Transgenics, toxicity and therapeutics in rodent models of mutant SOD1-mediated familial ALS

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Abstract

Gain-of-function mutations in the Cu,Zn-superoxide dismutase (SOD1) gene are implicated in progressive motor neuron death and paralysis in one form of inherited amyotrophic lateral sclerosis (ALS). At present, transgenic expression of 12 human SOD1 mutations driven by the endogenous promoter is disease-causative and uniformly lethal in mice and rats, despite tremendous biochemical and biophysical variation between the mutants tested. This contrasts with the subclinical motor neuron disease phenotypes of wild-type SOD1 transgenic and knockout mice. Molecular mechanisms such as glutamate-induced excitotoxicity, axonal transport blockade, mitochondrial dysfunction, neuroinflammation and apoptosis triggered by mutant SOD1 catalysed oxidative reactions and/or protein misfolding are proposed to drive ALS pathogenesis. Around 100 genetic cross-breeding experiments with transgenic mutant SOD1 mice have been performed to verify these mechanisms in vivo. Furthermore, mounting evidence from mice with cell restrictive, repressible or chimeric expression of mutant SOD1 transgenes and bone marrow transplants supports non-neuronal origins of neuroprotection in ALS. Transgenic mutant SOD1 rodents have also provided the benchmark preclinical tool for evaluation of over 150 potential therapeutic anti-oxidant, anti-aggregation, anti-glutamatergic, anti-inflammatory, anti-apoptotic and neurotrophic pharmacological agents. Recent promising findings from gene and antisense therapies, cell replacement and combinatorial drug approaches in transgenic mutant SOD1 rodents are also emerging, but await successful translation in patients. This review summarises the wealth of known genetic and therapeutic modifiers in rodent models with SOD1 mutations and discusses these in the wider context of ALS pathoetiology and treatment.

Introduction

Almost 140 years have elapsed since the commonest adult-onset motor neuron disease (MND) was termed amyotrophic lateral sclerosis (Charcot and Joffroy, 1869). Widely recognised as Lou Gehrig's disease in America, Charcot's disease in Europe and MND in the UK and Australia, ALS is a progressive and terminal neurodegenerative disorder characterised by paralysis of motor function due to a combination of voluntary muscle weakness, atrophy and spasticity. The disease derives its name from the combined degeneration of upper and lower motor neurons projecting from the spinal cord, brainstem and cortex (Rowland and Shneider, 2001). ALS primarily targets the large caliber alpha motor neurons of 35–100 μm diameter in the spinal anterior horns (Ravits et al., 2007a). Clinically, lower motor neuron loss usually appears focal and asymmetric in onset and radiates through contiguous anatomical segments especially evident in caudal spinal cord (Ravits et al., 2007b). ALS is strongly associated with middle to late age with a slight male-to-female bias of 1.3 and current estimates suggest a worldwide incidence of 2 per 100,000 and prevalence of 4 per 100,000, in keeping with the average short-term survival of 3–5 years (Hirtz et al., 2007). At present, there exists no prophylactic or curative treatment for ALS and the single prescribed anti-glutamatergic drug Rilutek (riluzole) may have a marginal and unsatisfactory effect in extending life by a few months (Bensimon et al., 1994). Thus, the need for significant disease-modifying therapies is paramount.

