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. 2010 Sep;17(9):1501-10.
doi: 10.1038/cdd.2010.54. Epub 2010 May 21.

Reduced expression of plasma membrane calcium ATPase 2 and collapsin response mediator protein 1 promotes death of spinal cord neurons

M P Kurnellas  1 , H Li, M R Jain, S N Giraud, A B Nicot, A Ratnayake, R F Heary, S Elkabes
Affiliations

Reduced expression of plasma membrane calcium ATPase 2 and collapsin response mediator protein 1 promotes death of spinal cord neurons

M P Kurnellas et al. Cell Death Differ. 2010 Sep.

Abstract

The mechanisms underlying neuronal pathology and death in the spinal cord (SC) during inflammation remain elusive. We previously showed the important role of plasma membrane calcium ATPases (PMCAs) in the survival of SC neurons, in vitro. We also postulated that a decrease in PMCA2 expression could cause neuronal death during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The current studies were undertaken to define the specific contribution of PMCA2 to degeneration of SC neurons, the effectors downstream to PMCA2 mediating neuronal death and the triggers that reduce PMCA2 expression. We report that knockdown of PMCA2 in SC neurons decreases collapsin response mediator protein 1 (CRMP1) levels. This is followed by cell death. Silencing of CRMP1 expression also leads to neuronal loss. Kainic acid reduces both PMCA2 and CRMP1 levels and induces neuronal death. Administration of an alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate receptor antagonist, at onset or peak of EAE, restores the decreased PMCA2 and CRMP1 levels to control values and ameliorates clinical deficits. Thus, our data link the reduction in PMCA2 expression with perturbations in the expression of CRMP1 and the ensuing death of SC neurons. This represents an additional mechanism underlying AMPA/kainate receptor-mediated excitotoxicity with relevance to neurodegeneration in EAE.

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Conflict of interest statement

