Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 is required for PARP1-dependent necrosis after H2O2 treatment. (a) Left panel: WT MEFs were transfected with the vector control (VC) or Flag-FAF1 plasmids. At 36 h after transfection, cells were pretreated with vehicle (DMSO), zVAD-fmk (100 μM) or DPQ (30 μM) for 1 h and were then treated with 500 μM H2O2 for 6 h in presence of individual compounds. Cell death was determined on the basis of LDH release (n=3). Right panel: representative immunoblots show the levels of FAF1, Flag and β-actin expression. (b) Immunoblot analysis of FAF1 expression in immortalized Faf1+/+ and Faf1gt/gt MEFs. β-actin expression was used as an endogenous control. (c) Faf1+/+ and Faf1gt/gt MEFs were treated with the indicated concentrations of H2O2 for 6 h. Cell death was determined on the basis of LDH release (n=3). (d) Faf1+/+ and Faf1gt/gt MEFs were treated with 500 μM H2O2 for the indicated times. Cell death was determined on the basis of LDH release (n=3). (e) Left panel: Faf1gt/gt MEFs were transfected with the VC or Flag-FAF1 plasmids. Thirty-six hours after transfection, the cells were treated with 500 μM H2O2 for 6 h. Cell death was determined on the basis of LDH release (n=3). Right panel: representative immunoblots show the levels of FAF1, Flag and β-actin expression. Data (a, c–e) are expressed as the mean±S.E.M. from three independent experiments. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD (a, c–e) post hoc analysis. ***P<0.001, **P<0.01 and *P<0.05

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Expressing

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 translocates to the nucleus and interacts with PARP1 during oxidative stress. (a) WT MEFs were treated with 500 μM H2O2 for the indicated times and were then fractionated into cytoplasmic and nuclear fractions. The fractions were analyzed by immunoblotting with anti-FAF1, anti-PARP1 (nuclear marker) and anti-β-actin (cytoplasmic marker) antibodies. (b) WT MEFs were treated with 500 μM H2O2 for the indicated times and were then immunostained with the anti-FAF1 antibody. The nuclei were stained using propidium iodide (PI) and analyzed by confocal microscopy. (c) WT MEFs were transfected with the indicated combination of V5-PARP1 and Flag-FAF1 plasmids. At 36 h after transfection, the cells were untreated or treated with 500 μM H2O2 for 30 min. The cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with the indicated antibodies. (d) WT MEFs were treated with 500 μM H2O2 for the indicated times. The cell lysates then were immunoprecipitated with the anti-FAF1 antibody. The interactions were determined by immunoblotting with the indicated antibodies

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Western Blot, Marker, Staining, Confocal Microscopy, Transfection, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 promotes the catalytic activity of PARP1 during oxidative stress. (a) Left panel: WT MEFs were transfected with the indicated concentration of Flag-FAF1 plasmids. At 36 h after transfection, the cells were treated with 500 μM H2O2 for 30 min. The cell lysates were immunoblotted with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblots (n=3). (b) Left panel: WT MEFs were transfected with the VC or Flag-FAF1 plasmids. At 36 h after transfection, the cells were treated with 500 μM H2O2 for the indicated times. The cell lysates were immunoblotted with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblots (n=3). (c) Left panel: Faf1+/+ and Faf1gt/gt MEFs were treated with 500 μM H2O2 for the indicated times. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblots (n=3). (d) Faf1+/+ and Faf1gt/gt MEFs were treated with 500 μM H2O2 for 30 min and were then immunostained with the anti-PAR antibody. The nuclei were stained using PI and the cells were analyzed by confocal microscopy. (e) Left panel: Faf1gt/gt MEFs were transfected with the VC or Flag-FAF1 plasmids. Thirty-six hours after transfection, the cells were treated with 500 μM H2O2 for the indicated times. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblot (n=3). (f) Left panel: GST-FAF1 or GST was incubated with recombinant PARP1 (1 unit), β-NAD+ (100 μM) and damaged DNA for 10 min at room temperature. After the in vitro poly(ADP-ribosyl)ation reactions, the samples were subjected to immunoblot analysis. Right panel: the graph shows the results of densitometric analysis of PAR immunoblots (n=3). Quantified data (a–c, e, f) are expressed as the mean±S.E.M. from three independent experiments. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD (a–c, e, f) post hoc analysis. **P<0.01 and *P<0.05

