The Role of Ca2 Influx and the Ca2Dependent Protease Calpain

1. Ca2+ Influx

Recent studies have implicated increases in intracellular Ca2+ levels by alterations in Ca2+ channels in the development of EAE. For instance, the pore-forming subunit of neu-ronal-type voltage-gated Ca2+ channels was upregulated in activated and inactivated lesions with active demyelination in both MS and EAE, suggesting that Ca2+ influx in EAE may be facilitated by this increase (Kornek et al., 2001). Studies have also demonstrated that upregulation of the sodium (Na+) channel Nav1.6 in EAE spinal cord, which results in the intracellular flow of Na+ and reversal of the Na+/Ca2+ exchanger, might allow damaging concentrations of Ca2+ into the demyelinated axon (Craner et al., 2004).

Ca2+ influx may also be increased in EAE as a result of glutamate neurotoxicity, which alters intracellular Ca2+ homeostasis (Pitt et al., 2000; Smith et al., 2000). Since glutamate AMPA receptor antagonists ameliorated disease severity (Pitt et al., 2000; Smith et al., 2000), restored density of motor neurons (Smith et al., 2000), increased oligodendrocytes survival (Pitt et al., 2000), and reduced dephospho-rylation of NFP (Pitt et al., 2000) in EAE animals.

The true intracellular Ca2+ concentration in the EAE lesion is currently unknown, and as activation of calpain is absolutely dependent on Ca2+, it is essential to determine the intracellular Ca2+ level in tissues from EAE and MS. Increased Ca2+ influx may promote calpain activation and expression, axonal damage, and cell death. In support of this, studies in our laboratory indicate that the intracellular Ca2+ levels are substantially increased in EAE lumbar spinal cord (Guyton et al., 2004b) and in optic nerve (Guyton et al., 2002) compared to controls. In addition, increased intracellular Ca2+ concentrations in cultured glioblastoma (Sur et al., 2003) and neuronal PC12 (Ray et al., 2000) cells treated with glutamate or oxidants have been found to activate calpain, leading to cell death, further supporting the hypothesis that calpain activation may be neurodegenerative in EAE.

2. Calpain Expression and Activity

Among the Ca2+-dependent proteases, calpain has been widely recognized as a prominent player in the pathogenesis of demyelinating diseases (Ray et al., 2003). Calpains exist as ubiquitous forms, microcalpain and millicalpain, requiring |M and mM calcium concentrations for activation, respectively (Saido et al., 1994; Shields and Banik, 1998a). The endogenous inhibitor, calpastatin, also is endogenously expressed and is specific only for calpain, suggesting the importance of calpain regulation in cell homeostasis. Generally, calpain exists as a proenzyme in an inactive form in the cytosol, where the normal Ca2+ levels are below concentrations needed for activation. During pathophysiological conditions, however, increased intracellular Ca2+ influx could potentially activate calpain (Ray and Banik, 2003; Saido et al., 1994). Calpain degrades myelin proteins (myelin-associated glycoproteins, proteolipid protein, MBP), NFP, cytoskeletal proteins (spectrin, talin, actin), and nuclear laminins on axons and cell membranes (Ray et al., 2003; Shields and Banik, 2001).

Calpain expression and activity has been demonstrated before disease onset in splenic cells (Shields et al., 1999a) and after disease onset in the spinal cord (Shields and Banik, 1998b) and optic nerve (Shields and Banik, 1998a), as well as in active brain lesions from patients with MS (Shields et al., 1999b). Studies have demonstrated that calpain is involved in the activation of T-cells, myelin degradation, axonal damage, and cell death mechanisms leading to apop-tosis (Ray and Banik, 2003; Schaecher et al., 2001).

a. The Role of Calpain in Immune Cells. About 28 years ago, detection of a neutral protease in inguinal and popliteal lymph nodes of rats with EAE which degraded MBP suggested a possible role for this enzyme in demyelination (Smith, 1976). Further support was demonstrated in studies showing that stimulated macrophages secreted several neutral proteases, which selectively degraded MBP (Norton et al., 1978). Increased neutral protease activity was found in leukocytes of patients with MS during relapse as compared to remission and controls (Cuzner et al., 1975). Later, neutral protease activity was also identified in lymphocytes and in serum of Lewis rats with EAE (Banik, 1979; Smith, 1979). It is now well known that this neutral protease is calpain. Activated lym-phoid cells are readily recognized in patients with MS. In vitro studies showed that calpain expression, both at the mRNA and protein levels, was increased in activated lym-phoid cells, and calpain secreted from activated human lymphoid cells could degrade MBP (Deshpande et al., 1995b).