Conflict of interest The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Viability of SC neurons after selective silencing of PMCA2 expression, in vitro. (a) Representative western blots probed with an anti-PMCA2 antibody showing a decrease in the intensity of a band corresponding to the molecular weight of PMCA2 (~129–133 kDa), at different times post-transfection. Total protein visualized by BLOT-FastStain was used to account for experimental variations. Lanes 1–2: untreated control neurons; 3–4: neurons transfected with scrambled siRNA (negative control siRNA); 5–6: neurons transfected with PMCA2-specific siRNA. (b) Graphic representation of the data obtained by western blot analysis. Results from two to three experiments were combined (n = 12 for each group except for 36 h n = 6). (c–e) PMCA2 immunoreactivity in pure neuronal cultures after transfection with PMCA2 siRNA. (c) A composite showing PMCA2-labeled cells in a control, untreated culture. (d) A composite showing PMCA2 immunoreactive cells in a culture transfected with scrambled siRNA (negative control) (e) A composite showing reduced PMCA2 staining in a culture transfected with PMCA2 siRNA. Arrows point at examples of labeled cells. Bar represents 50 μm. (f) PMCA1 levels, 24 and 72 h after transfection of neurons with PMCA2 siRNA (n = 3 for each group). (g) MTT-positive viable cell number at various times after transfection of neurons with PMCA2 siRNA (results are obtained from two experiments combined, n = 6). Values in the graph represent mean ± S.E.M. *P<0.001, significantly different from control and negative control siRNA by two-way ANOVA, Bonferroni post hoc test
Figure 2
Figure 2
Alterations in CRMP1 levels after silencing of PMCA2 expression. (a) 2DE gel patterns of lysates obtained from the control (top) and PMCA2 siRNA transfected (bottom) SC neuronal cultures. Arrows point at the spot representing CRMP1. (b) Quantification of the signal intensity shows a decrease in the CRMP1 level. (c) A representative tandem mass spectrum of a rat CRMP1 peptide. Peptide sequences of CRMP1 were deduced from the observation of continuous series of either N-terminal (b-series) or C-terminal (y-series) ions. (d) A representative western blot showing CRMP1 levels 24 h after transfection of SC neurons with PMCA2 siRNA. Two bands with molecular weight of ~70 and ~62 kDa were detected. Lanes 1–2: untreated control neurons; 3–4: neurons transfected with scrambled siRNA (negative control siRNA); 5–6: neurons transfected with PMCA2 specific siRNA. (e) The quantitative analysis of the bands at ~70 and ~62 kDa from two independent experiments (n = 6). *P<0.0336, significantly different from control and negative control siRNA by one-way ANOVA, Tukey’s post hoc test. (f) Composite showing CRMP1 positive cells and neurites in control, non-manipulated pure neuronal cultures. (g) Composite showing CRMP1 immunoreactive cells and processes in cultures transfected with scrambled siRNA (negative control) (h) Composite showing decreased CRMP1 staining in cultures transfected with PMCA2 siRNA. Arrows point at examples of labeled cells and arrowheads illustrate the examples of immunoreactive neurites. Bar represents 50 μm
Figure 3
Figure 3
Effects of selective silencing of CRMP1 expression on SC neurons. (a) Representative western blots showing a decrease in the intensity of the bands corresponding to the molecular weight of CRMP1 after transfection of neurons with a CRMP1 specific siRNA. Lanes 1–2: two individual cultures of untreated neurons; 3–4 neurons transfected with scrambled siRNA (negative control siRNA; 5–6 neurons transfected with CRMP1 siRNA. (b) Graphic representation of the data obtained by the western blots at 24 h (n = 6/group). (c) Graphic representation of the data obtained by western blots at 48 h (n = 6/group). (d) A composite showing CRMP1 labeling in neuronal cultures. (e) CRMP1-stained cells in neuronal cultures transfected with scrambled siRNA. (f) A composite showing decreased CRMP1 levels in cultures transfected with CRMP1 siRNA. Arrows point at examples of labeled cells and arrowheads illustrate examples of labeled neurites. Bar represents 50 μm. (g) MTT-positive cell number at 48 h following transfection of neurons with CRMP1 siRNA (n = 8). (h) Representative western blots showing unaltered PMCA2 levels 24 or 48 h after knockdown of CRMP1 in neuronal cultures. Total protein shows similar loading. (i) Graphic representation of the results obtained by the western blots (n = 6 for 24 h and n = 3 for 48 h). Values in the graph represent mean ± S.E.M. *P<0.0091, **P<0.0001 significantly different from control and negative control siRNA by one-way ANOVA, Tukey’s post hoc test. Results from two experiments were combined
Figure 4
Figure 4
