Contribution of calpains to photoreceptor cell death in N-methyl-N-nitrosourea-treated rats

https://doi.org/10.1016/j.expneurol.2006.09.011Get rights and content

Abstract

The purpose of the present study was to determine if proteolysis by the calcium-dependent enzyme calpains (EC 3.4.22.17) contributed to retinal cell death in a rat model of photoreceptor degeneration induced by intraperitoneal injection of N-methyl-N-nitrosourea (MNU). Retinal degeneration was evaluated by H&E staining, and cell death was determined by TUNEL assay. Total calcium in retina was measured by atomic absorption spectrophotometry. Activation of calpains was determined by casein zymography and immunoblotting. Proteolysis of α-spectrin and p35 (regulator of Cdk5) were evaluated by immunoblotting. Calpain inhibitor SNJ-1945 was orally administrated to MNU-treated rats to test drug efficacy. MNU decreased the thickness of photoreceptor cell layer, composed of the outer nuclear layer (ONL) and outer segment (OS). Numerous cells in the ONL showed positive TUNEL staining. Total calcium was increased in retina after MNU. Activation of calpains and calpain-specific proteolysis of α-spectrin were observed after MNU injection. Oral administration of SNJ-1945 to MNU-treated rats showed a significant protective effect against photoreceptor cell loss, confirming involvement of calpains in photoreceptor degeneration. Conversion of p35 to p25 was well correlated with calpain activation, suggesting prolonged activation of Cdk5/p25 as a possible downstream mechanism for MNU-induced photoreceptor cell death. SNJ-1945 reduced photoreceptor cells death, even though MNU is one of the most severe models of photoreceptor cell degeneration. Oral calpain inhibitor SNJ-1945 may be a candidate for testing as a medication against retinal degeneration in retinitis pigmentosa.

Introduction

Retinitis pigmentosa (RP) affects approximately 1.5 million people worldwide (Berson, 1993). RP is a group of hereditary retinal dystrophies, characterized by the early onset of night blindness followed by a progressive loss of the visual field. RP is phenotypically and genetically very heterogeneous (van Soest et al., 1999). More than 25 gene mutations have been associated with RP. The primary defect underlying RP is malfunctioning of the rod photoreceptor cells, followed by apoptotic degeneration (Li and Milam, 1995). Many models show photoreceptor degeneration, including retinal degeneration (rd) mice and the Royal College of Surgeons (RCS) rats (Pierce, 2001). rd mice contain a defect in the β subunit of rod cGMP phosphodiesterase (β-PDE). RCS rats have a deletion in the receptor tyrosine kinase, mertk. Mutations of both genes have been detected in patients with RP.

N-methyl-N-nitrosourea (MNU) causes DNA methylation (Christmann and Kaina, 2000), and it has been used extensively to study retinal photoreceptor cell degeneration in a variety of animals (Nakajima et al., 1996, Yuge et al., 1996, Taomoto et al., 1998). MNU-induced photoreceptor degeneration is characterized by apoptosis. Apoptosis is induced by the caspase family of cysteine proteinases. Inhibition of caspases protected structures and functional properties of photoreceptors (Yoshizawa et al., 2000, Petrin et al., 2003).

Calpains (EC 3.4.22.17) are also known to be activated during cell death in many types of cells (Utz and Anderson, 2000, Wang, 2000). Calpains are cytoplasmic, calcium-activated, cysteine proteases, comprising a superfamily of 15 genes in mammals (Branca, 2004). Calpains 1 (μ-calpain) and 2 (m-calpain) are the best characterized and are ubiquitously expressed in the animal kingdom. Enzymatic activities of calpains 1 and 2 are regulated by an endogenous inhibitory protein calpastatin and calcium (Saido et al., 1992). Uncontrolled and prolonged calpain-mediated proteolysis has been suggested in the pathogenesis of neuronal cell death, such as in Alzheimer's and Parkinson's diseases (Nixon, 2003). Calpain activation was also observed in hereditary models associated with photoreceptor degeneration in WBN/Kob rats and rd mice (Azuma et al., 2004, Doonan et al., 2005). Calpains were also involved in calcium-induced cell death in a murine photoreceptor-derived cell line (Sanvicens et al., 2004, Gomez-Vicente et al., 2005).

