**6. Experimental evidence of complement involvement in ALS**

Many studies in animal models of ALS have shown the involvement of the complement system during disease progression, supporting findings in ALS patients (Table 2). Although the SOD1 gene mutation only accounts for 2% of total ALS cases, mouse models carrying over-expression of mutant SOD1 enzyme are widely used, as it leads to progressive symptoms which are very similar to the human condition.


Table 2. Experimental evidence of complement activation in animal models of ALS

The first study to demonstrate experimentally the involvement of complement factors in a SOD1 transgenic mouse model was performed by Perrin and colleagues in 2005. They isolated the ventral motor neurons from the lumbar spinal cord of SOD1G93A transgenic mouse using laser-capture micro-dissection and then using microarray analysis they detected increased levels of all subcomponents of C1q in these mice at early symptomatic and end stage when compared to motor neurons from wild-type mice (~5 and ~8 fold respectively) (Perrin et al., 2005).

Subsequent studies in two distinct SOD1 transgenic mouse models also used laser-capture micro-dissection to isolate lumbar motor neurons from SOD1G37R and SOD1G85R transgenic mice which showed upregulation of genes for all three C1q subcomponents when compared to SOD1WT mice 2 months prior to clinical onset (P105) (Lobsiger et al., 2007). In addition, this group demonstrated that the complement regulatory molecule, decay accelerating factor (DAF) also decreased at this time point (Lobsiger et al., 2007). Furthermore they showed that C1q protein was expressed by motor neurons using immunohistochemistry on spinal cord sections of both SOD1G37R and SOD1G85R transgenic mice but absent in the agematched control mice (Lobsiger et al., 2007).

Many studies in animal models of ALS have shown the involvement of the complement system during disease progression, supporting findings in ALS patients (Table 2). Although the SOD1 gene mutation only accounts for 2% of total ALS cases, mouse models carrying over-expression of mutant SOD1 enzyme are widely used, as it leads to progressive

**factors mRNA/Protein Transgenic model Reference** 

**C1q mRNA Mouse SOD1 G93A (Perrin et al., 2005)** 

**C1q, C4 mRNA/Protein Mouse SOD1 G93A (Ferraiuolo et al., 2007)** 

**C1q mRNA Mouse SOD1 L126delTT (Fukada et al., 2007)** 

**CD88 mRNA/Protein Rat SOD1 G93A (Woodruff et al., 2008a)** 

**CD88 mRNA/Protein Mouse NFL -/- (Humayun et al., 2009)** 

**C1q, C3 mRNA/Protein Mouse SOD1 G93A (Heurich et al., 2011)** 

The first study to demonstrate experimentally the involvement of complement factors in a SOD1 transgenic mouse model was performed by Perrin and colleagues in 2005. They isolated the ventral motor neurons from the lumbar spinal cord of SOD1G93A transgenic mouse using laser-capture micro-dissection and then using microarray analysis they detected increased levels of all subcomponents of C1q in these mice at early symptomatic and end stage when compared to motor neurons from wild-type mice (~5 and ~8 fold

Subsequent studies in two distinct SOD1 transgenic mouse models also used laser-capture micro-dissection to isolate lumbar motor neurons from SOD1G37R and SOD1G85R transgenic mice which showed upregulation of genes for all three C1q subcomponents when compared to SOD1WT mice 2 months prior to clinical onset (P105) (Lobsiger et al., 2007). In addition, this group demonstrated that the complement regulatory molecule, decay accelerating factor (DAF) also decreased at this time point (Lobsiger et al., 2007). Furthermore they showed that C1q protein was expressed by motor neurons using immunohistochemistry on spinal cord sections of both SOD1G37R and SOD1G85R transgenic mice but absent in the age-

Table 2. Experimental evidence of complement activation in animal models of ALS

**SOD1 G85R (Lobsiger et al., 2007)** 

**6. Experimental evidence of complement involvement in ALS** 

**C1q, DAF mRNA Mouse SOD1 G37R and** 

symptoms which are very similar to the human condition.