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Activity Assay, Transfection, Concentration Assay, Western Blot, Staining, Confocal Microscopy, Incubation, Recombinant, In Vitro

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1-mediated PARP1 activation induces energy collapse, mitochondrial depolarization and AIF translocation during oxidative stress. (a and b) Faf1+/+ and Faf1gt/gt MEFs were pretreated with vehicle (DMSO) or DPQ (30 μM) for 1 h and then treated with 500 μM H2O2 for 1 h. Depletion of intracellular energy was determined by measuring the levels of NAD+ (a; n=3) and ATP (b; n=3). (c) Faf1+/+ and Faf1gt/gt MEFs were pretreated with vehicle (DMSO) or DPQ (30 μM) for 1 h and then treated with 500 μM H2O2 for 4 h. The cells were analyzed for mitochondrial membrane depolarization with a Muse analyzer (n=3). (d) Faf1+/+ and Faf1gt/gt MEFs were treated with 500 μM H2O2 for 4 h and subsequently immunostained with the anti-AIF antibody. The nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI) and the cells were analyzed by confocal microscopy. Data (a–c) are expressed as the mean±S.E.M. from three independent experiments. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD post hoc analysis. ***P<0.001, **P<0.01 and *P<0.05

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Activation Assay, Translocation Assay, Membrane, Staining, Confocal Microscopy

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 mediates PARP1-dependent necrosis in SH-SY5Y cells during oxidative stress. (a) Left panel: SH-SY5Y cells were transfected with the VC or FAF1 plasmids. Thirty-six hours after transfection, the cells were pretreated with vehicle (DMSO) or DPQ (30 μM) for 1 h and then treated with 500 μM H2O2 for 6 h. Cell death was determined on the basis of LDH release (n=3). Right panel: representative immunoblots show the levels of FAF1 and β-actin expression. (b) Left panel: SH-SY5Y cells were transfected with scRNA or FAF1 siRNA. At 48 h after transfection, the cells were treated with 500 μM H2O2 for 6 h. Cell death was determined on the basis of LDH release (n=3). Right panel: representative immunoblots show the levels of FAF1 and β-actin expression. (c) SH-SY5Y cells were treated with 500 μM H2O2 for the indicated times and were then fractionated into cytoplasmic and nuclear fractions. The fractions were analyzed by immunoblot analysis with anti-FAF1, anti-PARP1 (nuclear marker) and anti-β-actin (cytosolic marker) antibodies. (d) SH-SY5Y cells were treated with 500 μM H2O2 for the indicated times and then the cell lysates were immunoprecipitated with the anti-FAF1 antibody followed by immunoblotting with the indicated antibodies. (e) Left panel: SH-SY5Y cells were transfected with the VC or Flag-FAF1 plasmids. At 36 h after transfection, the cells were treated with 500 μM H2O2 for the indicated times. The cell lysates were immunoblotted with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblot (n=3). Quantified data (a, b, e) are expressed as the mean±S.E.M. from three independent experiments. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD post hoc analysis. ***P<0.001, **P<0.01 and *P<0.05

