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Dysregulated expression of death, stress and mitochondrion related genes in the sciatic nerve of presymptomatic SOD1(G93A) mouse model of Amyotrophic Lateral Sclerosis.

Alves CJ, Maximino JR, Chadi G - Front Cell Neurosci (2015)

Bottom Line: Despite the involvement of Schwann cells in early neuromuscular disruption in ALS, detailed molecular events of a dying-back triggering are unknown.DAVID demonstrated the deregulated genes related to death, stress and mitochondrion, which allowed the identification of Cell cycle, ErbB signaling, Tryptophan metabolism and Rig-I-like receptor signaling as the most representative KEGG pathways.The protein-protein interaction networks based upon deregulated genes have identified the top hubs (TRAF2, H2AFX, E2F1, FOXO3, MSH2, NGFR, TGFBR1) and bottlenecks (TRAF2, E2F1, CDKN1B, TWIST1, FOXO3).

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Neuroregeneration Center, University of São Paulo School of Medicine São Paulo, Brazil.

ABSTRACT
Schwann cells are the main source of paracrine support to motor neurons. Oxidative stress and mitochondrial dysfunction have been correlated to motor neuron death in Amyotrophic Lateral Sclerosis (ALS). Despite the involvement of Schwann cells in early neuromuscular disruption in ALS, detailed molecular events of a dying-back triggering are unknown. Sciatic nerves of presymptomatic (60-day-old) SOD1(G93A) mice were submitted to a high-density oligonucleotide microarray analysis. DAVID demonstrated the deregulated genes related to death, stress and mitochondrion, which allowed the identification of Cell cycle, ErbB signaling, Tryptophan metabolism and Rig-I-like receptor signaling as the most representative KEGG pathways. The protein-protein interaction networks based upon deregulated genes have identified the top hubs (TRAF2, H2AFX, E2F1, FOXO3, MSH2, NGFR, TGFBR1) and bottlenecks (TRAF2, E2F1, CDKN1B, TWIST1, FOXO3). Schwann cells were enriched from the sciatic nerve of presymptomatic mice using flow cytometry cell sorting. qPCR showed the up regulated (Ngfr, Cdnkn1b, E2f1, Traf2 and Erbb3, H2afx, Cdkn1a, Hspa1, Prdx, Mapk10) and down-regulated (Foxo3, Mtor) genes in the enriched Schwann cells. In conclusion, molecular analyses in the presymptomatic sciatic nerve demonstrated the involvement of death, oxidative stress, and mitochondrial pathways in the Schwann cell non-autonomous mechanisms in the early stages of ALS.

No MeSH data available.


Related in: MedlinePlus

Flow cytometry cell sorting (A–D) and PCR analysis of Schwann cell and fibroblast markers (E) from sciatic nerve of 60-day-old presymptomatic SOD1G93A and the wild-type controls. FITC-conjugated p75NGF Receptor and PE-Cy5-conjugated Thy1 antibodies were employed in the two-color immunolabeling of Schwann cells and fibroblasts, respectively (A,B). Dot plots indicate the total number of events in the sciatic nerve cell suspension, and the dots inside the box represent the excluded doublet and dead profiles, which have been eliminated by morphological criteria, according to previous descriptions (Herzenberg et al., 2006) (A). After morphological criteria, dot plots of Schwann cell and fibroblast profiles (B) were obtained using respective fluorescence filters and the blots inside the boxes represent the specific profiles after discounting the unspecific labeling. Positive p75NGF Receptor and Thy1 cell profiles are shown in corresponding boxes after FITC vs. PE-Cy5 fluorescence intensity plotting (B). Specific profiles-based on morphological criteria were further analyzed in relation to fluorescence criteria and the specific p75NGF Receptor positive Schwann cells profiles were identified in wild-type (median of FITC = 1142; C), and in SOD1G93A mice (median of FITC = 1305; D). Representative bands of PCRs for specific gene markers of Schwann cells (S100), fibroblasts (Thy1), and actin b (Actb) were searched in Schwann cells and fibroblasts enriched samples obtained by flow cytometry sorting of SOD1G93A mice. Mouse whole sciatic nerve sample was used as a positive control (E).
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Figure 1: Flow cytometry cell sorting (A–D) and PCR analysis of Schwann cell and fibroblast markers (E) from sciatic nerve of 60-day-old presymptomatic SOD1G93A and the wild-type controls. FITC-conjugated p75NGF Receptor and PE-Cy5-conjugated Thy1 antibodies were employed in the two-color immunolabeling of Schwann cells and fibroblasts, respectively (A,B). Dot plots indicate the total number of events in the sciatic nerve cell suspension, and the dots inside the box represent the excluded doublet and dead profiles, which have been eliminated by morphological criteria, according to previous descriptions (Herzenberg et al., 2006) (A). After morphological criteria, dot plots of Schwann cell and fibroblast profiles (B) were obtained using respective fluorescence filters and the blots inside the boxes represent the specific profiles after discounting the unspecific labeling. Positive p75NGF Receptor and Thy1 cell profiles are shown in corresponding boxes after FITC vs. PE-Cy5 fluorescence intensity plotting (B). Specific profiles-based on morphological criteria were further analyzed in relation to fluorescence criteria and the specific p75NGF Receptor positive Schwann cells profiles were identified in wild-type (median of FITC = 1142; C), and in SOD1G93A mice (median of FITC = 1305; D). Representative bands of PCRs for specific gene markers of Schwann cells (S100), fibroblasts (Thy1), and actin b (Actb) were searched in Schwann cells and fibroblasts enriched samples obtained by flow cytometry sorting of SOD1G93A mice. Mouse whole sciatic nerve sample was used as a positive control (E).

