Adaptation to G93A superoxide dismutase 1 in a motor neuron cell line model of amyotrophic lateral sclerosis. The role of glutathione

Motor neuron degeneration in amyotrophic lateral sclerosis involves oxidative damage. Glutathione (GSH) is critical as an antioxidant and a redox modulator. We used a motor neuronal cell line (NSC-34) to investigate whether wild-type and familial amyotrophic lateral sclerosis-linked G93A mutant Cu,Zn superoxide dismutase (wt ⁄G93ASOD1) modified the GSH pool and glutamate cysteine ligase (GCL), the rate-limiting enzyme for GSH synthesis. We studied the effect of various G93ASOD1 levels and exposure times. Mutant Cu,Zn superoxide dismutase induced an adaptive process involving the upregulation of GSH synthesis, even at very low expression levels. However, cells with a high level of G93ASOD1 cultured for 10 weeks showed GSH depletion and a decrease in expression of the modulatory subunit of GCL. These cells also had lower levels of GSH and GCL activity was not induced after treatment with the pro-oxidant tertbutylhydroquinone. Cells with a low level of G93ASOD1 maintained higher GSH levels and GCL activity, showing that the exposure time and the level of the mutant protein modulate GSH synthesis. We conclude that failure of the regulation of the GSH pathway caused by G93ASOD1 may contribute to motor neuron vulnerability and we identify this pathway as a target for therapeutic intervention

Characterization of Detergent-Insoluble Proteins in ALS Indicates a Causal Link between Nitrative Stress and Aggregation in Pathogenesis

BACKGROUND:Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease, and protein aggregation has been proposed as a possible pathogenetic mechanism. However, the aggregate protein constituents are poorly characterized so knowledge on the role of aggregation in pathogenesis is limited. METHODOLOGY/PRINCIPAL FINDINGS:We carried out a proteomic analysis of the protein composition of the insoluble fraction, as a model of protein aggregates, from familial ALS (fALS) mouse model at different disease stages. We identified several proteins enriched in the detergent-insoluble fraction already at a preclinical stage, including intermediate filaments, chaperones and mitochondrial proteins. Aconitase, HSC70 and cyclophilin A were also significantly enriched in the insoluble fraction of spinal cords of ALS patients. Moreover, we found that the majority of proteins in mice and HSP90 in patients were tyrosine-nitrated. We therefore investigated the role of nitrative stress in aggregate formation in fALS-like murine motor neuron-neuroblastoma (NSC-34) cell lines. By inhibiting nitric oxide synthesis the amount of insoluble proteins, particularly aconitase, HSC70, cyclophilin A and SOD1 can be substantially reduced. CONCLUSION/SIGNIFICANCE:Analysis of the insoluble fractions from cellular/mouse models and human tissues revealed novel aggregation-prone proteins and suggests that nitrative stress contribute to protein aggregate formation in ALS

Amyotrophic Lateral Sclerosis-Linked Mutant VAPB Inclusions Do Not Interfere with Protein Degradation Pathways or Intracellular Transport in a Cultured Cell Model

<div><p>VAPB is a ubiquitously expressed, ER-resident adaptor protein involved in interorganellar lipid exchange, membrane contact site formation, and membrane trafficking. Its mutant form, P56S-VAPB, which has been linked to a dominantly inherited form of Amyotrophic Lateral Sclerosis (ALS8), generates intracellular inclusions consisting in restructured ER domains whose role in ALS pathogenesis has not been elucidated. P56S-VAPB is less stable than the wild-type protein and, at variance with most pathological aggregates, its inclusions are cleared by the proteasome. Based on studies with cultured cells overexpressing the mutant protein, it has been suggested that VAPB inclusions may exert a pathogenic effect either by sequestering the wild-type protein and other interactors (loss-of-function by a dominant negative effect) or by a more general proteotoxic action (gain-of-function). To investigate P56S-VAPB degradation and the effect of the inclusions on proteostasis and on ER-to-plasma membrane protein transport in a more physiological setting, we used stable HeLa and NSC34 Tet-Off cell lines inducibly expressing moderate levels of P56S-VAPB. Under basal conditions, P56S-VAPB degradation was mediated exclusively by the proteasome in both cell lines, however, it could be targeted also by starvation-stimulated autophagy. To assess possible proteasome impairment, the HeLa cell line was transiently transfected with the ERAD (ER Associated Degradation) substrate CD3δ, while autophagic flow was investigated in cells either starved or treated with an autophagy-stimulating drug. Secretory pathway functionality was evaluated by analyzing the transport of transfected Vesicular Stomatitis Virus Glycoprotein (VSVG). P56S-VAPB expression had no effect either on the degradation of CD3δ or on the levels of autophagic markers, or on the rate of transport of VSVG to the cell surface. We conclude that P56S-VAPB inclusions expressed at moderate levels do not interfere with protein degradation pathways or protein transport, suggesting that the dominant inheritance of the mutant gene may be due mainly to haploinsufficiency.</p></div

Transport of VSVG to the cell surface occurs normally in cells expressing P56S-VAPB inclusions.

<p><b>A:</b> HeLa-TetOff cells, induced (−Dox) or not induced (+Dox) to express <i>myc</i>-P56S-VAPB, were transfected with VSVG-EGFP at 39.3°C. After 24 h, cells were shifted to 32°C. At the indicated times, the cells were chilled and incubated with anti-lumenal domain of VSVG under non-permeabilizing conditions (red). The cells were then permeabilized and stained with anti-VAPB antibodies (blue in merge panel - see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113416#s2" target="_blank">Methods</a>). Total VSVG (intracellular+surface) was revealed by GFP fluorescence (green). Maximum intensity projections of z-stacks are shown. The acquisition parameters were the same in all images. Scale bar, 10 µm. <b>B:</b> Time course (means ± SD) of VSVG surface labeling normalized to total EGFP fluorescence. Significant differences between induced or non-induced samples were not detected by Student's t-test.</p

P56S-VAPB is degraded by the proteasome and by activated, but not basal, autophagy.