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Transfection, Western Blot, Expressing, Marker, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 promotes dopaminergic neuronal cell death via PARP1 activation in an MPTP mouse model of PD. (a and b) The mice were administered four intraperitoneal injections of MPTP-HCl or saline at 2 h intervals. The mice were killed 24 h after the last injection. (a) Left panel: the subcellular localization of FAF1 was measured by immunofluorescence staining in TH-positive neurons of substantia nigra of mice that were treated with saline or MPTP. The nuclei were stained using DAPI and the tissues were analyzed by confocal microscopy. The white arrows indicate the presence of nuclear FAF1 in TH-positive neurons after MPTP treatment. Right panel: enlarged images of TH-positive neurons were taken from the white box in the merged images. (b) Brain lysates were prepared from the ventral midbrain of saline and MPTP-treated mice and were then subjected to immunoprecipitation with the anti-FAF1 antibody followed by immunoblotting with the indicated antibodies (n=3 per group). (c) The experimental scheme for panel d–f. The mice were injected unilaterally into the substantia nigra (SN) with adeno-associated virus type 1 (AAV1)-FAF1. Two weeks after AAV1-FAF1 injection, the mice were administered MPTP-HCl or saline, at 2 h intervals. The mice were killed 1 day and 7 days after the last MPTP injection and their brain tissues were prepared for western blot (WB) analysis or immunohistochemistry. (d) Left panel: at day 1 after the last MPTP injection, brain lysates were prepared from the ventral midbrain of the AAV1-FAF1-injected (inj.) or non-injected (non-inj.) side of saline- and MPTP-treated mice and were then subjected to immunoblot analysis using the indicated antibodies. Right panel: the graphs show the results of densitometric analysis of PAR and FAF1 immunoblots in left panel (n=3 per group). (e and g) At day 7 after the last MPTP injection, the dopaminergic neurodegeneration was measured by histological analysis for TH-positive (e) and Nissl-positive cells (g) in SN of the AAV1-FAF1 injected (inj.) or non-injected (non-inj.) side of saline- and MPTP-treated mice. Dashed lines represent a region of SN. (f) The graph shows the results of densitometric analysis of TH-stained neurons in e (n=5 per group). (h) The graph shows the counts of Nissl-stained cells in g (n=5 per group). Quantified data (d, f, h) are expressed as the mean±S.E.M. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD post hoc analysis. ***P<0.001, **P<0.01

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Activation Assay, Saline, Injection, Immunofluorescence, Staining, Confocal Microscopy, Immunoprecipitation, Western Blot, Virus, Immunohistochemistry

Journal: Journal of Pain Research

Article Title: Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain

doi: 10.2147/JPR.S441706

Figure Lengend Snippet: Infusion of virus expressing Nrxn3 shRNA reduced the number of Nrxn3α positive cells. The central amygdala of Long Evans rats was infused with AAV1 virus containing the construct AAV1-GFP-mNRXN3-shRNA or a scrambled shRNA. Four weeks after infusion the whisker pad was injected with MeWo cells without varicella zoster virus (no VZV) or MeWo cells containing varicella zoster virus. Six weeks after infusion the brain was isolated and the sections imaged. ( A ) is a low magnification image of a brain slice indicating green GFP fluorescence from expression of the virus construct in the central amygdala region (white oval). A high magnification image of the GFP positive cells (green) within the central amygdala is shown in ( B ). Bar = 20 micrometers. ( C ) shows a histogram for Nrxn3 expression within the central amygdala after infusion of AAV1 and injection of the whisker pad. Each point is from an individual animal. An asterisk indicates a significant difference of α=0.05.

Article Snippet: In a separate group of Long Evans rats, the central amygdala was infused with 1 µL of 1 × 10 13 TU/mL AAV1 containing a scrambled shRNA sequence (AAV1-GFP-U6-shRNA, Vector Biolabs) mixed 1 to 1 with the synaptophysin construct.