Mentions: Schwann cells and fibroblasts were isolated by means of flow cytometry sorting from sciatic nerve explants of presymptomatic SOD1G93A mice and their age-paired wild-type controls as described in our previous publication (Maximino et al., 2014). The sciatic nerve-derived cell suspension was submitted to a double immunolabeling to identify Schwann cells and fibroblasts by means of a fluorescein isothiocyanate (FITC)-conjugated mouse p75NGF Receptor antibody (Abcam, USA) and a fluorescein phychoerythrin (PE-Cy5)-conjugated monoclonal antibody against Thy-1 (Abcam, USA), respectively. The p75NGF Receptor labeling was employed in the cell sorting experiments because it is a well-characterized surface marker for Schwann cells (Niapour et al., 2010). Cells were then analyzed for type and specificity as well as separated on a FACSAria III Cell Sorter (BD Biosciences, USA). A maximum of 106 cells were ressuspended in 500 μl of buffer. Flow cytometry dot plot Schwann cell and fibroblast profiles are shown in Figures 1A–D. Of note, the flow cytometry sorting Schwann cells of ALS mice did not show morphological differences (cell size and cytoplasmic granules) compared to control mice (Figures 1C,D). Also, the Schwann cells of ALS mice expressed high levels of mutant human SOD1, while no signal was seen in the Schwann cells of wild-type mice, as evidenced by PCR (Figure 1E).


Dysregulated expression of death, stress and mitochondrion related genes in the sciatic nerve of presymptomatic SOD1(G93A) mouse model of Amyotrophic Lateral Sclerosis.

Alves CJ, Maximino JR, Chadi G - Front Cell Neurosci (2015)

Flow cytometry cell sorting (A–D) and PCR analysis of Schwann cell and fibroblast markers (E) from sciatic nerve of 60-day-old presymptomatic SOD1G93A and the wild-type controls. FITC-conjugated p75NGF Receptor and PE-Cy5-conjugated Thy1 antibodies were employed in the two-color immunolabeling of Schwann cells and fibroblasts, respectively (A,B). Dot plots indicate the total number of events in the sciatic nerve cell suspension, and the dots inside the box represent the excluded doublet and dead profiles, which have been eliminated by morphological criteria, according to previous descriptions (Herzenberg et al., 2006) (A). After morphological criteria, dot plots of Schwann cell and fibroblast profiles (B) were obtained using respective fluorescence filters and the blots inside the boxes represent the specific profiles after discounting the unspecific labeling. Positive p75NGF Receptor and Thy1 cell profiles are shown in corresponding boxes after FITC vs. PE-Cy5 fluorescence intensity plotting (B). Specific profiles-based on morphological criteria were further analyzed in relation to fluorescence criteria and the specific p75NGF Receptor positive Schwann cells profiles were identified in wild-type (median of FITC = 1142; C), and in SOD1G93A mice (median of FITC = 1305; D). Representative bands of PCRs for specific gene markers of Schwann cells (S100), fibroblasts (Thy1), and actin b (Actb) were searched in Schwann cells and fibroblasts enriched samples obtained by flow cytometry sorting of SOD1G93A mice. Mouse whole sciatic nerve sample was used as a positive control (E).
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Related In: Results  -  Collection

