Mutations in superoxide dismutase 1 (SOD1): the first and hitherto most common cause of familial ALS
Mutations in the SOD1 gene (chromosome 21) remain the most common cause of familial ALS (Rosen et al., 1993). They are found in ∼20% of the families and thus account for ∼2% of all ALS.
SOD1 is an enzyme of 153 amino acid residues, ubiquitously expressed and active as a homodimer. It catalyses the conversion of superoxide free radicals to hydrogen peroxide, which can be further detoxified to water and oxygen by glutathione peroxidase or catalase. It should be distinguished from SOD2 (a mitochondrial SOD) and SOD3 (an extracellular SOD).
Missense mutations, affecting almost every amino acid residue of the protein (and a few small deletions and insertions, in addition to rare C-terminal truncating non-sense mutations) are known to give rise to familial ALS, irrespective of their effect on dismutase activity (Borchelt et al., 1994; Robberecht et al., 1994; Rosen et al., 1994). Transgenic mice or rats overexpressing mutant SOD1 develop motor neuron degeneration with progressive muscle weakness, muscle wasting and reduced life span (Gurney et al., 1994; Ripps et al., 1995; Wong et al., 1995; Bruijn et al., 1997; Howland et al., 2002). In addition, spontaneous mutations in the SOD1 gene give rise to a recessively inherited age-dependent motor neuron disease in dogs (Awano et al., 2009). More recently, mutant SOD1 models have been generated in zebrafish (Lemmens et al., 2007) and Caenorhabditis elegans (Witan et al., 2008; Wang et al., 2009a), suitable for genetic and small compound screening (Fig. 1).
Figure 1. Animal models of mutant SOD1-induced motor neuron degeneration. (A) Mutant SOD1G93A mouse with weakness and muscle atrophy of the lower limbs, which is accompanied by (B and C) loss of large motor neuron cell bodies in the ventral horn of the spinal cord (C, compared to a control B). (D and E) Zebrafish embryos injected with mutantSOD1A4V RNA have reduced motor axon outgrowth with abnormal branching (F, compared to wild-type injected embryos G). An SV2-staining (in red) was used to visualize the motor axons.
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Not surprisingly, dysfunction of the axon, containing 99% of the motor neuron cytoplasm, is among the earliest manifestations of the mutant SOD1-induced degenerative process. This dysfunction appears as retraction of motor axons from neuromuscular junctions resulting in denervation and muscle weakness (Fischer et al., 2004). The pivotal significance of the axonal compartment explains the finding that preserving the cell body by interfering with the later stages of the degenerative process is insufficient to affect the clinical disease (Gould et al., 2006; Dewil et al., 2007a).
A toxic gain-of-function of the mutant protein underlies motor neuron toxicity, as these rodent models retain their endogenous SOD1 activity and SOD1-deficient mice have no overt phenotype of motor neuron degeneration (Reaume et al., 1996). The expression level of the SOD1 mutant protein for a given mutation determines disease severity, higher levels yielding a more aggressive phenotype. This has been well documented for the G93A-mutant SOD1 mouse model (Alexander et al., 2004; Fig. 2).
The mechanism through which mutant SOD1 induces motor neuron degeneration remains incompletely understood, even nearly two decades after their discovery, but most probably involves several (interacting) pathways rather than a single pathogenic mechanism.
A gain-of-function for mutant SOD1
SOD1 is an important enzyme in the defence against superoxide anions, most of which are inadvertent reaction products in the mitochondria due to incomplete efficiency (‘leakiness’) of oxidative phosphorylation. Many studies have reported the presence of oxidative damage to proteins, lipids or DNA in patients with familial or sporadic ALS as well as in several mutant SOD1 mice (Barber & Shaw, 2010). It remains uncertain whether these changes are primary or secondary in nature. Two oxidation-modified proteins are particularly worth mentioning. SOD1 itself was found to be heavily oxidized (Andrus et al., 1998); this may at least contribute to the newly acquired toxic property of the protein (Ezzi et al., 2007). Another interesting protein damaged by oxidation is the glial glutamate transporter EAAT2, which provides a direct link between mutant SOD1-induced toxicity and excitotoxic motor neuron death (see below; also Trotti et al., 1999).