Techniques: Virus, Expressing, shRNA, Construct, Whisker Assay, Injection, Isolation, Slice Preparation, Fluorescence

Journal: Journal of Pain Research

Article Title: Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain

doi: 10.2147/JPR.S441706

Figure Lengend Snippet: Synaptophysin was expressed in GABAergic cells of the central amygdala. The central amygdala of Gad1-iCre Long Evans rats was infused with AAV1 containing pAAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby. During this same surgery the central amygdala was infused with the shRNA viral construct. Image is from a representative rat infused with control shRNA and injected with no VZV. ( A ) Six weeks after infusion mRuby fluorescent signal (red) was detected within the central amygdala (CeA). White dotted line shows the borders of the central amygdala. Arrow points to the injection site ( A and B ). Enlarged image of synaptophysin positive cell (red, arrow) within the central amygdala is shown in ( C ). Hoechst 33342 stain of the nuclei from the same cell (arrow) is shown in blue ( D ). Bar= 100 µm.

Article Snippet: In a separate group of Long Evans rats, the central amygdala was infused with 1 µL of 1 × 10 13 TU/mL AAV1 containing a scrambled shRNA sequence (AAV1-GFP-U6-shRNA, Vector Biolabs) mixed 1 to 1 with the synaptophysin construct.

Techniques: shRNA, Construct, Control, Injection, Staining

Journal: Journal of Pain Research

Article Title: Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain

doi: 10.2147/JPR.S441706

Figure Lengend Snippet: Synaptophysin positive terminals colocalize with excitable cells in the lateral parabrachial nucleus. Atlas image of coronal brain section from a rat with the lateral parabrachial region outlined with a black dotted line in ( A ). The central amygdala of Gad1-iCre Long Evans rats was infused with AAV1 containing pAAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby. Excitable cells within the lateral parabrachial nucleus were labeled by infusing the lateral parabrachial nucleus with AAV5 virus containing pAAV-CaMKIIa-EGFP. Six weeks after infusion EGFP positive cells (green, ( B ) were present within the lateral parabrachial region (white dotted line). In ( B ), a low magnification image is shown on the left and a higher magnification image is shown on the right. EGFP positive cells (green) are shown in ( C, F, I and L ) and synaptophysin (red) is shown in ( C (through ( N )). Cell nuclei are labeled blue with Hoechst 33342 stain in ( B–D, F, G, I, J, L and M ). ( F ) through ( N ) are images through the z plane of the lateral parabrachial tissue. ( F, I and L ) are three different z plane cross sections, respectively, through the cell in ( C ) (open arrow). ( F–H ) show one z plane slice through the cell, ( I–K ) show the second slice and ( L–N ) are the third slice through the cell. Synaptophysin labeled puncta on the CaMKII positive cell was indicated by small white arrows in ( I–N ). Bar = 50 µm in ( B ) and 5 µm in ( C ).

Article Snippet: In a separate group of Long Evans rats, the central amygdala was infused with 1 µL of 1 × 10 13 TU/mL AAV1 containing a scrambled shRNA sequence (AAV1-GFP-U6-shRNA, Vector Biolabs) mixed 1 to 1 with the synaptophysin construct.

