a. STX-1a expression levels have been analyzed by using One-way ANOVA with post-hoc Tukey test: STX-1a significantly increase in PBMCs of IS patients with respect to HC (p ≤ 0.0001) and Y-HC (p ≤ 0.003) subjects, while no difference is observed between the two control groups (p = 0.7). (c) Representative Immunofluorescence analysis of STX-1a in PBMCs of IS patients and HC subjects. PBMCs have been labeled with β-actin (green) and STX-1a (red). The STX-1a fluorescence intensity has been normalized by dividing the measure of its intensity by that of the β-actin subtracted from that measured for the nucleus (DAPI immunostaining). (d) Quantitative analysis STX-1a levels in PBMCs. The normalized STX-1a expression levels have been analyzed by using One-way ANOVA with post-hoc Tukey test: STX-1a levels are significantly increased in PBMCs of IS patients with respect to HC subjects (p = 0.005). Original magnification: 60X. Bars correspond to 10 µm. For WB analysis 4 µL of each fraction (Nc and Cyto) and 100 µg of whole PBMCs have been loaded in each lane. For IF analysis at least 15 cells for each subject have been analyzed. In quantitative graphs (panels b and d) each point in a frame depicts the value for a single subject (n = 5–10 IS patients, 5–10 HC subjects and 0–5 young HC subjects, respectively in (b) and (d) while bars represent the median value ± SD. **p ≤ 0.005; ***p ≤ 0.001. Uncropped WB in a) have been reported in Fig. S8 online.CC BY · PubMed Central
Figure 6 Analysis of SNARE in extracellular vesicles of IS and HC subjects. (a, c, f) Representative WB analysis of the expression levels of STX-1a and of SNAP-25 in extracellular vesicles derived from sera. Representative WB analysis of total (T) extracellular vesicles (a), NDE (N) (c) and total extracellular vesicles depleted of NDE (T-N) (f) fractions. Each sample has been tested against CD9 (total exosomes marker), NSE (NDE marker), STX-1a and SNAP-25. The SNAP-25 densitometric values in total extracellular vesicles fraction have been normalized to that of CD9 (a), while the STX-1a and SNAP-25 densitometric values in NDE have been normalized to that of NSE (c). (b, d, e) Densitometric analysis of STX-1a and SNAP-25 in extracellular vesicles The normalized SNAP-25 levels in T extracellular vesicles (b) and those of both STX-1a (d) and SNAP-25 (e) in NDE have been analyzed by using One-way ANOVA with post-hoc Tukey test. Both STX-1a and SNAP-25 expression levels are significantly increased in NDE of IS patients with respect to HC subjects (p ≤ 0.0001 and p ≤ 0.02 for STX-1a (d) and SNAP-25 (e) respectively), while no differences is observed for SNAP-25 in the T fraction (p = 0.91) (b). (g, h) Pearson’s correlation analysis between expression of STX-1a in IS NDEs and the clinical stroke. STX-1a levels in NDE of IS patients present a moderate positive correlation with NIH-SS (p = 0.003) (g) and a moderate negative correlation with BI (p = 0.02) (h). For WB analysis 50 µg of each extracellular vesicles fraction and 0.5 µg of mouse brain cortex lysate have been loaded in each lane. In quantitative graphs (panels b, d and e) each point in a frame depicts the value for a single subject (n = 10 IS patients and 10 HC subjects) while bars represent the median value ± SD. *p ≤ 0.05; ***p ≤ 0.001. In panels (b), (d) and (e) blue points and histograms correspond to HC subjects, red points and histograms correspond to IS patients. Uncropped WB in (a), (c) and (f) have been reported in Fig. S9 online.
Figure 6 Analysis of SNARE in extracellular vesicles of IS and HC subjects. (a, c, f) Representative WB analysis of the expression levels of STX-1a and of SNAP-25 in extracellular vesicles derived from sera. Representative WB analysis of total (T) extracellular vesicles (a), NDE (N) (c) and total extracellular vesicles depleted of NDE (T-N) (f) fractions. Each sample has been tested against CD9 (total exosomes marker), NSE (NDE marker), STX-1a and SNAP-25. The SNAP-25 densitometric values in total extracellular vesicles fraction have been normalized to that of CD9 (a), while the STX-1a and SNAP-25 densitometric values in NDE have been normalized to that of NSE (c). (b, d, e) Densitometric analysis of STX-1a and SNAP-25 in extracellular vesicles The normalized SNAP-25 levels in T extracellular vesicles (b) and those of both STX-1a (d) and SNAP-25 (e) in NDE have been analyzed by using One-way ANOVA with post-hoc Tukey test. Both STX-1a and SNAP-25 expression levels are significantly increased in NDE of IS patients with respect to HC subjects (p ≤ 0.0001 and p ≤ 0.02 for STX-1a (d) and SNAP-25 (e) respectively), while no differences is observed for SNAP-25 in the T fraction (p = 0.91) (b). (g, h) Pearson’s correlation analysis between expression of STX-1a in IS NDEs and the clinical stroke. STX-1a levels in NDE of IS patients present a moderate positive correlation with NIH-SS (p = 0.003) (g) and a moderate negative correlation with BI (p = 0.02) (h). For WB analysis 50 µg of each extracellular vesicles fraction and 0.5 µg of mouse brain cortex lysate have been loaded in each lane. In quantitative graphs (panels b, d and e) each point in a frame depicts the value for a single subject (n = 10 IS patients and 10 HC subjects) while bars represent the median value ± SD. *p ≤ 0.05; ***p ≤ 0.001. In panels (b), (d) and (e) blue points and histograms correspond to HC subjects, red points and histograms correspond to IS patients. Uncropped WB in (a), (c) and (f) have been reported in Fig. S9 online.CC BY · PubMed Central

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Metadata

Authors
Cappelletti, P., Filareti, M., Masuelli, L., Bei, R., Hassanzadeh, K., Corbo, M., & Feligioni, M.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2022-08-25
Volume
12
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-022-18719-2
PMC
PMC9411545
Cited by
12
References
4
Language
en
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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

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