It has been suggested that oxidative damage is caused by aberrant oxidation reactions catalysed by mutant SOD1. However, expression of a mutant SOD1 without any oxidoreductive activity (obtained by mutating the histidine residues that are necessary for copper loading of the protein) still results in motor neuron degeneration in the mouse (Wang et al., 2003). This suggests that its enzymatic activity is not needed for the protein to be pathogenic. Alternative mechanisms have been suggested. Mutant SOD1 may bind with greater affinity to Rac1 than wildtype SOD1 does. Rac1 is a protein that regulates Nox2, an active subunit of the NADPH oxidase complex (Harraz et al., 2007). Inappropriate activation of Nox2 results in hazardous production of superoxide anions. Of notice, deletion of Nox2 slowed disease progression and improved survival of mutant SOD1 mice (Marden et al., 2007).
Alternatively, oxidative stress may be induced by mitochondrial dysfunction caused by abnormal recruitment of mutant SOD1 to the mitochondrial compartment (Shi et al., 2010). In the mutant SOD1 mouse, mitochondria undergo vacuolar degeneration in motor neurons (Jaarsma et al., 2001; Liu et al., 2004; Pasinelli et al., 2004). Misfolded mutant SOD1 has been found to bind to the outer mitochondrial membrane in a cell- and tissue-specific manner (Liu et al., 2004; Vande Velde et al., 2008). This may result in increased leakiness of the mitochondria (with reduced energy production and increased free radical generation), interfere with their calcium-buffering capacity (important in excitotoxicity; see below) or initiate apoptosis.
Evidence for an unexpected and newly discovered function for mutant SOD1 came from the finding that this protein is aberrantly secreted by motor neurons. Mutant SOD1 interacts with chromogranin (CHB)A and B, and is shuttled into the secretory pathway (Urushitani et al., 2006). The extracellular mutant protein was found to be toxic for motor neurons (Zhao et al., 2010). Because of this finding, a possible association between (non-hereditary) ALS and the CHBA and -B genes has been investigated. One study has shown the P413L CHGB variant to be associated with sporadic ALS and to determine age at onset (Gros-Louis et al., 2009).
Mutant SOD1-induced motor neuron degeneration: a foldopathy?
The most generally accepted hypothesis on the pathobiology of mutant SOD1 relates to its propensity to aggregate (Shaw & Valentine, 2007). ALS-causing mutations in SOD1 often result in decreased protein stability or net repulsive charge, which affect the folding and assembly of SOD1 dimers (Nordlund & Oliveberg, 2008). When synthesized, a protein has to be folded properly, a complex process in which several chaperone systems aid. Failure of this process results in protein misfolding and accumulation. The cell attempts to correct this by activating the so-called unfolded protein response (UPR), which includes the upregulation of a variety of chaperone proteins. If no refolding of the protein can be obtained, the protein is exported from the endoplasmic reticulum (ER) to the cytosol, where the ubiquitinated protein is degraded by the proteasome system. This process is thought to be at play in ALS (Kanekura et al., 2009). Mutant SOD1 has been found to accumulate in the ER and to inhibit derlin-1, the protein that transports proteins destined to be degraded from the ER to the cytosol (Nishitoh et al., 2008). Furthermore, a decrease in proteasome activity has been found in mutant SOD1-overexpressing cells and tissue (Urushitani et al., 2002; Kabashi et al., 2004, 2008a; Cheroni et al., 2009). Mutant SOD1 thus induces ER stress, and may overload the UPR response and the proteasome system. Upregulation of ER stress molecules has been correlated with the vulnerability of motor neurons in mutant SOD1 mice (Saxena et al., 2009). Overexpression of heat-shock proteins (HSPs) in vitro rescues the cell from mutant SOD1-induced toxicity (Patel et al., 2005). Disappointingly, neither HSP27 nor HSP70 overexpression in vivo affected motor neuron degeneration in mutant SOD1 mice (Liu et al., 2005; Krishnan et al., 2008). It is obvious that overexpressing one component of this sophisticated system may be insufficient.
