Journal: Antioxidants & Redox Signaling

Article Title: Nitration and Glycation Turn Mature NGF into a Toxic Factor for Motor Neurons: A Role for p75 NTR and RAGE Signaling in ALS

doi: 10.1089/ars.2016.6966

Figure Lengend Snippet: Methylglyoxal-treated NGF induces p75NTR-dependent motor neuron death. (A) Motor neuron cultures maintained with GDNF (1 ng/mL) were treated with vehicle (control), NGF (100 ng/mL), glycated human albumin (HGA; 50 ng/mL), or the indicated concentrations of methylglyoxal-treated NGF (NGF-MG). NGF was treated with MG (2 μM), and residual MG was eliminated by ultrafiltration. To rule out direct toxic effects of potential residual MG, a mock sample lacking NGF was processed as mentioned and used as a control (ctl-MG). It had no effect on motor neuron survival. Motor neuron death induced by trophic factor deprivation (NONE) was used as a control of the maximal motor neuron death observed in this culture model. (B) Motor neuron death induced by NGF-MG (10 ng/mL) was prevented by the addition of the general caspase inhibitor Z-VAD-FMK (10 μM; Z-VAD) or p75NTR-blocking antibodies (a-p75; 1:1000). Nonimmune serum was devoid of effect (not shown). For (A and B), cultures were treated with NGF-MG 16 h after plating. p75NTR-blocking antibodies were added once, 30 min before NGF-MG treatment. Z-VAD-FMK was added every 24 h. Motor neuron survival was determined 48 h after treatment. Data are expressed as percentage of control (mean ± SEM). *Significantly different from control (p ≤ 0.05). GDNF, cell-derived neurotrophic factor; NGF-MG, methylglyoxal-treated NGF

Article Snippet: Blocking antibodies were rabbit polyclonal anti-NGF (#AB1526SP; EMD Millipore-Chemicon), goat polyclonal anti-RAGE antibody (AF1179; R&D Systems), and rabbit polyclonal anti-p75 NTR (#AB-N01AP; Advanced Targeting Systems).

Techniques: Control, Blocking Assay, Derivative Assay

Journal: Antioxidants & Redox Signaling

Article Title: Nitration and Glycation Turn Mature NGF into a Toxic Factor for Motor Neurons: A Role for p75 NTR and RAGE Signaling in ALS

doi: 10.1089/ars.2016.6966

Figure Lengend Snippet: NGF present in hSOD1G93A lumbar spinal cord extracts induces the death of cultured motor neurons by signaling through p75NTR and RAGE. (A) Western blot analysis of RAGE expression in spinal cord protein extracts from early symptomatic hSOD1G93A mice (G93A) or age-matched nontransgenic (non-Tg) littermates. Each lane corresponds to a different mouse. Lower panel: quantification of RAGE levels after correction by tubulin levels. Data are expressed as percentage of non-Tg (mean ± SEM). *Significantly different from non-Tg (p ≤ 0.05). (B) Primary motor neurons were isolated from the spinal cord of E12.5 hSOD1G93A embryos. Motor neuron cultures were maintained with GDNF (1 ng/mL). Cultures were treated 2 h after plating with vehicle (control) or spinal cord extracts (0.5 μg protein/mL) from early symptomatic hSOD1G93A mice (G93A) or aged-matched nontransgenic littermates (non-Tg). Blocking antibodies to NGF (a-NGF; 2 μg/mL), p75NTR (a-p75; 1:1000), or RAGE (a-RAGE; 15 μg/mL) were added 30 min before treatment. Neuronal survival was determined 48 h after treatment. Motor neuron death induced by hSOD1G93A spinal cord extracts was prevented by either NGF, p75NTR, or RAGE-blocking antibodies. Preimmune IgG had no effect (not shown). Data are expressed as percentage of control (mean ± SEM). *Significantly different from control (p ≤ 0.05).

Article Snippet: Blocking antibodies were rabbit polyclonal anti-NGF (#AB1526SP; EMD Millipore-Chemicon), goat polyclonal anti-RAGE antibody (AF1179; R&D Systems), and rabbit polyclonal anti-p75 NTR (#AB-N01AP; Advanced Targeting Systems).

