**2. Factors controlling SOD1 translocation to mitochondria and SOD1 activity in mitochondrial intermembrane space**

Although the majority of SOD1 is present in the cytosol (Okado-Matsumoto & Fridovich, 2001), a fraction of SOD1 is translocated into the mitochondrial intermembrane space (IMS)

Mutant Cu/Zn-Superoxide Dismutase Induced

Mitochondrial Dysfunction in Amyotrophic Lateral Sclerosis 251

oxidative modification of its critical thiol groups is necessary for the activation (Iñarrea et al., 2005, 2011; Goldsteins et al., 2008). This activation, at least partly, depends on protein disulphide isomerase (PDI) activity (Iñarrea et al., 2005). On the other hand, the toxicity of mutant SOD1 is not correlated with its aggregation potential but with the ability to form active dimeric molecules (Witan et al., 2008). These findings are in concert with a concept that mitochondrial dysfunction and cell damage are paradoxically induced by SOD1-

Fig. 1. Import and activation of SOD1 in IMS. CCS is imported into mitochondria through formation of a complex with Mia40 (I). Disulphide-reduced SOD1 monomer enters IMS and acquires copper ion (Cu2+) with a help of CCS (II). Formation of intramolecular disulphide bound and dimerisation of SOD1 creates an active SOD1 molecule retained in IMS (III).

Mitochondrial abnormalities and degeneration of motor neurons are early signs of ALS disease (Wong et al., 1995; Dal Canto & Gurney, 1997; Kong & Xu, 1998). They also represent pathological hallmarks in mutant SOD1 transgenic animal models for FALS as well as in patients with sporadic ALS (Kong & Xu, 1998; Mattiazzi et al., 2002; Manfredi & Xu, 2005). Mitochondrial toxicity may thus be an important factor in the degeneration of motor neurons. The pathology, demonstrated in sporadic ALS cases includes mitochondrial aggregates, mitochondrion swelling and increased calcium levels in mitochondria (Atsumi, 1981; Siklós et al., 1996). In G93A-SOD1 transgenic mice the disease onset is associated with a remarkable increase of vacuolated mitochondria in motor neurons (Kong & Xu, 1998). It

**3. Proposed mechanisms for mutant SOD1 toxicity in mitochondria** 

mediated hydroperoxide production in the IMS (Goldsteins et al., 2008).

(Sturtz et al., 2001; Higgins et al., 2002). Since SOD1 does not contain mitochondrial targeting sequence, the true physiological function of SOD1 in the IMS remains mostly enigmatic.

In mammalian cells the mitochondrial localization of SOD1 is regulated by the folding state of this enzyme, depending on the intracellular distribution of copper chaperone for SOD1 (CCS), which in turn is regulated by oxygen concentration. Redox status of the cysteine residues in human SOD1 is critical for its retention in mitochondria. The cysteine residues form intramolecular disulphide bonds and interact with CCS (Kawamata & Manfredi, 2008). This regulation appears to be impaired for SOD1 mutants, which can lead to misfolding and aggregation of mutant SOD1 and eventually result in SOD1 accumulation inside the mitochondria. In animal models the mitochondrial association of mutant SOD1 is apparent even before the disease onset (Liu et al., 2004), indicating a causative link of mitochondrial SOD1 to the initiation of pathology.

Even though SOD1 has been suggested to be an important part of the mitochondrial superoxide scavenging system, as previously demonstrated in the yeast (Sturtz et al., 2001) and rat (Iñarrea et al., 2005) mitochondrion IMS, SOD1 activity is kept under redox control in this compartment and undergoes activation upon increased hydroperoxide concentration (Iñarrea et al., 2005).

There are 4 cysteines in the human SOD1 molecule, located at 6, 57, 111 and 144 position of the sequence. The intramolecular disulphide bridge between Cys57 and Cys146 is required for the proper tertiary and quaternary structure and enzymatic activity of SOD1 (Arnesano et al., 2004). Diminished copper loading and reduced intramolecular disulphide bound has been thought to be responsible for increased aggregation potential of G93A and D90A mutant SOD1 (Jonsson et al., 2006).

The maturation and activation of SOD1 in the cytosol is controlled by a number of factors and can be divided in several principal steps. Upon post-translational activation, an SOD1 monomer binds a Zn2+ ion. Next ,CCS transiently binds to SOD1 monomer and inserts a Cu2+ ion in the molecule (Culotta et al., 1997; Casareno et al., 1998). After the dissociation of SOD1 from CCS, oxidative formation of disulphide bounds takes place (Arnesano et al., 2004; Ding & Dokholyan, 2008), which is followed by dimerisation yielding an active SOD1 molecule (Vonk et al., 2010).

Active SOD1 dimers are not capable of entering mitochondria, in contrast to disulphide reduced apo-SOD1. According to the model proposed (Kawamata & Manfredi, 2008; Reddehase et al., 2009), CCS is first imported into mitochondria by interaction with Mia40, an IMS component critical for protein import to mitochondria. The CCS-Mia40 complex is formed through an intermolecular disulphide bound (Fig. 1.). Further disulphide rearrangement generates oxidized CCS, preventing its escape from the IMS. The activation of SOD1 in IMS is thought to be similar to the activation of SOD1 in cytosol, where SOD1 binds to CCS in the presence of Cu2+ ions and oxygen generating an active enzyme retained in IMS (Leitch et al., 2009).