<p><b>A:</b> Immunoblotting analysis of degradation of P56S-VAPB in the presence or absence of proteasome or autophagy inhibitors. 3 h after the inhibition of transcription of the P56S-VAPB transgene by addition of Dox to the media (lanes 2 and 7), cells were either left untreated (lanes 3 and 8), treated with the autophagy inhibitor Bafilomycin (Baf) or with the proteasome inhibitors MG132 (MG) or Lactacystin (Lact) for 6–7 h, as indicated. Control (Ctl) cells were grown in the presence of Dox. Equal aliquots of each sample were loaded (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113416#s2" target="_blank">Methods</a>). The lower panel shows Ponceau staining of the blotted gel region, as loading control. The vertical white line (here and in panel D) juxtaposes lanes deriving from the same blot exposure. The position of the 25 kDa size marker is indicated. <b>B:</b> Quantification (means from 2–5 experiments +SEM) of P56S-VAPB remaining at 10 h after Dox addition in the presence or absence of drugs, as indicated, compared to levels measured at 3 h *: p = 0.013 and 0.025 for MG132 and lactacystin treated samples <i>vs</i> untreated by Student's t test. respectively. The difference between 3-MA or bafilomycin-treated samples and untreated was non-significant (ns). <b>C:</b> Equal amounts of protein of the samples of lanes 3 and 4 of panel A were analyzed for p62 by immunoblotting, to control for inhibition of autophagy by bafilomycin. Actin was probed as loading control. <b>D:</b> Effect of starvation on clearance of P56S-VAPB. 3 h after addition of Dox to the media (lane 2), cells were either left untreated (lane 3), or treated with bafilomycin (Baf) or MG132 (MG), as indicated, for 6 h; the samples of lanes 6–8 were also starved during the incubation with or without the drugs. Control (Ctl) cells were cultured in presence of Dox. Ponceau staining of the blotted region is shown in the lower panel. <b>E:</b> Quantification of three experiments (means +S.E.M.) of P56S-VAPB remaining 9 h after Dox addition under the indicated conditions compared to levels measured before drug treatment and/or starvation at 3 h after Dox addition. *: p = 0.036 by Student's t test; ns, non significant.</p

P56S-VAPB inclusions in a model motoneuronal cell line are degraded by the proteasome.

<p><b>A:</b> Immunofluorescence analysis of NSC34 Tet-Off cells induced to express <i>myc</i>-wt-VAPB (left) or <i>myc</i>-P56S-VAPB (right). The upper panel shows anti-<i>myc</i> immunofluorescence, the lower one the superposition of <i>myc</i> staining with phase contrast. The inset of the upper left panel shows a 2 fold enlargement of the boxed area, and illustrates the web-like distribution of wt VAPB typical of an ER protein. Scale bar: 15 µm. <b>B:</b> Degradation of P56S-VAPB stably expressed in NSC34 cells. Induced cells were supplemented with Dox; 3 h thereafter the cells were either left untreated or treated with MG132 (MG) or Bafilomycin (Baf) for 7 h. Control (Ctl) cells were grown in the presence of Dox. Equal aliquots of each sample were loaded. The lower panel shows Ponceau staining of the blotted gel region; the positions of the 25 and 37 kDa size marker are indicated. The vertical white line indicates removal of irrelevant lanes form the image. The levels of P56S-VAPB, as percentage of values in untreated cells at 3 h after Dox addition, are indicated below the lanes. p62 immunoblotting was performed to check the efficacy of bafilomycin to inhibit autophagy (upper). <b>C:</b> Confocal analysis (single sections are shown) of P56S-VAPB inclusions stained with anti-<i>myc</i> antibody (red) at 3 h after Dox addition (left) and 7 h later in the presence or absence of the indicated drugs. Nuclei were stained with DAPI. The number and size of the inclusions decreased in the absence of drugs or in the presence of Bafilomycin, but remained similar to the 3 h cells when MG132 was present. Scale bar, 10 µm.</p

Close relationship between P56S-VAPB inclusions and the Golgi Complex.

<p>Induced HeLa Tet-Off cells were doubly immunostained with anti-<i>myc</i> antibodies, to reveal P56S-VAPB, and antibodies against the Golgi proteins GM130 or giantin, as indicated. Nuclei, stained with DAPI, are shown in the merge panel. Shown are maximum intensity projections of z-stacks. Scale bars: upper row, 10 µm; middle and lower row 5 µm.</p

Transport of VSVG to the Golgi Complex occurs normally in cells expressing P56S-VAPB inclusions.

<p><b>A:</b> HeLa-TetOff cells, induced (−Dox, right) or not induced (+Dox, left) to express <i>myc</i>-P56S-VAPB, were transfected with VSVG-EGFP at 39.3°C. After 24 h, one coverslip of each sample was fixed (0 min), while the others were shifted to 32°C and fixed after incubation for the indicated times. Cells were stained with anti-Giantin (red) and anti-<i>myc</i> (blue) antibodies. Maximum intensity projections of z-stacks are shown. The cell boundaries at the 30 min time point are indicated by the white line in the merge panel. Acquisition parameters were the same in all images. Scale bar, 10 µm. <b>B:</b> Time course (means ± SD) of VSVG transport through the Golgi. Significant differences between induced or non-induced samples were not detected by Student's t-test.</p