Techniques: Cell Culture, Western Blot, Expressing, Isolation, Control, Blocking Assay

Journal: Antioxidants & Redox Signaling

Article Title: Nitration and Glycation Turn Mature NGF into a Toxic Factor for Motor Neurons: A Role for p75 NTR and RAGE Signaling in ALS

doi: 10.1089/ars.2016.6966

Figure Lengend Snippet: A role for NGF, p75NTR, and RAGE signaling in the motor neuron death induced by hSOD1G93A astrocytes. (A) Nontransgenic embryonic motor neurons were plated on top of nontransgenic (non-Tg) or hSOD1G93A (G93A) astrocyte monolayers. Blocking antibodies to NGF (a-NGF; 2 μg/mL), p75NTR (a-p75; 1:1000), or RAGE (a-RAGE; 15 μg/mL) were added 2 h after motor neuron plating. Neuronal survival was determined 72 h later. Motor neuron death induced by hSOD1G93A astrocytes was prevented by NGF, p75NTR, or RAGE-blocking antibodies. Preimmune IgG had no effect (not shown). Data are expressed as percentage of motor neuron survival on top of non-Tg control astrocytes (mean ± SEM). *Significantly different from non-Tg control (p ≤ 0.05). (B) Motor neurons isolated from the spinal cord of E12.5 nontransgenic (RAGE+/+) or RAGE knockout (RAGE−/−) embryos were plated on top of nontransgenic (non-Tg) or hSOD1G93A (G93A) astrocyte monolayers. Neuronal survival was determined 72 h later. On average, a similar number of motor neurons RAGE+/+ and RAGE−/− were counted on top of nontransgenic astrocytes. hSOD1G93A astrocytes induced the death of nontransgenic motor neurons but did not alter the survival of RAGE knockout motor neurons. For each neuronal genotype, data are expressed as percentage of motor neuron survival on top of non-Tg control astrocytes (mean ± SEM). *Significantly different from its respective non-Tg control (p ≤ 0.05). (C) Schematic representation of the main results and conclusions. As supported by previous literature, reactive astrocytes in the spinal cord of hSOD1G93A mice display increased production of NGF in the presence of oxidative and glycating stress. Overwhelming of antioxidant defenses leads to increased production and accumulation of peroxynitrite (ONOO−) and MG, which in turn, induce NGF nitration and glycation, respectively. Because MG and peroxynitrite can diffuse across cellular membranes, post-translational modification of NGF could occur intra- or extracellularly. Both nitration and glycation induce oligomerization and confer upon NGF the ability to signal through a RAGE–p75NTR signaling complex. Signaling through RAGE–p75NTR leads to motor neuron cell death by a mechanism involving p38MAPK activation, increased production of nitric oxide, and peroxynitrite formation. The possibility of independent p75NTR and RAGE signaling in response to post-translational modified NGF was not ruled out (dashed arrows).

Article Snippet: Blocking antibodies were rabbit polyclonal anti-NGF (#AB1526SP; EMD Millipore-Chemicon), goat polyclonal anti-RAGE antibody (AF1179; R&D Systems), and rabbit polyclonal anti-p75 NTR (#AB-N01AP; Advanced Targeting Systems).

Techniques: Blocking Assay, Control, Isolation, Knock-Out, Nitration, Modification, Activation Assay

Journal: Antioxidants & Redox Signaling

Article Title: Nitration and Glycation Turn Mature NGF into a Toxic Factor for Motor Neurons: A Role for p75 NTR and RAGE Signaling in ALS

doi: 10.1089/ars.2016.6966

Figure Lengend Snippet: RAGE and p75NTR interact at the cell surface. Cells were incubated for 30 min at room temperature with the membrane-impermeable reducible crosslinker DTSSP. After lysis, RAGE or p75NTR was immunoprecipitated. Precipitated complexes were analyzed by reducing SDS-PAGE and Western blot (WB) using antibodies against p75NTR, c-Myc, RAGE, GAPDH, or tubulin. (A) Immunoprecipitation (IP) in extracts from H4 neuroglioma cells co-overexpressing c-Myc-tagged RAGE and His-tagged p75NTR. Left: IP c-Myc-tag; WB p75NTR. Right: IP His tag; WB RAGE. (B and C) IP p75NTR; WB RAGE in primary cortical neurons (B) or NSC-34 cells (C). IP in two independent biological replicates is shown. For (A–C), IP specificity was confirmed using antibodies against GAPDH (A) or tubulin (B and C). These abundant proteins were not pulled down after immunoprecipitation and were detected only in the sample before IP (INPUT). Ab Ctl: antibody control, mock sample including the antibody in the absence of protein extract. This control was included to confirm that the RAGE immunoreactive band does not correspond to the unspecific detection of the immunoglobulin heavy chain, both of similar apparent molecular weight. Full-length images of the membranes are shown in Supplementary Figure S2. (D) Image of RAGE–p75NTR colocalization as determined by immunofluorescence using antibodies against the extracellular domain of p75NTR and RAGE. Nuclei were counterstained with DAPI. The right panel shows the distribution of fluorescence intensity of p75NTR (red) and RAGE (green) staining across the indicated path (red line). The distribution of peak fluorescence intensity corresponding to p75NTR and RAGE staining suggests spatial colocalization. Scale bar, 20 μm. (E) Images showing RAGE–p75NTR interaction in primary motor neurons by in situ PLA. Motor neuron cultures were stained with antibodies against the extracellular domain of p75NTR and RAGE (same antibodies as in D) and developed using DuoLink kit. Nuclei were counterstained with DAPI. The signal from each detected pair of PLA probes is seen as an individual red fluorescent spot. A representative image showing RAGE–p75NTR interaction in the soma and neurites of motor neurons is shown in (a) and (b). A representative image of the negative control omitting one primary antibody is shown in (c) and (d). Scale bar, 20 μm.