Techniques: Labeling, Virus, Staining

Journal: Journal of Pain Research

Article Title: Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain

doi: 10.2147/JPR.S441706

Figure Lengend Snippet: Synaptophysin and CaMKII expression in the lateral parabrachial nucleus. In these rats the central amygdala was infused with virus expressing a scrambled shRNA or a Nrxn3 shRNA. The central amygdala of Gad1-iCre Long Evans rats was infused with AAV1 containing pAAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby. Excitable cells within the lateral parabrachial nucleus were labeled by infusing this nucleus with AAV5 virus containing pAAV-CaMKIIa-EGFP. After four weeks post-surgery the whisker pad of the infused rats were injected with MeWo cells without varicella zoster virus (no VZV) or MeWo cells containing VZV. Two weeks after injection the brain was isolated and imaged. ( A ) shows the average number of synaptophysin terminals or puncta localized to each CaMKII positive cell within the lateral parabrachial nucleus. Representative images of rats treated with control shRNA/no VZV ( B–E ) or control shRNA/VZV ( F–I ) or Nrxn3 shRNA/no VZV ( J–M ) or Nrxn3 shRNA/VZV ( N–Q ) are shown. Hoechst 33342 nuclear stain ( B, F, J and N ) and CaMKII stain ( C, G, K and O ) and synaptophysin stain ( D, H, L and P ) is represented in several cells . Individual CaMKII positive cells (green) are outlined with a white dotted line. Arrows point to synaptophysin positive puncta (red, D, H, L and P ) colocalizing with CaMKII staining ( E, I, M and Q ). Bar = 10 µm. Panel R shows the number of CaMKII positive cells in the lateral parabrachial nucleus that colocalized with synaptophysin. Each point is from an individual animal in panels A and R. An asterisk indicates a significant difference of α=0.05. Representative images of rats treated with control shRNA/no VZV ( S ) or control shRNA/VZV ( T ) or Nrxn3 shRNA/no VZV ( U ) or Nrxn3 shRNA/VZV ( V ) show cells with synaptophysin stain colocalizing with CaMKII stain (yellow, arrows). Bar = 50 µm.

Article Snippet: In a separate group of Long Evans rats, the central amygdala was infused with 1 µL of 1 × 10 13 TU/mL AAV1 containing a scrambled shRNA sequence (AAV1-GFP-U6-shRNA, Vector Biolabs) mixed 1 to 1 with the synaptophysin construct.

Techniques: Expressing, Virus, shRNA, Labeling, Whisker Assay, Injection, Isolation, Control, Staining

Journal: Journal of Pain Research

Article Title: Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain

doi: 10.2147/JPR.S441706

Figure Lengend Snippet: Prodynorphin cells within the lateral parabrachial nucleus. The central amygdala of Gad1-iCre Long Evans rats was infused with AAV1 containing pAAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby. Excitable cells within the lateral parabrachial nucleus were labeled by infusing this nucleus with AAV5 virus containing pAAV-CaMKIIa-EGFP. Four weeks after infusion the whisker pad was injected with either no VZV or VZV. Six weeks after infusion brain sections of the treated rats were immunostained for prodynorphin. In ( A and B ) multiple prodynorphin positive (red) cells were imaged in the lateral parabrachial nucleus (LPB). Prodynorphin is a marker for neurons involved in pain. Cell nuclei are labeled blue with Hoechst 33342 in ( A – C ). ( B ) shows only the prodynorphin cells (red) from the same region and ( C ) shows only the EGFP positive cells (green). A higher magnification image of the LPB is shown in ( D ) and cells that colocalize prodynorphin and EGFP are yellow. Insert in ( D ) is an image through the z plane of the lateral parabrachial nucleus after staining for prodynorphin. Prodynorphin is red, EGFP is green and synaptophysin terminals are in yellow for the insert image in ( D ). Images are from a representative rat that was treated with Nrxn3 shRNA and VZV. scp = superior cerebellar peduncle. Bar= 20 µm. The histogram in ( E ) shows the number of EGFP/prodynorphin positive cells that colocalized with synaptophysin in the lateral parabrachial nucleus after knockdown of Nrxn3α in the central amygdala. Each point is from an individual animal. An asterisk indicates a significant difference of α=0.05. Representative images of rats treated with control shRNA/no VZV ( F ) or control shRNA/VZV ( G ) or Nrxn3 shRNA/no VZV ( H ) or Nrxn3 shRNA/VZV ( I ) show cells with synaptophysin stain colocalizing with prodynorphin stain (yellow, arrows). Bar = 50 µm.

Article Snippet: In a separate group of Long Evans rats, the central amygdala was infused with 1 µL of 1 × 10 13 TU/mL AAV1 containing a scrambled shRNA sequence (AAV1-GFP-U6-shRNA, Vector Biolabs) mixed 1 to 1 with the synaptophysin construct.

Techniques: Labeling, Virus, Whisker Assay, Injection, Marker, Staining, shRNA, Knockdown, Control