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Figure 1: Flow cytometry cell sorting (A–D) and PCR analysis of Schwann cell and fibroblast markers (E) from sciatic nerve of 60-day-old presymptomatic SOD1G93A and the wild-type controls. FITC-conjugated p75NGF Receptor and PE-Cy5-conjugated Thy1 antibodies were employed in the two-color immunolabeling of Schwann cells and fibroblasts, respectively (A,B). Dot plots indicate the total number of events in the sciatic nerve cell suspension, and the dots inside the box represent the excluded doublet and dead profiles, which have been eliminated by morphological criteria, according to previous descriptions (Herzenberg et al., 2006) (A). After morphological criteria, dot plots of Schwann cell and fibroblast profiles (B) were obtained using respective fluorescence filters and the blots inside the boxes represent the specific profiles after discounting the unspecific labeling. Positive p75NGF Receptor and Thy1 cell profiles are shown in corresponding boxes after FITC vs. PE-Cy5 fluorescence intensity plotting (B). Specific profiles-based on morphological criteria were further analyzed in relation to fluorescence criteria and the specific p75NGF Receptor positive Schwann cells profiles were identified in wild-type (median of FITC = 1142; C), and in SOD1G93A mice (median of FITC = 1305; D). Representative bands of PCRs for specific gene markers of Schwann cells (S100), fibroblasts (Thy1), and actin b (Actb) were searched in Schwann cells and fibroblasts enriched samples obtained by flow cytometry sorting of SOD1G93A mice. Mouse whole sciatic nerve sample was used as a positive control (E).
Mentions: Schwann cells and fibroblasts were isolated by means of flow cytometry sorting from sciatic nerve explants of presymptomatic SOD1G93A mice and their age-paired wild-type controls as described in our previous publication (Maximino et al., 2014). The sciatic nerve-derived cell suspension was submitted to a double immunolabeling to identify Schwann cells and fibroblasts by means of a fluorescein isothiocyanate (FITC)-conjugated mouse p75NGF Receptor antibody (Abcam, USA) and a fluorescein phychoerythrin (PE-Cy5)-conjugated monoclonal antibody against Thy-1 (Abcam, USA), respectively. The p75NGF Receptor labeling was employed in the cell sorting experiments because it is a well-characterized surface marker for Schwann cells (Niapour et al., 2010). Cells were then analyzed for type and specificity as well as separated on a FACSAria III Cell Sorter (BD Biosciences, USA). A maximum of 106 cells were ressuspended in 500 μl of buffer. Flow cytometry dot plot Schwann cell and fibroblast profiles are shown in Figures 1A–D. Of note, the flow cytometry sorting Schwann cells of ALS mice did not show morphological differences (cell size and cytoplasmic granules) compared to control mice (Figures 1C,D). Also, the Schwann cells of ALS mice expressed high levels of mutant human SOD1, while no signal was seen in the Schwann cells of wild-type mice, as evidenced by PCR (Figure 1E).

Bottom Line: Despite the involvement of Schwann cells in early neuromuscular disruption in ALS, detailed molecular events of a dying-back triggering are unknown.DAVID demonstrated the deregulated genes related to death, stress and mitochondrion, which allowed the identification of Cell cycle, ErbB signaling, Tryptophan metabolism and Rig-I-like receptor signaling as the most representative KEGG pathways.The protein-protein interaction networks based upon deregulated genes have identified the top hubs (TRAF2, H2AFX, E2F1, FOXO3, MSH2, NGFR, TGFBR1) and bottlenecks (TRAF2, E2F1, CDKN1B, TWIST1, FOXO3).

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Neuroregeneration Center, University of São Paulo School of Medicine São Paulo, Brazil.

ABSTRACT
Schwann cells are the main source of paracrine support to motor neurons. Oxidative stress and mitochondrial dysfunction have been correlated to motor neuron death in Amyotrophic Lateral Sclerosis (ALS). Despite the involvement of Schwann cells in early neuromuscular disruption in ALS, detailed molecular events of a dying-back triggering are unknown. Sciatic nerves of presymptomatic (60-day-old) SOD1(G93A) mice were submitted to a high-density oligonucleotide microarray analysis. DAVID demonstrated the deregulated genes related to death, stress and mitochondrion, which allowed the identification of Cell cycle, ErbB signaling, Tryptophan metabolism and Rig-I-like receptor signaling as the most representative KEGG pathways. The protein-protein interaction networks based upon deregulated genes have identified the top hubs (TRAF2, H2AFX, E2F1, FOXO3, MSH2, NGFR, TGFBR1) and bottlenecks (TRAF2, E2F1, CDKN1B, TWIST1, FOXO3). Schwann cells were enriched from the sciatic nerve of presymptomatic mice using flow cytometry cell sorting. qPCR showed the up regulated (Ngfr, Cdnkn1b, E2f1, Traf2 and Erbb3, H2afx, Cdkn1a, Hspa1, Prdx, Mapk10) and down-regulated (Foxo3, Mtor) genes in the enriched Schwann cells. In conclusion, molecular analyses in the presymptomatic sciatic nerve demonstrated the involvement of death, oxidative stress, and mitochondrial pathways in the Schwann cell non-autonomous mechanisms in the early stages of ALS.

No MeSH data available.


Related in: MedlinePlus