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Plasma membrane calcium ATPase deficiency causes neuronal pathology in the spinal cord: a potential mechanism for neurodegeneration in multiple sclerosis and spinal cord injury

Michael P. Kurnellas^*,, Arnaud Nicot, Gary E. Shull^¶ and Stella Elkabes||^*,,1

^* Neurology and Neuroscience, UMDNJ-New Jersey Medical School, Newark, New Jersey, USA;
Neurology Service, Veterans Affairs, East Orange, New Jersey, USA;
INSERM EMI 0350, Hôpital St. Antoine, Paris, France; and
^¶ Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio, USA

¹Correspondence: Neurology and Neuroscience, NJMS/UMDNJ, 185 S. Orange Ave., MSB H-506, Newark, NJ 07103, USA. E-mail: elkabest{at}umdnj.edu

SPECIFIC AIMS

Neuronal/axonal dysfunction and loss in the spinal cord are critical determinants of neurological deficits in various pathological conditions of the CNS, including multiple sclerosis (MS) and spinal cord injury (SCI). Yet the molecular mechanisms underlying neuronal pathology remain undefined. We have previously shown a dramatic decrease in the levels of plasma membrane calcium ATPase isoform 2 (PMCA2), a major pump extruding calcium from cells, in spinal cord neurons during experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and after spinal cord trauma. A reduction in the transcript levels of various PMCA isoforms in postmortem MS brain has also been reported. However, it was not clear whether these changes are the cause or the consequence of neuronal damage. The present studies investigate whether the deficiency in PMCA expression or activity is the cause of neuronal pathology and loss in the spinal cord in vivo and in vitro.

PRINCIPAL FINDINGS

1. Blockade of PMCA activity induces neuronal pathology and death
Initially we investigated whether calcium pump inhibitors affect neuronal function and survival in vitro. We first ensured that PMCA2 mRNA and protein were expressed in purified spinal cord neuronal cultures by use of RT-PCR and immunocytochemistry, respectively (Fig. 1 A, B). We then exposed cells to different concentrations of sodium orthovanadate (Na₃VO₄), a general inhibitor of P-type ATPases, including PMCAs and sarco(endo)plasmic reticulum Ca²⁺ ATPases (SERCA). We assessed immunoreactivity to nonphosphorylated neurofilament H using the SMI-32 antibody, a marker of neuronal/axonal damage in MS and other pathological conditions of the CNS, including trauma. Thirty µM Na₃VO₄ significantly increased the number of SMI-32 positive cells within 4 h (Fig. 1C ). A lower dose (10 µM) was effective when cells were exposed to the inhibitor for 8 h. As Na₃VO₄ can interfere with the activity of SERCA pumps, we investigated whether cyclopiazonic acid (CPA; 10 µM) and thapsigargin (250 nM), specific SERCA pump inhibitors, induce a similar response. We observed no significant changes in the SMI-32 positive cell number (Fig. 1D ). These results suggested that Na₃VO₄-induced neuronal pathology might primarily be due to inhibition of PMCAs.

View larger version (35K): [in this window] [in a new window]   Figure 1. Effects of PMCA and SERCA inhibitors on spinal cord neurons in vitro. A) RT-PCR showing expression of PMCA2 in neuronal cultures. Lane 1: molecular weight marker; bp, base pairs; lane 2: RT-PCR product (arrow) at the predicted molecular weight (1014 bp). The identity of the band was further verified by sequence analysis. B) PMCA2 immunoreactivity in spinal cord neurons. Arrows and arrowheads point to immunopositive cells and processes, respectively. C) SMI-32 positive cell number after exposure of cultures to 10, 30, and 100 µM Na3VO4. C: control. D) Effects of SERCA inhibitors on SMI-32 positive cell number. CPA: cyclopiazonic acid. E) SMI-32 positive cells after exposure of cultures to 5 µM 5-(and-6)-carboxyeosin diacetate, succinimidyl ester (CE). F) Effects of CE on cell survival. G) Induction of activated caspase-3 in cells treated with CE. Experiments were repeated at least twice and yielded similar results. Values are presented as mean ± SE. Significantly different from control *P< 0.003, **P < 0.0001 by ANOVA; + significantly different from control by t test, P< 0.02; n = 6–12.

To further ascertain that changes in SMI-32 immunopositive neuron number were indeed due to inhibition of PMCA activity, we used 5-(and-6)-carboxyeosin (CE), a more selective and potent PMCA inhibitor. Exposure to 5 µM CE for 4 h induced a 1.8-fold increase in SMI-32 positive cells (Fig. 1E ). Surprisingly, SMI-32 positive cell number in CE-treated cultures was not significantly different from controls at 8 h. Since this could be due to loss of affected neurons, we investigated cell survival by use of MTT assay. Indeed, a reduction in MTT positive cells was observed by 8 h (Fig. 1F ). Thus, prolonged exposure to CE affects neuronal survival.

