Metallothionein proteins expression, copper and zinc concentrations, and lipid peroxidation level in a rodent model for amyotrophic lateral sclerosis

Abstract

It has been hypothesized that copper-mediated oxidative stress contributes to the pathogenesis of familial amyotrophic lateral sclerosis (ALS), a fatal motor neuron disease in humans. To verify this hypothesis, we examined the copper and zinc concentrations and the amounts of lipid peroxides, together with that of the expression of metallothionein (MT) isoforms in a mouse model [superoxide dismutase1 transgenic (SOD1 Tg) mouse] of ALS. The expression of MT-I and MT-II (MT-I/II) isoforms were measured together with Western blotting, copper level, and lipid peroxides amounts increased in an age-dependent manner in the spinal cord, the region responsible for motor paralysis. A significant increase was already seen as early as 8-week-old SOD1 Tg mice, at which time the mice had not yet exhibited motor paralysis, and showed a further increase at 16 weeks of age, when paralysis was evident. Inversely, the spinal zinc level had significantly decreased at both 8 and 16 weeks of age. The third isoform, the MT-III level, remained at the same level as an 8-week-old wild-type mouse, finally increasing to a significant level at 16 weeks of age. It has been believed that a mutant SOD1 protein, encoded by a mutant SOD1, gains a novel cytotoxic function while maintaining its original enzymatic activity, and causes motor neuron death (gain-of-toxic function). Copper-mediated oxidative stress seems to be a probable underlying pathogenesis of gain-of-toxic function. Taking the above current concepts and the classic functions of MT into account, MTs could have a disease modifying property: the MT-I/II isoform for attenuating the gain-of-toxic function at the early stage of the disease, and the MT-III isoform at an advanced stage.

Introduction

Metallothionein (MT) is a family of low-molecular-weight, cystein-rich, heat-stable and metal binding proteins (Kägi and Schäffer, 1988). MT participates in a broad range of physiological functions such as detoxification of heavy metals like mercury (Hg) and cadmium (Cd), homeostasis of essential metals including copper (Cu) and zinc (Zn), and scavenging reactive oxygen species (ROS) (Aschner et al., 1997, Hidalgo et al., 2001). Four isoforms are identified in mammals, three of which, MT-I, MT-II and MT-III are found in the central nervous system (CNS) (Aschner et al., 1997, Palmiter et al., 1992, Uchida et al., 1991). The localization and induction patterns between the MT-I and MT-II (MT-I/II) isoforms and the MT-III isoform in the CNS appear to be distinct. The expression of MT-I/II is mainly localized in glias (Aschner et al., 1997, Blaauwgeers et al., 1993) and is induced by exposure to metals including Hg, Cd, Cu and Zn, cytokines and ROS (Aschner et al., 1997, Hidalgo et al., 2001). On the other hand, the MT-III isoform is mainly present in neurons (Masters et al., 1994, Uchida et al., 1991), and is not easily induced by exposure to the above agents (Zheng et al., 1995).

Amyotrophic lateral sclerosis (ALS) is a lethal motor neuron disease characterized by selective degeneration of motoneurons, resulting in muscular atrophy including respiratory and bulbar muscles, complete paralysis, and death (Rowland and Shneider, 2001). Approximately 90% of ALS is sporadic and the remaining 10% or so is familial (Cleveland and Rothstein, 2001). Rosen et al. found that about 20% of familial ALS is linked with mutations of the gene encoding Cu/Zn superoxide dismutase (SOD1) (Rosen et al., 1993). It has been believed that abnormal SOD1 proteins encoded by mutant SOD1 do not lose their original enzymatic function but gain a novel cytotoxic function in motoneurons (gain-of-toxic function theory) (Bruijn et al., 2004, Gurney et al., 1994). A presumable mechanism of the gain-of-toxic function is a Cu-mediated oxidative stress. A point mutation in SOD1 causes chemical structure changes of SOD1 proteins (misfolding proteins), while retaining its original activity and with subsequent clumsy handling of Cu and Zn (Beckman et al., 2001, Bruijn et al., 2004, Valentine and Hart, 2003). As a result of decreased Zn-binding affinity and higher affinity for Cu (Crow et al., 1997, Lyons et al., 1996), Cu-mediated oxidative stress is enhanced, and leads to neuronal death (Said Ahmed et al., 2000, Wiedau-Pazos et al., 1996).

Mouse carrying a human mutant SOD1 develops an ALS-like disease, and is a good animal model for ALS research. The mice do no exhibit any clinical signs of motor paralysis up to the age of about 12 weeks, and develop paralysis at the age of 14–16 weeks, dying of respiratory failure by 17–18 weeks old (Bruijn et al., 1997, Gurney et al., 1994, Ripps et al., 1995).

In this way, all physiological functions of MT are likely to be associated with the presumable current pathogenesis of ALS. In order to investigate the possible role of the changes in Cu and Zn concentrations and lipid peroxides (LPO) products, we therefore measured temporal changes in the Cu and Zn levels and the amount of LPO together with MT-I/II and MT-III proteins in a rodent model for ALS.