The misfolded mutant SOD1 that escapes the cellular degradation system may interact with aberrant binding partners (such as mitochondrial membranes or chromogranins; see above) or form oligomers which then may proceed to the formation of higher molecular species and finally aggregate into intracellular inclusions (Johnston et al., 2000; Rakhit et al., 2002; Ezzi et al., 2007; Teilum et al., 2009). It is thought but not certain that this process is toxic for the neuron. Which stage of formation of inclusions is responsible for toxicity is uncertain, as is the question whether wildtype SOD1, which can form heterodimers with mutant SOD1 or can be recruited to coaggregate with it, contributes to this toxicity (Bruijn et al., 1998; Jaarsma et al., 2000; Fukada et al., 2001; Lemmens et al., 2007; Wang et al., 2009b). Aggregates may deplete the cell of essential constituents by coaggregation, or may physically disturb cellular processes such axonal transport (axonal strangulation; De Vos et al., 2007). However, just like for many other neurodegenerative diseases, it remains unknown whether aggregation of mutant protein is a hazardous or a protective phenomenon. It may well be that the first stages of the process (oligomerisation) are toxic (Johnston et al., 2000; Wang et al., 2002) while the aggregates themselves are essentially inert.
Non-cell autonomous motor neuron death
The convergence of damage in non-neuronal cells surrounding motor neurons has a larger impact on motor neuron survival then initially anticipated (Ilieva et al., 2009). Several types of non-neuronal cells, such as microglia and astrocytes, are activated in the course of the neurodegenerative process in ALS (Hall et al., 1998). This neuroinflammatory response, probably provoked by signals from diseased motor neurons, has neurotrophic and neurotoxic properties.
The contribution of non-neuronal cells to the pathogenesis of motor neuron degeneration has been studied in mutant SOD1 mice, in which the transgene was excised in specific cell types. It was found that deleting mutant SOD1 from microglia slowed motor neuron degeneration but did not affect disease onset (Boillee et al., 2006). Interestingly, the activation of microglial cells was not affected, showing that this reaction itself is not harmful, a finding that is consistent with the observation that preventing T-lymphocyte activation in the ALS spinal cord reduces microglial activation but accelerates disease (Beers et al., 2008). Replacement of mutant SOD1 microglia with transplanted wildtype microglia had a beneficial effect (Beers et al., 2006), but inhibition of microglial proliferation had no effect on disease progression (Gowing et al., 2008). This shows that, at least in this mouse model, microglial cells containing the mutant protein have a detrimental effect. On the other hand, wildtype microglia appear to be protective (Chiu et al., 2008). The role of microglia as pathogenic and/or protective cells is complicated by the question whether hematogenic macrophages populate the adult spinal cord. Several of the conclusions drawn from earlier experiments are indeed questioned by recent experiments using parabiosis (Ajami et al., 2007; Mildner et al., 2007).
Deletion of mutant SOD1 from astrocytes also slowed disease progression in the mutant SOD1 mouse model (Yamanaka et al., 2008). Interestingly, microglial activation was reduced in this experiment, suggesting an interaction between the two cell types. The nature of the interaction between motor neurons and astrocytes is likely to be multifactorial (Van Den Bosch & Robberecht, 2008). Astrocytes may release toxic factors (Nagai et al., 2007) or provide surrounding cells with less trophic support. Few of the astrocytic factors or motor neuron targets have been identified to date. Reduced expression of the glutamate transporter EAAT2 in astrocytes (Rothstein et al., 1992, 1995) and a reduced astrocyte-induced upregulation of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit GluR2 (Van Damme et al., 2007) may enhance excitotoxic motor neuron death (see below). The transcription factor Nrf2, which regulates the expression of antioxidant enzymes containing an ARE element (antioxidant response element) was able to counteract the toxicity of mutant SOD1-containing astrocytes and prolong survival of mutant SOD1 mice (Vargas et al., 2008). Of major interest is the finding that transplanting wildtype astrocytes into the mutant SOD1 spinal cord delayed disease (Lepore et al., 2008).
