**Protein Aggregates in Pathological Inclusions of Amyotrophic Lateral Sclerosis**

Yoshiaki Furukawa

*Department of Chemistry, Keio University Japan* 

### **1. Introduction**

334 Amyotrophic Lateral Sclerosis

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Anne Sahawneh, John J. Shacka, Marion Kirk, Stephen Barnes, Mary Ann Accavitti-Loper, Rafael Radi, Joseph S. Beckman, and Alvaro G. Estevez. 2007. Prevention of peroxynitrite-induced apoptosis of motor neurons and PC12 cells by tyrosineAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that is characterized by a progressive loss of upper and/or lower motor neurons (Bruijn et al., 2004). Dysfunction and death of these neurons lead to muscle weakness, atrophy and spasticity. A fatal event for the majority of patients is a failure of the respiratory muscles, which generally occurs within one to five years of disease onset. The typical age of onset is between 50 and 60 years, and the prevalence rate is 5 – 10 cases per 100,000 populations (de Belleroche et al., 1996). No effective cures for this disease are currently available, and the pathomechanism still remains controversial. The majority of ALS cases have no genetic component (sporadic ALS, sALS), while about 10 % are inherited in a dominant manner (familial ALS, fALS).

Historically, ALS has been described by Charcot and Joffroy in 1869 (Charcot & Joffroy, 1869), and linkage analysis of fALS families was performed in 1991, by which the genetic locus was identified to be linked to chromosome 21q (Siddique et al., 1991). In 1993, Rosen et al. (Rosen et al., 1993) and Deng et al. (Deng et al., 1993) have found that mutations in the Cu,Zn-superoxide dismutase (SOD1) gene, which lies on chromosome 21q, are associated with fALS. Because SOD1-related fALS exhibited several clinicopathological similarities to sALS, various animal models including rodents, worms and flies have been constructed, in which mutant forms of SOD1 are expressed. Using these models, furthermore, various drugs have been continuingly tested to cure or alleviate ALS. In 2001, ALS2 (or called alsin) has been also identified as a new gene associated with a rare, recessively inherited and slowly progressed juvenile onset form of ALS, which is, however, significantly different from the disease phenotypes of sALS (Hadano et al., 2001; Yang et al., 2001). Accordingly, studies on mutant SOD1 have served as a "gold standard" for a long time and provided valuable insight into molecular pathomechanisms of ALS.

Recent progress on genetic analysis has fuelled the identification of other genes responsible for fALS: for example, **T**AR **D**NA-binding **p**rotein-43 (TDP-43) gene reported in 2008 (Gitcho et al., 2008; Kabashi et al., 2008; Sreedharan et al., 2008; Van Deerlin et al., 2008), **Fu**sed in **S**arcoma (FUS) gene in 2009 (Kwiatkowski et al., 2009; Vance et al., 2009). Each of TDP-43 and FUS mutations describes approximately 4 % of total fALS cases, which is a smaller number than that of SOD1 mutations (~20 % of total fALS cases). Unlike SOD1, however, TDP-43 and/or FUS pathologies are observed in many of sALS patients (Deng et al., 2011b; Mackenzie et al., 2007) and also in other neurodegenerative diseases (Lagier-

Protein Aggregates in Pathological Inclusions of Amyotrophic Lateral Sclerosis 337

whether SOD1 aggregation is really toxic or even protective. Neuronal hyaline inclusions in the spinal cord are pathological hallmark of mutant SOD1-linked fALS (Shibata et al., 1996b), and those inclusions are ultrastructurally composed of the granule-coated fibrils with a diameter of 15 – 25 nm that can be labeled by anti-SOD1 antibodies (Kato et al., 2000). Such fibrillar morphologies of protein aggregates are reminiscent of the amyloid formation observed in many neurodegenerative diseases (Ross & Poirier, 2004). Pathological inclusions containing mutant SOD1 seem, however, not fulfill some of the criteria for identification as amyloid; that is, neither Congo Red staining with apple-green birefringence nor Thioflavin T/S staining with significant enhancement of fluorescence has been observed in the fALS patient with SOD1A4V (Kerman et al., 2010). Given that these staining methods for amyloid diagnosis have not been routinely performed in the tissue sections from fALS patients with SOD1 mutations, more investigations will be required on the tissue samples of fALS patients

Until recently, involvement of wild-type SOD1 (SOD1WT) in pathogenesis of sALS had been considered to be limited (Shibata et al., 1994; Shibata et al., 1996a; Watanabe et al., 2001). Indeed, an antibody called SEDI (**S**OD1-**e**xposed-**d**imer-**i**nterface) that only labels monomer/misfolded forms of SOD1 were failed to immunostain the motor neurons from sALS patients (Liu et al., 2009). Nonetheless, novel antibodies raised against 4 – 20, 57 – 72, and 131 – 153 of an SOD1 amino acid sequence were found to specifically detect unfolded SOD1 and also immunostain inclusions in motor neurons of the spinal cord from all the 29 sporadic and 8 familial ALS patients (Forsberg et al., 2010). Furthermore, motor neurons in the spinal cord of sALS patients (four out of nine cases) are found to be immunoreactive to another monoclonal antibody called C4F6, which specifically recognizes the misfolded conformation of SOD1 (Bosco et al., 2010). The staining patterns of C4F6 in sALS are, however, diffusive, and solubility of SOD1WT is also not different between sALS patients and controls. Pathological SOD1WT in sALS would, therefore, be relatively soluble, compared to mutant SOD1 within the inclusions in fALS patients. Although it appears that SOD1 is not involved in fALS cases without SOD1 mutations (Bosco et al., 2010), more prevailing roles of SOD1 proteins in the pathogenesis of ALS

Transgenic mice expressing human SOD1 with several fALS-associated mutations have been published, and phenotypes in each model mouse have been concisely summarized in (Turner & Talbot, 2008). These transgenic mice well reproduce the formation of ubiquitinpositive SOD1 inclusions, which is the pathological hallmark of SOD1-related fALS diseases (Watanabe et al., 2001). Biochemical analysis on the inclusions has further suggested the ubiquitination of mutant SOD1, based upon which the molecular pathogenesis of ALS has been proposed to include the inhibition of intracellular ubiquitin-proteasome system clogged by insoluble SOD1 aggregates (Basso et al., 2006). Retardation of proteasomal activity thus decelerates degradation of misfolded mutant SOD1, which further promotes the SOD1 aggregation. Recent mass analysis on inclusions in SOD1G93A, SOD1G37R, and SOD1H46R/H48Q transgenic mice has, however, not supported any direct modifications on SOD1, and ubiquitinated mutant SOD1 may be a minor constituent of inclusions (Shaw et al., 2008). Immunohistochemical analysis has also shown that the inclusions in transgenic mice are immunostained with antibodies to a copper chaperone for SOD1 (see below) and

with several different types of SOD1 mutations.

have been recently considered.

**2.1.1 SOD1 aggregates in mouse models** 

Tourenne et al., 2010); much attention has thus been increasingly paid on TDP-43/FUS genes. FALS cases with SOD1/TDP-43/FUS mutations exhibit distinct disease phenotypes such as site and age of onset (Millecamps et al., 2010), and the pathomechanism of fALS might be dependent upon which of the genes is mutated. Common to all ALS cases, nonetheless, inclusions can be found in motor neurons of the affected spinal cord; therefore, molecular characterization of such pathological inclusions will be important in understanding the ALS diseases.