The majority of ALS cases are of unknown aetiology and classed as sporadic (SALS). A genetic contribution possibly underlies SALS as a number of susceptibility or modifier genes have been identified by association studies including angiogenin, apolipoprotein E, apurinic endonuclease, neurofilament heavy chain (NFH), peripherin, survival motor neuron and vascular endothelial growth factor (VEGF) as discussed elsewhere (Simpson and Al-Chalabi, 2006). However, the majority of association studies in ALS have not been replicated across all populations, suggesting that population specific oligogenetic susceptibility may operate or that these apparent associations are false positives. The 10% remaining cases are familial (FALS) and 6 disease-causative genes from 11 loci have been identified by linkage analysis and positional cloning as reviewed recently (Gros-Louis et al., 2006). Mutations in Cu,Zn-superoxide dismutase (SOD1) occur in autosomal dominant adult-onset ALS (ALS1) and account for 2–3% of ALS cases overall when considering familial disease (Rosen et al., 1993). It is also noteworthy that SOD1 mutations are reported in up to 7% of SALS cases (Andersen, 2006), though this is in the setting of a specialist ALS clinic population. Autosomal recessive juvenile-onset ALS (ALS2) is caused by loss-of-function mutations in the Rab5 guanine nucleotide exchange factor protein ALS2 or alsin (Hadano et al., 2001, Yang et al., 2001), while autosomal dominant juvenile-onset ALS (ALS4) is linked to DNA/RNA helicase senataxin (Chen et al., 2004). A single mutation in vesicle-associated membrane protein/synaptobrevin-associated membrane protein B (VAPB) was also described in a form of autosomal-dominant late-onset ALS (ALS8) (Nishimura et al., 2004). Lastly, mutations in tau and the p150 dynactin subunit were reported in ALS-dementia and lower MND, respectively (Hutton et al., 1998, Puls et al., 2003). It should be emphasised that only FALS patients with SOD1 mutations present with classic adult-onset Charcot-type ALS, while the other five genes are mutated in atypical ALS syndromes. SALS and FALS have long been regarded clinically and pathologically similar suggesting a unified pathogenesis; however, one biochemical difference appears to be the absence of pathologic TDP-43 in FALS due to SOD1 mutation, a hallmark of affected neurons in SALS and frontotemporal dementia, in one study (Mackenzie et al., 2007), but not another (Robertson et al., 2007). However, the significance of abnormal TDP-43 species and their nuclear export in motor neuron degeneration remains uncertain.

Section snippets

SOD1 mutations

In 1993, a landmark discovery of 11 missense mutations in the antioxidant SOD1 gene in 13 FALS families was made (Rosen et al., 1993). This number has increased considerably to over 150 mutations at present, building on previous accounts (Andersen et al., 2003, Andersen, 2006). SOD1 is a ubiquitously expressed cytosolic metalloenzyme of 153 amino acids encoded by 5 exons. The mutations encompass all coding regions of the gene affecting over 70 positions with preponderance for exons 4 and 5 (

SOD1 mouse models

Ultimately, the evidence for disease causation of SOD1 mutations in ALS rested on functional modeling and disease recapitulation in animals. This approach with transgenic mice expressing mutant SOD1 is described below; however, the interpretation of these models requires some consideration of wild-type SOD1 overexpressing and knockout mice.

Genetic modifiers of the transgenic ALS mouse phenotype

SOD1 represents one of the most intensely characterised proteins in biochemistry, yet only the power of genetic linkage analysis established a pathogenic role for SOD1 in inherited motor neuron degeneration. The dominant age-penetrant disease phenotype of transgenic ALS mice strongly supports a neurotoxic gain-of-function property of mutant SOD1 that accumulates damage over time. Fourteen years after these discoveries, the biochemical nature of this property and the reason underlying its

Therapeutic modulation of the transgenic ALS mouse phenotype

Mice expressing mutated SOD1 transgenes on identical backgrounds have the same aetiology and are isogenic, predicting reliable disease onset, duration and endpoint times. In theory, this makes them ideal animal models for preclinical and experimental evaluation of potential ALS therapeutics. Currently, over 150 different potential therapeutic agents or strategies have been tested in transgenic ALS mice according to published trials (Table 6). This list quite literally spans from (a)spirin to

SOD1 rat models

Despite the intense focus on genetic mouse models of ALS in the last decade, transgenic rats overexpressing SOD1 mutations were recently developed with the obvious advantage of increased size for experimental manipulation. Reassuringly, these rats developed progressive neuromuscular symptoms suggestive of MND and are discussed below.

Therapeutic modulation of the transgenic ALS rat phenotype

At present, eight different therapeutic agents have been evaluated in transgenic ALS rats according to publication (Table 8). Chronic intraspinal infusion of human SOD1WT protein into transgenic SOD1G93A rats significantly slowed disease duration and endpoint, but not onset (Turner et al., 2005). This sharply contrasts with the neutral or deleterious effects of SOD1WT overexpression on the phenotype of transgenic ALS mice (Bruijn et al., 1998, Deng et al., 2006, Jaarsma et al., 2000),

Conclusion

ALS has been described as one of the most incapacitating diseases of our species (Ludolph, 2006), which is no understatement. Current estimates of ALS epidemiology do the disease burden little justice. In most cases, it strikes sporadically without obvious cause and paralyses with terminal ferocity. Despite almost 140 years since its initial clinical description, the reason for its selective lethality to motor neurons remains frustratingly elusive. The discovery of SOD1 mutations in 20% of

Acknowledgments

Work for this review was supported by grants from the Australian NHMRC to BJT (359269), the ALS Association and the MND Association.

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