Counterintuitively, deletion of mutant SOD1 from Schwann cells aggravated disease (Lobsiger et al., 2009), possibly via the dismutase effect of SOD1 in this cell type. Deletion from endothelial cells had no effect (Zhong et al., 2008) (see below). Influencing mutant SOD1 synthesis in muscle cells did not affect motor neuron degeneration in the mutant SOD1 mouse (Miller et al., 2006; Towne et al., 2008). However, overexpression of insulin-like growth factor isoforms exclusively in muscle did slow down progression (Dobrowolny et al., 2005). Therefore, the exact role of muscle in ALS remains an interesting topic of research.
Lowering mutant SOD1 as a therapeutic option
The removal of mutant SOD1, the primary cause of motor neuron toxicity, is an obvious therapeutic strategy. This has been achieved by the viral delivery of RNAi against SOD1 (Ralph et al., 2005; Raoul et al., 2005), by intracerebroventricular administration of antisense oligonucleotides (Smith et al., 2006) and by crossbreeding mutant SOD1 mice with mice that express an shRNA against mutant SOD1 (Xia et al., 2006). Hence, gene silencing holds great promise as a therapy for ALS (and in fact for many neurodegenerative diseases; Maxwell, 2009). The first clinical studies investigating the feasibility of these approaches in humans are under way.
As toxicity from aberrant secretion of mutant SOD1 is likely to play a role, targeting this pool of mutant SOD1 may be of interest. The burden of extracellular SOD1 could be reduced using an active or a passive immunization strategy, and this led to a slower disease progression in mutant SOD1 mice (Urushitani et al., 2007).
The SOD1 mouse as a model for studying ALS therapy
The mutant SOD1 mouse (and rat) has been used extensively to study compounds or approaches with possible therapeutic value (Turner & Talbot, 2008). The validity of this model has been questioned because some of the compounds with a positive effect in the mouse were negative in human studies. There may be other explanations. The effects observed in the mouse were often small, and may be easily missed in a clinically and genetically heterogenous human ALS population. Furthermore, the differences in pharmacokinetics between mice and humans were often largely neglected. In addition, the ‘positive’ results obtained in mice often came from (inadequately powered) studies in which administration of the compound began before disease onset, while in humans therapeutic trials are done in patients who have had ALS for at least one, sometimes even several, years.
The question is whether the mutant SOD1 mouse is a good model in which to study sporadic ALS. Obviously it is not ideal: sporadic ALS is definitely etiologically different from monogenic mutant SOD1-related familial ALS. Recent studies on transactivation response DNA-binding protein with molecular weight 43 kDa (TDP-43) suggest that there may also be a pathogenic difference, which will be discussed below.
TDP-43: a new player shifts attention to RNA and links familial with sporadic ALS
TDP-43 is a widely-expressed 414-amino-acid protein encoded by the TARDBP gene on chromosome 1 (Pesiridis et al., 2009; Geser et al., 2010). It has two RNA-binding domains and a glycine-rich domain in the C-terminal part, with which it binds to various heterogenous nuclear nucleoproteins (hnRNPs). It is more abundantly present in the nucleus than in the cytoplasm. The exact role of TDP-43 is incompletely understood, but it is thought to play a role in a variety of processes such as processing, stabilisation and transport of RNA (Buratti & Baralle, 2009; Geser et al., 2010). A well known example is its role in the splicing of cystic fibrosis transmembranous conductance regulator mRNA (Buratti et al., 2001). Of interest is the finding that another target for the action of TDP-43 in mRNA processing is the protein SMN, deficiency of which results in spinomuscular atrophy, an infantile or juvenile onset motor neuron disorder (Burghes & Beattie, 2009). Overexpression of TDP-43 enhances exon 7 inclusion during SMN splicing, a crucial event in yielding fully active SMN protein (Bose et al., 2008). SMN deficiency in its turn is thought to cause spinomuscular atrophy through defective RNA processing or transport (Burghes & Beattie, 2009). The possible link between SMN and TDP-43 is of major interest when thinking of a common pathway for motor neuron degeneration.