Surprisingly, CCS overexpression in G93A-SOD1 mouse, a widely used transgenic mouse model of ALS, produces severe mitochondrial pathology and accelerates disease course (Son et al., 2007). According to the model above, the potentiation of mutant SOD1 toxicity by CCS overexpression can be explained by the CCS-mediated increase in SOD1 mitochondrial import, leading to enhanced SOD1 aggregation.

In contrast to the model of CCS-dependent activation of mitochondrial SOD1, a number of recent studies suggest that SOD1 in the IMS of intact mitochondria is mostly inactive and an

(Sturtz et al., 2001; Higgins et al., 2002). Since SOD1 does not contain mitochondrial targeting sequence, the true physiological function of SOD1 in the IMS remains mostly

In mammalian cells the mitochondrial localization of SOD1 is regulated by the folding state of this enzyme, depending on the intracellular distribution of copper chaperone for SOD1 (CCS), which in turn is regulated by oxygen concentration. Redox status of the cysteine residues in human SOD1 is critical for its retention in mitochondria. The cysteine residues form intramolecular disulphide bonds and interact with CCS (Kawamata & Manfredi, 2008). This regulation appears to be impaired for SOD1 mutants, which can lead to misfolding and aggregation of mutant SOD1 and eventually result in SOD1 accumulation inside the mitochondria. In animal models the mitochondrial association of mutant SOD1 is apparent even before the disease onset (Liu et al., 2004), indicating a causative link of mitochondrial

Even though SOD1 has been suggested to be an important part of the mitochondrial superoxide scavenging system, as previously demonstrated in the yeast (Sturtz et al., 2001) and rat (Iñarrea et al., 2005) mitochondrion IMS, SOD1 activity is kept under redox control in this compartment and undergoes activation upon increased hydroperoxide concentration

There are 4 cysteines in the human SOD1 molecule, located at 6, 57, 111 and 144 position of the sequence. The intramolecular disulphide bridge between Cys57 and Cys146 is required for the proper tertiary and quaternary structure and enzymatic activity of SOD1 (Arnesano et al., 2004). Diminished copper loading and reduced intramolecular disulphide bound has been thought to be responsible for increased aggregation potential of G93A and D90A

The maturation and activation of SOD1 in the cytosol is controlled by a number of factors and can be divided in several principal steps. Upon post-translational activation, an SOD1 monomer binds a Zn2+ ion. Next ,CCS transiently binds to SOD1 monomer and inserts a Cu2+ ion in the molecule (Culotta et al., 1997; Casareno et al., 1998). After the dissociation of SOD1 from CCS, oxidative formation of disulphide bounds takes place (Arnesano et al., 2004; Ding & Dokholyan, 2008), which is followed by dimerisation yielding an active SOD1

Active SOD1 dimers are not capable of entering mitochondria, in contrast to disulphide reduced apo-SOD1. According to the model proposed (Kawamata & Manfredi, 2008; Reddehase et al., 2009), CCS is first imported into mitochondria by interaction with Mia40, an IMS component critical for protein import to mitochondria. The CCS-Mia40 complex is formed through an intermolecular disulphide bound (Fig. 1.). Further disulphide rearrangement generates oxidized CCS, preventing its escape from the IMS. The activation of SOD1 in IMS is thought to be similar to the activation of SOD1 in cytosol, where SOD1 binds to CCS in the presence of Cu2+ ions and oxygen generating an active enzyme retained

Surprisingly, CCS overexpression in G93A-SOD1 mouse, a widely used transgenic mouse model of ALS, produces severe mitochondrial pathology and accelerates disease course (Son et al., 2007). According to the model above, the potentiation of mutant SOD1 toxicity by CCS overexpression can be explained by the CCS-mediated increase in SOD1 mitochondrial

In contrast to the model of CCS-dependent activation of mitochondrial SOD1, a number of recent studies suggest that SOD1 in the IMS of intact mitochondria is mostly inactive and an

enigmatic.

SOD1 to the initiation of pathology.

mutant SOD1 (Jonsson et al., 2006).

molecule (Vonk et al., 2010).

in IMS (Leitch et al., 2009).

import, leading to enhanced SOD1 aggregation.

(Iñarrea et al., 2005).

oxidative modification of its critical thiol groups is necessary for the activation (Iñarrea et al., 2005, 2011; Goldsteins et al., 2008). This activation, at least partly, depends on protein disulphide isomerase (PDI) activity (Iñarrea et al., 2005). On the other hand, the toxicity of mutant SOD1 is not correlated with its aggregation potential but with the ability to form active dimeric molecules (Witan et al., 2008). These findings are in concert with a concept that mitochondrial dysfunction and cell damage are paradoxically induced by SOD1 mediated hydroperoxide production in the IMS (Goldsteins et al., 2008).

Fig. 1. Import and activation of SOD1 in IMS. CCS is imported into mitochondria through formation of a complex with Mia40 (I). Disulphide-reduced SOD1 monomer enters IMS and acquires copper ion (Cu2+) with a help of CCS (II). Formation of intramolecular disulphide bound and dimerisation of SOD1 creates an active SOD1 molecule retained in IMS (III).