Previous studies in our laboratory have demonstrated increased calpain expression in splenic inflammatory cells before disease onset in EAE. Calpain inhibition blocked T-cell activation in vitro, suggesting that calpain may play a role in the production by autoreactive T-cells of factors that are involved in myelin destruction in EAE (Schaecher et al.,

2001). Subsequent work demonstrated that calpain expression and activity were also increased in activated T-cells and in macrophages that infiltrated into the spinal cord of EAE animals, beginning with disease onset (Schaecher et al.,

2002). This suggested that calpain expression and activity in peripheral immune cells occurs early after disease induction but is not detected in the CNS until after clinical symptoms develop.

b. The Role of Calpain in the CNS. In addition to increased calpain expression in inflammatory cell infiltrates such as OX42+ mononuclear phagocytes and activated CD25+ T-cells, calpain was also intensely increased in astro-cytes in EAE spinal cord and optic nerve. Calpain-induced degradation of 68 kD NFP and spectrin demonstrated increased calpain activity in EAE (Shields and Banik, 2001). Studies from our laboratory have demonstrated increased calpain activity and expression with degradation of myelin and axonal proteins, such as NFP, occurring in EAE spinal cord, as compared to control (Shields and Banik, 2001). Increased calpain expression in activated GFAP+ astrocytes and infiltrating IFN7+ inflammatory cells (Fig. 1) coincided with increased calpain activity and the appearance of clinical symptoms. These activated inflammatory cells (T-cells, macrophages) and reactive cells (astrocytes and microglia), which express more calpain, are the major source of calpain activity, confirming our earlier finding(s) that calpain activity is upregulated in EAE spinal cord and optic nerve, thus leading to degradation of NFP and other cytoskeletal proteins.

Subsequent studies in postmortem tissue from patients with MS revealed increased calpain activity as measured by

Figure 1 Double immunofluorescent labeling using antibodies for calpain (green) and cell-specific markers (red). (A—B) Antibodies specific for astrocytes (GFAP). (C-D) Antibodies specific for IFN-y in control (A, C) and EAE (B, D) spinal cord white matter from Lewis rats. Arrows indicate co-localization shown in yellow (x 200). (Reproduced with permission from Shields et al., Proc. Natl. Acad. Sci. U. S. A. 95, 5768-5772, 1998.)

Figure 1 Double immunofluorescent labeling using antibodies for calpain (green) and cell-specific markers (red). (A—B) Antibodies specific for astrocytes (GFAP). (C-D) Antibodies specific for IFN-y in control (A, C) and EAE (B, D) spinal cord white matter from Lewis rats. Arrows indicate co-localization shown in yellow (x 200). (Reproduced with permission from Shields et al., Proc. Natl. Acad. Sci. U. S. A. 95, 5768-5772, 1998.)

antibody specific for the calpain-cleaved fodrin fragment in MS white matter, compared to white matter tissue from controls and patients with Alzheimer's disease or Parkinson's disease (Fig. 2). Double immunofluorescent labeling, using calpain antibody and cell-specific marker antibodies, demonstrated substantial increases in calpain expression in MHC-II+ cells and mononuclear phagocytes (Fig. 3) in MS lesions. Similarly, calpain expression was also upregulated in reactive astrocytes and CD4+ T-cells from MS plaque and normal appearing white matter, as compared to tissue from patients with other neurological disorders, and control white matter (Shields et al., 1999b). In the lesion, there was extensive upregulation of calpain expression in CD4+ cells. It is important to note that in Alzheimer's disease and Parkinson's disease, gray matter is affected, and this is where calpain activation would potentially be involved in the neurodegeneration of these diseases. These findings, that cal-pain expression and activity are upregulated in EAE and MS, provide support for calpain's role in axonal damage and neuronal cell death in the pathology of these diseases.

Figure 2 Calpain activity as measured by Western blot detection of the 145 kD calpain-dependent fodrin breakdown product in CNS white matter from normal control, Parkinson's, Alzheimer's, normal-appearing white matter (NAWM), and MS plaque samples (n = 8-10). Western blots were quantified via densitometry and analyzed by one-way ANOVA (±SEM). (Reproduced with permission from Shields et al., Proc. Natl. Acad. Sci. U.S. A. 96,11486-11491, 1999.)

Figure 2 Calpain activity as measured by Western blot detection of the 145 kD calpain-dependent fodrin breakdown product in CNS white matter from normal control, Parkinson's, Alzheimer's, normal-appearing white matter (NAWM), and MS plaque samples (n = 8-10). Western blots were quantified via densitometry and analyzed by one-way ANOVA (±SEM). (Reproduced with permission from Shields et al., Proc. Natl. Acad. Sci. U.S. A. 96,11486-11491, 1999.)

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