The more than 25 mutations found in the TARDBP gene are, primarily, missense mutations and are almost exclusively located in the C-terminal (glycine-rich) part of the protein (Lagier-Tourenne & Cleveland, 2009). There is also a truncating mutation in this gene (Daoud et al., 2009). TARDBP mutations are rare: they probably account for < 5% of familial ALS, i.e. < 1% of all ALS (Ticozzi et al., 2009a). The major interest in them comes from the finding mentioned above, that wildtype TDP-43 containing inclusions are found in the majority of sporadic ALS patients (Neumann et al., 2006; Fig. 3). Here, we will refer to this abnormal form of TDP-43 as TDP-43SALS/FTLD in contrast to ‘normal’ TDP-43, reminiscent of the naming in prion disease, where PrPC refers to the normal PrP and PrPSc refers to the pathogenic form of PrP in sporadic and infectious Creutzfeldt–Jakob disease; it does not differ from normal PrPC in its amino acid sequence. Mutant TDP-43 refers to the mutant proteins causing the hereditary forms of ALS, just as with mutant PrP and Creutzfeldt–Jakob disease, and will be referred to as TDP-43mutant.
Figure 3. TDP-43 containing inclusions in a motor neuron in the spinal cord of a sporadic ALS patient (courtesy of T Tousseyn and R Sciot, Pathology, University Hospital Leuven).
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An overwhelming number of papers on the role of TDP-43 in neurodegeneration have been published over the last 2 years. A common finding seems to be that TDP-43mutant and TDP-43SALS/FTLD are mislocated, hyperphosphorylated, abnormally processed and ubiquitinated. The distribution of TDP-43 pathology spreads far beyond the motor system, suggesting the biological multisystem nature of the condition we clinically know as ALS (Geser et al., 2009).
TDP-43mutant and TDP-43SALS/FTLD are mainly present in the cytoplasm and appear to be depleted in the nucleus (Neumann et al., 2006; Winton et al., 2008; Sumi et al., 2009; Barmada et al., 2010). It therefore has been suggested that depletion of TDP-43 in the nucleus results in failure of RNA metabolism in this compartment, possibly resulting in the generation of abnormal splice variants. Alternatively, mRNA species in the cytoplasm that require the action of TDP-43 may be mistargeted or even degraded. Of interest in this regard is the finding that TDP-43 interacts with NF-L (neurofilament-light) mRNA, which may play a pathogenic role in ALS (Strong et al., 2007; Strong, 2010). Ongoing studies aim to identify RNA abnormities in TDP-43SALS/FTLD and TDP-43mutant cells and to establish their pathogenic role. This is obviously not easy given the large number of RNA species and the need to use unbiased approaches. In addition, it should be noted that these studies should not be limited to mRNAs, as recent studies have identified a role for microRNAs in neurodegeneration in general and in ALS in particular (Williams et al., 2009).
Mislocation may also result in pathogenicity due to a cytoplasmic gain-of-function rather than nuclear depletion (loss-of-function). There appears to be a correlation between cytoplasmic expression of TDP-43 or its C-terminal fragments and toxicity in vitro (Johnson et al., 2009; Nonaka et al., 2009; Zhang et al., 2009; Barmada et al., 2010), but it remains to be demonstrated that this is a causal correlation.
TDP-43mutant and TDP-43SALS/FTLD also appear to be abnormally processed, as C-terminal small molecular weight species, and in particular a fragment with a molecular weight of 25 kDa, are found in disease conditions (Neumann et al., 2006; Hasegawa et al., 2008). It has been suggested that caspase-3 is a TDP-43-processing enzyme (Zhang et al., 2007, 2009; Dormann et al., 2009). Expression of C-terminal fragments results in aggregate formation in vitro (Igaz et al., 2009), but the specificity of this processing and its significance for the pathogenesis remains to be shown (Dormann et al., 2009; Nishimoto et al., 2010). Of interest, the cleavage appears to be region-specific. In spinal cord, most of the TDP-43 recovered is full length (Igaz et al., 2008). TDP-43mutant and TDP-43SALS/FTLD are also hyperphosphorylated (the S409/410 sites are best characterized; Hasegawa et al., 2008; Inukai et al., 2008; Kametani et al., 2009; Neumann et al., 2009). Again, it is unclear whether these are primary or secondary modifications (Dormann et al., 2009).