Article Snippet: The following primary antibodies were used for Western blot experiments: mouse monoclonal anti-β-Actin (clone AC-15; #A5441; Sigma-Aldrich); mouse monoclonal anti-AGE-4 (clone 14B5; #KAL-KG133; Cosmo Bio USA, Inc.); rabbit polyclonal anti-GAPDH (#ab9485; Abcam); mouse monoclonal anti-6-Histidine epitope tag (clone AD1.1.10; #NB100-64768; Novus Biologicals); mouse monoclonal anti-c-Myc (clone 9E10; #M4439; Sigma-Aldrich); rabbit polyclonal anti-NGF (#AB1526SP; EMD Millipore-Chemicon); rabbit polyclonal anti-NGF (#AN-240; Alomone Labs); rabbit monoclonal anti-p75 (clone EP1039Y; #NB110-58000 Novus Biologicals or #GTX61425; or GeneTex); rabbit polyclonal anti-proNGF (#ANT-005; Alomone Labs); rabbit polyclonal anti-RAGE (#R5278; Sigma-Aldrich); rabbit polyclonal anti-RAGE (#ab37647; Abcam); mouse monoclonal anti-α-Tubulin (clone DM1A; #3873; Cell Signaling Technology).

Techniques: Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Molecular Weight, Immunofluorescence, Fluorescence, Staining, In Situ, Negative Control

Journal: Antioxidants & Redox Signaling

Article Title: Nitration and Glycation Turn Mature NGF into a Toxic Factor for Motor Neurons: A Role for p75 NTR and RAGE Signaling in ALS

doi: 10.1089/ars.2016.6966

Figure Lengend Snippet: NGF present in hSOD1G93A lumbar spinal cord extracts induces the death of cultured motor neurons by signaling through p75NTR and RAGE. (A) Western blot analysis of RAGE expression in spinal cord protein extracts from early symptomatic hSOD1G93A mice (G93A) or age-matched nontransgenic (non-Tg) littermates. Each lane corresponds to a different mouse. Lower panel: quantification of RAGE levels after correction by tubulin levels. Data are expressed as percentage of non-Tg (mean ± SEM). *Significantly different from non-Tg (p ≤ 0.05). (B) Primary motor neurons were isolated from the spinal cord of E12.5 hSOD1G93A embryos. Motor neuron cultures were maintained with GDNF (1 ng/mL). Cultures were treated 2 h after plating with vehicle (control) or spinal cord extracts (0.5 μg protein/mL) from early symptomatic hSOD1G93A mice (G93A) or aged-matched nontransgenic littermates (non-Tg). Blocking antibodies to NGF (a-NGF; 2 μg/mL), p75NTR (a-p75; 1:1000), or RAGE (a-RAGE; 15 μg/mL) were added 30 min before treatment. Neuronal survival was determined 48 h after treatment. Motor neuron death induced by hSOD1G93A spinal cord extracts was prevented by either NGF, p75NTR, or RAGE-blocking antibodies. Preimmune IgG had no effect (not shown). Data are expressed as percentage of control (mean ± SEM). *Significantly different from control (p ≤ 0.05).

Article Snippet: The following primary antibodies were used for Western blot experiments: mouse monoclonal anti-β-Actin (clone AC-15; #A5441; Sigma-Aldrich); mouse monoclonal anti-AGE-4 (clone 14B5; #KAL-KG133; Cosmo Bio USA, Inc.); rabbit polyclonal anti-GAPDH (#ab9485; Abcam); mouse monoclonal anti-6-Histidine epitope tag (clone AD1.1.10; #NB100-64768; Novus Biologicals); mouse monoclonal anti-c-Myc (clone 9E10; #M4439; Sigma-Aldrich); rabbit polyclonal anti-NGF (#AB1526SP; EMD Millipore-Chemicon); rabbit polyclonal anti-NGF (#AN-240; Alomone Labs); rabbit monoclonal anti-p75 (clone EP1039Y; #NB110-58000 Novus Biologicals or #GTX61425; or GeneTex); rabbit polyclonal anti-proNGF (#ANT-005; Alomone Labs); rabbit polyclonal anti-RAGE (#R5278; Sigma-Aldrich); rabbit polyclonal anti-RAGE (#ab37647; Abcam); mouse monoclonal anti-α-Tubulin (clone DM1A; #3873; Cell Signaling Technology).