Reductions in PMCA activity may lead to abnormal increases in intracellular calcium levels ([Ca²⁺]_i), which promote mitochondrial cytochrome c release. This in turn activates the caspase cascade mediating apoptotic cell death. To determine whether inhibition of PMCA activity causes induction of apoptotic mechanisms, we quantified the number of cells expressing activated caspase-3, a key component of apoptosis. There was a 2-fold increase in the number of caspase-3 positive cells in cultures treated with CE for 8 h, suggesting that cell death may partially be due to apoptosis (Fig. 1G ).

2. Inhibition of PMCA activity affects morphology of neurites
CE affected the morphology of neurites in vitro. Previous reports have shown that the SMI-32 antibody labels motor neurons in vitro and in vivo even in the absence of insult. In control cultures, the somata and neurites of a small subpopulation of cells with motor neuron-like morphology were immunopositive for SMI-32. The processes of these cells were smooth and showed no significant morphological abnormalities. However, addition of 5 µM CE to sister cultures initiated swelling and beading of neurites within 2 h, becoming further pronounced at 4 h (results not shown). Whereas in controls only 10.2 ± 2.63% of all SMI-32 positive cells showed neuritic swelling, in CE-treated cultures 42.4 ± 2.39% of SMI-32 positive cells had neurites with beading and swellings. Spherical structures resembling axonal retraction bulbs could be observed at the end of many processes.

3. Inhibition of PMCA activity delays clearance of depolarization-evoked calcium transients
Since PMCAs play a major role in calcium extrusion, we hypothesized that a reduction in PMCA activity may lead to an increase in [Ca²⁺]_i, which in turn induces injury cascades. To determine the effects of CE on intracellular calcium balance, we performed Fura-2 imaging on spinal cord neuronal cultures exposed to vehicle or CE for 1 h, a time that precedes the appearance of morphological changes in neurites. This treatment elevated the resting cytosolic calcium levels and increased the amplitudes of depolarization-induced [Ca²⁺]_i peaks. Clearance of depolarization-evoked calcium transients was at least 5-fold slower in CE-treated cells than controls (results not shown).

4. Loss of motor neurons in the spinal cord of PMCA2 null mice and deafwaddler(dfw^2J) mutant mice
To determine whether a lack of PMCA2 causes neuronal loss in vivo, we quantified the number of motor neurons in PMCA2 (+/+) and (–/–) mice. We focused on motor neurons because they are highly vulnerable to Ca²⁺-mediated injury. We labeled spinal cord sections with SMI-32 or an antibody against peripherin, markers of motor neurons, and quantified immunopositive cells. There was a 37.5% decrease in the number of SMI-32 positive cells in PMCA2 null mice compared with controls (Fig. 2 A). We analyzed the results by calculating the SMI-32 positive cell number/mm² gray matter (Fig. 2B ), as the total gray matter area is 13% smaller in PMCA2 (–/–) than PMCA2 (+/+) mice (Fig. 2C ). There was a 26.5% decrease in SMI-32 immunoreactive cells/mm² gray matter in PMCA2 knockout mice.

View larger version (34K): [in this window] [in a new window]   Figure 2. Loss of SMI-32 and peripherin positive neurons in the spinal cord of PMCA2 knockout (KO) mice. The number of SMI-32 positive cells/spinal cord section is decreased in KO compared with wild-type (WT) mice (A). SMI-32 positive cells/mm2 gray matter is lower in KO mice (B) when the reduction in gray matter area is considered (C). Peripherin positive cells/spinal cord section (D) and peripherin positive cells/mm2 gray matter (E) are significantly reduced in KO compared with WT mice. Peripherin immunoreactive motor neurons in the anterior horn of WT (F, arrows) and KO mouse (G). Four WT and KO mice were used and 7 sections collected from each lumbar spinal cord at 100 µm intervals were analyzed. Values are presented as mean ± SE. Significantly different from WT *P < 0.03, **P < 0.01, ***P < 0.0002 by t test. Bar = 200 µm.

In agreement, we found a significant decrease in the number of peripherin immunoreactive cells and peripherin positive cells/mm² gray matter (37.4 and 27.7%, respectively) in PMCA2 (–/–) vs. PMCA2 (+/+) mice (Fig. 2D-G ).

To further ensure that the lack of PMCA2 activity leads to motor neuron loss, we quantified SMI-32 positive cells in the spinal cord of dfw^2J mice. A 2 bp deletion in the PMCA2 gene of dfw^2J causes a frameshift leading to a truncated, inactive protein. In agreement with studies of PMCA2 (–/–) mice, we found a 41% decrease in the number of SMI-32 positive cells in dfw^2J mice vs. their wild-type controls (results not shown).

CONCLUSIONS AND SIGNIFICANCE

These studies define new roles for PMCAs in neuronal pathology and establish a causal link between these calcium pumps and neuronal damage in vivo and in vitro. Together with our previous reports showing a decrease in the levels of PMCA2 in animal models of MS and SCI, these investigations provide novel insight into potential molecular events that underlie neuronal injury in the aforementioned pathological conditions. The mechanisms described here may be relevant to other CNS disorders leading to neurodegeneration.

View larger version (28K): [in this window] [in a new window]   Figure 3. Mechanism of PMCA-mediated neuronal injury in spinal cord neurons.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2549fje;

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