Overexpression of TDP-43mutant in zebrafish results in a phenotype resembling that seen with overexpression of mutant SOD1 (Lemmens et al., 2007; Kabashi et al., 2010). Knockdown of TDP-43 results in a similar motor neuron phenotype (Kabashi et al., 2010), which suggests an essential role of endogenous TDP-43 for motor axon development, as has also been suggested in Drosophila (Feiguin et al., 2009; Li et al., 2010), but which is unlikely to be a model for nuclear depletion through cytoplasmic sequestration. The essential role of TDP-43 for early embryonic development in mammals has recently been shown using an elegant gene-trap approach, demonstrating early lethality of TARDBP-knockout mice (Sephton et al., 2010). TDP-43 is a developmentally regulated protein essential for early embryonic development. Loss of murine TDP-43 disrupts motor function and plays an essential role in embryogenesis (Kraemer et al., 2010). Interestingly, the heterozygous knockout mice (TARDBP+/−) showed signs of motor dysfunction, although no abnormalities in their motor neurons were apparent.
Overexpression of mutant TDP-43A315T driven by the prion promoter in mouse yielded expression of the transgene in neurons and glial cells throughout the nervous system and resulted in degeneration of motor neurons and of layer V cortical neurons (Wegorzewska et al., 2009). Expression of the TDP-43A315T was about three-fold that of endogenous TDP-43. These mice developed a paralyzing disease characterised by loss of upper and lower motor neurons. Interestingly, degenerating neurons contained ubiquitinated aggregates which, in spite of thorough investigation, did not contain the mutant TDP-43A315T. Loss of TDP-43 immunoreactivity from the nucleus was seen occasionally but did not seem to be a prominent finding. On the other hand, 25-kDa fragments appeared early in the disease. Unfortunately, this study did not report the findings in wildtype TDP-43-overexpressing mice.
Not surprisingly, based on the effects seen in other models such as Drosophila (Feiguin et al., 2009; Li et al., 2010), overexpression of human wildtype TDP-43 driven by the Thy1 promotor in mice gave rise to a phenotype as well. This promoter results in postnatal neuronal expression of the transgene. Expression of wildtype TDP-43 to a degree similar to that of TDP-43A315T in the previous study resulted in no phenotype. When increasing the level of wildtype TDP-43 expression, animals developed gait abnormalities and showed evidence for degeneration of motor neurons and neurons in layer V of the frontal cortex (Wils et al., 2010). The severity of the phenotype was parallel to the degree of TDP-43wt expression. In the diseased neurons, nuclear and cytoplasmic aggregates of ubiquitinated and phosphorylated TDP-43 were found. In this study, C-terminal 25-kDa fragments were found in the nuclear fraction. In this report, no TDP-43mutant was overexpressed.
It is difficult to compare these two models. Overexpression of TDP-43 seems to be toxic and may switch TDP-43 into TDP-43SALS/FTLD. The presence of a mutation favours this switch, although it needs to be taken into account that, in the TDP-43mutant study, glial cells also expressed the transgene and this may contribute to the process of neuronal degeneration, as we have learnt from the SOD1 model. The wildtype TDP-43 study, however, suggests that expression limited to neurons appears sufficient to induce degeneration.
The question is how to interpret the many findings in terms of pathogenic mechanism at play in vivo, and thus in non-overexpression conditions. It remains uncertain whether the cleavage, phosphorylation and ubiquination of TDP-43 are important for pathogenicity or not. Propensity of TDP-43 to aggregate, further enhanced by the presence of mutations, is an almost universal finding (Johnson et al., 2009; Nonaka et al., 2009; Zhang et al., 2009), although the most relevant model generated hitherto did not contain TDP-43-containing aggregates (Wegorzewska et al., 2009). Furthermore, the significance of the depletion of TDP-43 from the nucleus (found in many but not in all studies) as an underlying ‘compartmental’ loss-of-function mechanism needs to be established. Alternatively, the sequestering of TDP-43 in the cytoplasm may be the underlying gain-of-function mechanism. Does cytoplasmic TDP-43 gain a toxic biochemical function? Is the formation of aggregates, or one of the (oligomeric) species that are a step in the dynamics of this process, the mechanism of disease? Are essential cellular constituents trapped into these aggregates, resulting in an ‘unrelated’ loss of function?