Restoration of PMCA2 and CRMP1 levels after blockade of AMPA/kainate receptors in EAE. (a) Clinical scores of mice with EAE following administration of NBQX or vehicle 24 h after onset of symptoms (early intervention). Bar represents the duration of the treatment. (b) Representative western blot (left panel) showing PMCA2 levels in the lumbar SC of individual control and EAE mice with and without NBQX administration at 24 h after onset of symptoms. Lanes 1–2: samples from two individual control mice; 3–4: samples from EAE mice which received vehicle and lanes 5–6: samples from EAE mice treated with NBQX. Graphic representation of the results obtained by western blot analysis (right panel). (c) Western blot probed with an anti-α-tubulin (~50 kDa) antibody to account for experimental variations. (d) Clinical scores of mice with EAE following administration of NBQX or vehicle at peak of symptoms (late intervention). Bar represents the duration of the treatment. (e) Representative western blot (left panel) showing PMCA2 levels in the lumbar SC of individual control and EAE mice with and without NBQX administration at peak of symptoms. Graphic representation of the results obtained by western blot analysis (right panel). (f) Western blot probed with an anti-α-tubulin antibody. (g) Western blot showing no changes in the level of PMCA1 (~129 kDa). The western blot shown in e was stripped and re-probed with the PMCA1 antibody. (h) Modulation of CRMP1 levels. The western blot shown in E was stripped and probed with an anti-CRMP1 antibody (~70 kDa, left panel). Graphic representation of the data (right panel). Values in graph represent mean ± S.E.M. *P<0.0056, ** P<0.0001, +P<0.0004 significantly different from other groups by one-way ANOVA, Tukey’s post hoc test. (n = 3–4 for a–c and g; n = 6 for d–f and h). The experiment was repeated twice and yielded similar results
Figure 5
Figure 5
Activation of AMPA/kainate receptors reduces neuronal PMCA2 and CRMP1 levels, in vitro. (a) A representative western blot probed with anti- PMCA2 and anti-CRMP1 antibodies showing a reduction in the levels of both proteins after treatment of neuronal cultures with 4 μM KA or vehicle (Control: C) for 36 h. Lowest panel (total protein) shows the BLOT-FastStain stained membrane, which was used as a control for the experimental variations. (b) PMCA2 levels following the exposure of neurons to 4 μM KA for 12 (n = 8), 24 (n = 11) or 36 (n = 10) hours. (c) PMCA2 levels following exposure of neurons to 20 μM KA for 12 (n = 7), 24 (n = 5) or 36 (n = 5) hours. (d) CRMP1 levels after the treatment of neuronal cultures with 4 μM KA for 36 h. Data from two independent experiments were combined (n = 6). (e) MTT-positive cell number following treatment of neurons with 4 μM KA for 36 or 48 h. (f) MTT-positive cell number following the treatment of neurons with 20 μM KA. Data from two experiments were combined (n = 6–8). (g) PMCA2 levels after co-administration of 10 μM NBQX with 20 μM KA (n = 6). (h) A representative western blot showing CRMP1 levels after co-administration of 10 μM NBQX with 20 μM KA. Lanes 1 and 2: controls; 3 and 4: NBQX; 5 and 6: KA; 7 and 8: KA + NBQX. Values in graph represent mean ± S.E.M. *P<0.05, **P<0.01, ***P<0.001 significantly different from the control and 12 h by two-way ANOVA, Bonferroni posttest; &P<0.001 significantly different from control and all the other groups; +P<0.02, + +P<0.0008 significantly different from control by Student’s t-test; #P<0.0026 significantly different from all the other groups by one-way ANOVA, Tukey’s post hoc test
Figure 6
Figure 6
The effects of calpastatin on PMCA2 and CRMP1 levels in KA-treated SC neuronal cultures. (a) Top panel: RT-PCR showing PMCA2 transcript levels (1014 bp) in KA-treated cultures. Control: C. Bottom panel: 18S RNA (489 bp) was used to standardize against experimental variation. (b) A representative western blot probed with an anti-PMCA2 antibody showing the effects of KA in the presence and absence of 1 μM calpastatin. Cultures were treated for 36 h. Total protein was used as a control for experimental variations. Lanes 1–2: control, 3–4: 1 μM CP, 5–6: 4 μM KA, 7–8: 20 μM KA; 9–10: 4 μM KA + 1 μM CP, 11–12: 20 μM KA + 1 μM CP. (c) Graphic representation of the data obtained by use of western blotting. Data from two independent experiments were combined (n = 6). Values in the graph represent mean ± S.E.M. *P<0.0006 significantly different from the control, 1 μM CP and 1 μM CP + 4 μM KA by one-way ANOVA, Tukey’s post hoc test. (d) Representative western blots probed with an anti-PMCA2 antibody showing the effects of 20 μM KA in the presence and absence of 10 μM calpastatin at 24 and 36 h. Lane 1: control; lane 2: 10 μM CP, lane 3: 20 μM KA, lane 4: 20 μM KA + 10 μM CP
Figure 7
Figure 7
Scheme representing the potential mechanism of PMCA2-mediated neurodegeneration in the SC during EAE. Inflammation of the CNS during EAE leads to excess glutamate from non-neuronal cells and injured neurons (1). Glutamate binds to AMPA/kainate receptors on neurons (2a) and non-neuronal cells (2b). Over-activation of neuronal AMPA/kainate receptors results in excess calcium influx (3a), while non-neuronal cells release effectors (3b) such as IL-1β in response to glutamate. Effectors released by non-neuronal cells could affect PMCA2 at the transcriptional level, whereas excessive intracellular calcium can activate calcium-dependent proteases such as calpain (4), which, in turn, degrade PMCA2. The combined outcome is a decrease in the PMCA2 levels (5). The reduction in PMCA2 can lead to a decrease in CRMP1 levels by mechanisms that are not yet determined (6). This can cause perturbations in microtubule assembly (7). On the other hand, excess intracellular calcium activates apoptotic mechanisms including caspase-3 (8). These changes lead to neuronal pathology and death (9)
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