Calpains have a large number of substrates, including signaling molecules, membrane proteins, intracellular enzymes, and structural proteins (Goll et al., 2003). However, most of these substrates were identified using in vitro cleavage site analysis and do not necessarily reflect calpain target proteins in vivo. Identification of intracellular calpain substrates during cell injury and death is needed to understand the role of calpains in photoreceptor degeneration. For example, the cytoskeletal protein α-spectrin is a well-known, sensitive substrate of calpain. Proteolysis of α-spectrin has been suggested in the pathogenesis of neuronal cell death in brain ischemia (Yokota et al., 2003). α-Spectrin supports the neuronal structures and tethers synaptic vesicles to the active zone of the synaptic plasma membrane (Sikorski et al., 2000). α-Spectrin breakdown products (SBDPs) have been used as biochemical markers for calpain activation in central neurons and cultured neuroblastoma cells (Saido et al., 1993, Nath et al., 1996). Our previous studies show that α-spectrin was proteolyzed in vivo and in vitro retinal degeneration models (Sakamoto et al., 2000, Tamada et al., 2002, Azuma et al., 2004, Oka et al., 2006). In mammalian retina, α-spectrin is ubiquitously localized in both of inner and outer retina (Isayama et al., 1991), suggesting that calpain-induced SBDPs are useful markers in the pathogenesis of photoreceptor cell death.

Other proteins could also be substrates for calpains in photoreceptor cells. In vertebrate outer segments, the two intracellular messengers cGMP and calcium regulate visual transduction and adaptation. Levels of intracellular calcium in photoreceptors are regulated by influx through cGMP-gated calcium channels and efflux through Na+/Ca2+,K+exchangers (McNaughton, 1995). Some Na+/Ca2+exchangers are cleaved by calpain, causing calcium overload and excitotoxicity in neuronal cells (Bano et al., 2005).

The photoreceptor cGMP-gated calcium channel is regulated by the concentration of cGMP, which is degraded by phosphodiesterase 6 (PDE6). PDE6 is composed of two homologous catalytic αβ subunits and two identical regulatory γ subunits. PDE6 is activated by removal of γ subunits from the αβγ2 complex after stimulation by upstream signals initiated by illuminated rhodopsin (Stryer, 1986). In rd mice, photoreceptor cell death is thought to be due to defects in the β subunit of PDE (β-PDE) causing lack of PDE6 activity, elevated cGMP, and sustained influx of calcium via the cGMP-gated channel (Yau and Baylor, 1989). Mutations in the gene for the β subunit of PDE (PDE6B) occur in some cases of human recessive RP (McLaughlin et al., 1993, Lolley, 1994).

Normally, the γ subunit of PDE6 is phosphorylated by cyclin-dependent kinase 5 (Cdk5). Phosphorylated γ subunits strongly bind to αβ subunits, leading to PDE6 inactivation (Matsuura et al., 2000). Cdk5 requires the regulatory subunit p35 for activity (Lew et al., 1994, Tsai et al., 1994). p35 is converted to p25 by calpains, leading to prolonged activation and mislocalization of Cdk5 found in neural degeneration in Alzheimer's disease (Kusakawa et al., 2000, Lee et al., 2000, Nath et al., 2000). This suggests that conversion of p35 to p25 by calpains could also lead to inactivation of PDE6 and calcium influx in photoreceptor cells. Further, p25 may be a useful marker for relating calpain activity to photoreceptor cell death.

The purpose of the present experiment was to determine if calpain-induced proteolysis of photoreceptor proteins contributed to retinal degeneration in MNU-treated rats.

Section snippets

Experimental animals

Female Sprague–Dawley rats at 7 weeks of age were used as previously reported (Yoshizawa et al., 1999). The rats were obtained from Charles River (Yokohama, Japan) and were maintained at room temperature on a 12-h light/dark schedule, with ad libitum access to food and water. All experimental animals were handled in accordance with the Declaration of Helsinki and The Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 80-23) and according to the tenets of the ARVO Statement

Histological changes in MNU-treated rats

H&E staining of retinal sections showed no substantial differences in the retinal structures 6 h after MNU injection (Fig. 1A). The first changes observed 12 h after injection of MNU were expansion of extracellular space with vacuoles in the outer nuclear layer (ONL) and the outer segment (OS). By day 1, the thicknesses of the ONL and OS were slightly thinner than those in retinas from rats before MNU treatment (Fig. 1A, 1 day vs. Time 0). These changes became more severe over 7 days. Numerous

Discussion

The major findings of the present study were that calpains could contribute to photoreceptor cell death in the ONL and OS from rats treated with MNU, and that oral administration of calpain inhibitor SNJ-1945 reduced cell death. These conclusions were based on increased total calcium, activation of calpains, appearance of calpain-specific proteolysis products, and inhibition of cell death by calpain inhibitor SNJ-1945.

Acknowledgments

Dr. Shearer is a paid consultant for Senju Pharmaceutical Co., Ltd., a company that may have a commercial interest in the results of this research and technology. Dr. Azuma is an employee of Senju Pharmaceutical Co., Ltd. These potential conflicts of interest have been reviewed and managed by the OHSU. We thank Ayumi Tochigi and Noriko Nada for technical assistance.

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