In summary, the finding of TDP-43 in ALS and FTLD neurons and the identification of TDP-43 mutations in familial ALS was a second leap forward in ALS research. It has drawn attention to the possible role of RNA processing in the pathogenic mechanism of these diseases, even though the involvement of RNA in the mechanism itself remains to be demonstrated (Lemmens et al., 2010). Of major importance is of course the possible involvement of TDP-43 in sporadic ALS. It looks as if TDP-43 may play a role similar to α-synuclein in Parkinson’s disease (PD) and amyloid precursor protein (APP) in Alzheimer’s disease (AD). α-Synuclein mutations are a rare cause of familial PD, and α–synuclein-containing inclusions are seen in the sporadic form of PD. APP mutations are a rare cause of AD, but abnormally processed APP under the form of Aβ is a hallmark of sporadic AD. APP and α-synclein overexpression give rise to AD and PD in humans. This has not been observed for TDP-43 in ALS yet.
Finally, it needs to be pointed out that, while TDP-43-containing aggregates are seen in the large majority of sporadic ALS patients, they were noted to be absent in many (Mackenzie et al., 2007; Robertson et al., 2007; Tan et al., 2007), but not all (Shan et al., 2009) studies on mutant SOD1 ALS. This may suggest that the mechanisms underlying mutant SOD1-induced motor neuron degeneration and that of sporadic ALS may be different. This still needs to be studied in depth but it has further fuelled the doubts about whether mutant SOD1 models are of use in studying sporadic ALS.
FUS/ TLS: further evidence for a central role of proteins involved in RNA processing
Recently, two studies identified mutations in FUS ( fused in sarcoma)/TLS (translocated in liposarcoma) as a cause of ALS in Cape Verde, the US, Australia and the UK (Kwiatkowski et al., 2009; Vance et al., 2009). FUS/TLS mutations were also found in other populations in Europe, Japan and the US and it is estimated that FUS/TLS mutations cause familial ALS in 4–5% of cases (Belzil et al., 2009; Blair et al., 2010; Chio et al., 2009b; Damme et al., 2009; Drepper et al., 2010; Ticozzi et al., 2009b; Corrado et al., 2010; Groen et al., 2010; Suzuki et al., 2010). In addition, one de novo truncation mutation was reported (Dejesus-Hernandez et al., 2010).
The FUS/TLS gene is located on chromosome 16. Also known as hnRNPP2, it belongs to the FET family of RNA-binding proteins and it is an hnRNP. The protein consists of an N-terminal region rich in glutamine, glycine, serine and tyrosine residues (QGSY region) immediately followed by a glycine-rich domain. It contains an RNA-recognition motif (RRM) and multiple arginine, glycine, glycine (RGG) repeats implicated in RNA binding, a zinc finger and a C-terminal region that is highly conserved (Lagier-Tourenne & Cleveland, 2009). FUS/TLS is involved in pre-mRNA splicing as well as in the export of fully processed mRNA to the cytoplasm and thus shuttles between the nucleus and the cytoplasm (Zinszner et al., 1997). It may also play an important role in transport of mRNA (Yoshimura et al., 2006). In addition, it is important in gene regulation and it was recently shown that FUS/TLS can serve as a transcriptional regulatory sensor of DNA damage signals leading to gene-specific repression of gene transcription (Wang et al., 2008). FUS/TLS is ubiquitously expressed and under normal conditions it is mainly localized in the nucleus (Hackl & Luhrmann, 1996). In cultured hippocampal pyramidal neurons, FUS/TLS was localized not only in the nucleus but also in the dendrites (Fujii et al., 2005). This punctuate dendritic localization was dependent on an intact microtubule and actin network, and activation of mGluR5 metabotropic glutamate receptors stimulated FUS/TLS accumulation at the spines of excitatory synapses (Fujii et al., 2005). FUS/TLS-knockout mice die immediately after birth (Hicks et al., 2000) or are rarely alive at weaning (Kuroda et al., 2000). In an outbred strain, FUS/TLS-knockout mice survived but showed male sterility and reduced fertility of females (Kuroda et al., 2000). It was reported that heterozygous FUS/TLS mice were indistinguishable from wildtype littermates (Kuroda et al., 2000). Neurons deficient in FUS/TLS showed abnormal spine morphology and lower spine density (Fujii et al., 2005).