Techniques: Cell Culture, Western Blot, Expressing, Isolation, Blocking Assay

Journal: Antioxidants & Redox Signaling

Article Title: Nitration and Glycation Turn Mature NGF into a Toxic Factor for Motor Neurons: A Role for p75 NTR and RAGE Signaling in ALS

doi: 10.1089/ars.2016.6966

Figure Lengend Snippet: RAGE and p75NTR interact at the cell surface. Cells were incubated for 30 min at room temperature with the membrane-impermeable reducible crosslinker DTSSP. After lysis, RAGE or p75NTR was immunoprecipitated. Precipitated complexes were analyzed by reducing SDS-PAGE and Western blot (WB) using antibodies against p75NTR, c-Myc, RAGE, GAPDH, or tubulin. (A) Immunoprecipitation (IP) in extracts from H4 neuroglioma cells co-overexpressing c-Myc-tagged RAGE and His-tagged p75NTR. Left: IP c-Myc-tag; WB p75NTR. Right: IP His tag; WB RAGE. (B and C) IP p75NTR; WB RAGE in primary cortical neurons (B) or NSC-34 cells (C). IP in two independent biological replicates is shown. For (A–C), IP specificity was confirmed using antibodies against GAPDH (A) or tubulin (B and C). These abundant proteins were not pulled down after immunoprecipitation and were detected only in the sample before IP (INPUT). Ab Ctl: antibody control, mock sample including the antibody in the absence of protein extract. This control was included to confirm that the RAGE immunoreactive band does not correspond to the unspecific detection of the immunoglobulin heavy chain, both of similar apparent molecular weight. Full-length images of the membranes are shown in Supplementary Figure S2. (D) Image of RAGE–p75NTR colocalization as determined by immunofluorescence using antibodies against the extracellular domain of p75NTR and RAGE. Nuclei were counterstained with DAPI. The right panel shows the distribution of fluorescence intensity of p75NTR (red) and RAGE (green) staining across the indicated path (red line). The distribution of peak fluorescence intensity corresponding to p75NTR and RAGE staining suggests spatial colocalization. Scale bar, 20 μm. (E) Images showing RAGE–p75NTR interaction in primary motor neurons by in situ PLA. Motor neuron cultures were stained with antibodies against the extracellular domain of p75NTR and RAGE (same antibodies as in D) and developed using DuoLink kit. Nuclei were counterstained with DAPI. The signal from each detected pair of PLA probes is seen as an individual red fluorescent spot. A representative image showing RAGE–p75NTR interaction in the soma and neurites of motor neurons is shown in (a) and (b). A representative image of the negative control omitting one primary antibody is shown in (c) and (d). Scale bar, 20 μm.

Article Snippet: The following primary antibodies were used for Western blot experiments: mouse monoclonal anti-β-Actin (clone AC-15; #A5441; Sigma-Aldrich); mouse monoclonal anti-AGE-4 (clone 14B5; #KAL-KG133; Cosmo Bio USA, Inc.); rabbit polyclonal anti-GAPDH (#ab9485; Abcam); mouse monoclonal anti-6-Histidine epitope tag (clone AD1.1.10; #NB100-64768; Novus Biologicals); mouse monoclonal anti-c-Myc (clone 9E10; #M4439; Sigma-Aldrich); rabbit polyclonal anti-NGF (#AB1526SP; EMD Millipore-Chemicon); rabbit polyclonal anti-NGF (#AN-240; Alomone Labs); rabbit monoclonal anti-p75 (clone EP1039Y; #NB110-58000 Novus Biologicals or #GTX61425; or GeneTex); rabbit polyclonal anti-proNGF (#ANT-005; Alomone Labs); rabbit polyclonal anti-RAGE (#R5278; Sigma-Aldrich); rabbit polyclonal anti-RAGE (#ab37647; Abcam); mouse monoclonal anti-α-Tubulin (clone DM1A; #3873; Cell Signaling Technology).

Techniques: Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Molecular Weight, Immunofluorescence, Fluorescence, Staining, In Situ, Negative Control