It is estimated that FUS/TLS mutations account for ∼5% of familial ALS and thus again for < 1% of total ALS (Lagier-Tourenne & Cleveland, 2009). FUS/TLS-linked ALS is a dominant disease, except in the original Cape Verdian family in which the FUS/TLS mutation is recessive (Kwiatkowski et al., 2009). Although in-frame deletions and insertions have been reported, most mutations are missense and the majority are located in the last exon. Mutant FUS/TLS accumulates in the cytoplasm of neurons (Kwiatkowski et al., 2009; Vance et al., 2009). Interestingly, FUS/TLS is also a component of nuclear polyQ aggregates in a cellular model of Huntington’s disease, as well as in patients with polyQ diseases, indicating that changing FUS/TLS to an insoluble form may be a common process in polyQ diseases and ALS (Doi et al., 2008, 2010).
Our knowledge on the role of FUS/TLS in the pathogenesis of ALS is still limited. Whether the RNA processing function of the protein is relevant or whether the mutant protein acquires an unrelated toxic function is not yet known and is an area of intensive research.
Other causes of familial ALS
Several other genes have been identified, mutations in which cause ALS, but these mutations occur in a very limited number of patients (Van Damme & Robberecht, 2009) (Table 1).
Mutations in vesicle-associated membrane protein-associated protein B (VAPB) are mainly found in Brazil (Nishimura et al., 2004). VAPB is involved in the unfolded protein ER response mentioned above (Kanekura et al., 2009). Mutant protein (P56S is the most studied mutation) looses this function and makes motor neurons vulnerable to ER stress induced by unfolded proteins (Suzuki et al., 2009). Studies in Drosophila showed that VAPB fragments interact with the ephrin system and that mutants are not correctly processed, resulting in a loss of function (Tsuda et al., 2008). However, VAPB-mutant protein is also prone to misfolding and aggregation (Teuling et al., 2007; Tsuda et al., 2008), again suggesting that aggregation is involved in the gain-of-function mechanism of these dominant mutations.
A surprising and exciting observation is the identification of variants in factor-induced gene 4 (FIG 4), a phosphoinositide 5-phosphatase in ALS patients (Chow et al., 2009). This enzyme regulates PI(3,5)P2 levels, which are involved in autophagy (Ferguson et al., 2009). FIG 4 is known to cause CMT4J if the two alleles are mutated (Chow et al., 2009). Heterozygous loss-of-function mutations in FIG 4 are found in 2% of sporadic and familial ALS patients (Chow et al., 2009).
Figure 4. Schematic overview of the mechanism of excitotoxic neuronal death. Glutamate (Glt) released from the presynaptic neuron stimulates AMPA receptors present on the postsynaptic neuron. If the GluR2 subunit is absent in the AMPA receptor complex, excessive intracellular calcium can induce neuronal death. Under normal conditions, glutamate is cleared from the synaptic cleft by glutamate transporters (EAAT2) present in the astrocytes, a process that is disturbed in ALS. See text for more details.
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Angiogenin (ANG) mutations are found in both familial and sporadic ALS patients and will be discussed later.
Finally, we mention alsin, mutations in which cause recessive motor neuron disease, probably more resembling an infantile ascending paraparesis, and senataxin (SETX), mutations in which cause ALS4, which actually is more similar to a distal hereditary motor neuropathy with some pyramidal findings (Valdmanis et al., 2009). Dynactin (DCTN1) variants have been found in sporadic ALS patients (Munch et al., 2004, 2005) after the identification of the G59S mutation in the p150Glued subunit (encoded by DCTN1) of the dynactin complex in a family with a lower motor neuron syndrome with vocal cord involvement (Puls et al., 2003). The latter mutation has been modeled in mice (Laird et al., 2008). The contribution of DCTN1 variants to ALS may be limited (Vilarino-Guell et al., 2009). Studies investigating the possible pathogenic role of the DCTN1 variants are underway.