**Complement** 

respectively) (Perrin et al., 2005).

matched control mice (Lobsiger et al., 2007).

A separate group also used laser-capture microdissection to isolate the lumbar motor neurons from SOD1G93A transgenic mice. Using microarray analysis and real time quantitative PCR, they showed there were increased levels of C1q (subcomponent B) and C4 mRNA at disease onset (P90) and late-stage disease (P120) (~7 and ~8 fold respectively) (Ferraiuolo et al., 2007). A similar study also used microarray analysis in a separate SOD1 transgenic mouse model using whole lumbar spinal cord homogenate (Fukada et al., 2007). This study used SOD1L126delTT transgenic mice and showed elevated levels of C1q (subcomponent B) mRNA in post-symptomatic (P154) mice compared to wild-type mice. A very recent study has shown increased levels of C1q in the neuromuscular junction of SOD1G93A transgenic mice compared to wild-type mice (Heurich et al., 2011).

By contrast to the above studies, which indicates a role for the classical complement pathway in the progression of pathology of the SOD1 transgenic mouse, a recent study has demonstrated that when SOD1G93A transgenic mice were bred onto a background deficient in complement C4 (a necessary component of the classical complement pathway, downstream of C1q), there was a difference in the macrophage levels and activation in the peripheral nervous system but no difference in the onset of motor symptoms and survival when compared to wild-type mice (Chiu et al., 2009). This study indicates that other molecular pathways such as the alternative or extrinsic pathway may play compensatory roles in immune activation and macrophage recruitment in the absence of the classical pathway in these mice. To support this, recent studies in SOD1G93A transgenic mouse showed increases in the C3 mRNA and protein levels in the spinal cord when compared to wild-type animals at symptomatic stage (P126) (Heurich et al., 2011). They also observed upregulation of C3 at the motor end plate and nerve terminals in the SOD1G93A transgenic mice at pre-symptomatic stage (P47) when compared to wild-type animal (Heurich et al., 2011).

To further validate the involvement of downstream components of the complement cascade in the disease progression of ALS, upregulation of C5a receptor CD88 mRNA and protein was observed in mice deficient in the low molecular weight neurofilament (NFL) subunit protein, a mouse model of motor neuron degeneration in which neurofilament aggregates in a similar fashion to that in ALS patients (Humayun et al., 2009). This study showed there was a 4 and 3 fold increase in CD88 mRNA expression level at 2 and 3 months respectively, a time which is early in the disease process (Humayun et al., 2009). There was also an increased immunoreactivity of CD88 in motor neurons of NFL deficient mice when compared to wild-type mice at 3, 4 and 5 months. Our own findings also support a pathogenic role for C5a in ALS (Woodruff et al., 2008a). Chronic administration of a specific C5a receptor antagonist, developed in our laboratories (Wong et al., 1998) in SOD1G93A transgenic rats, markedly delayed the onset of motor symptoms and increased survival, compared to untreated animals (Woodruff et al., 2008a). We also showed upregulation of CD88 in the lumbar spinal cord of SOD1G93A transgenic rats, which increased as disease progressed (Woodruff et al., 2008a).

These findings of upregulated complement components in different animal models of ALS suggest that the activation of complement system is critically linked with disease progression in ALS. Whilst inhibition of one component of the classical and lectin complement pathway, C4, failed to ameliorate disease in SOD1G93A transgenic mice, inhibition of the classical receptor for C5a, CD88, reduced disease pathology in SOD1G93A transgenic rats. It should be noted that C5a is expressed following activation of all

Innate Immunity in ALS 403

a likely side effect of other inhibitors of complement which act more upstream in the system, were they are to be used chronically. Finally, PMX53, an analogue to PMX205 has already been shown to be safe when administered to humans, successfully completing three Phase I/IIa clinical trials, thus promoting the safety of these classes of drugs in humans (Woodruff

In addition to anti-complement agents, combined therapies targeting multiple and disparate pathways will most likely be needed to effectively treat ALS. Extensive controlled clinical trials will need to be conducted in order to ascertain any potential therapeutic benefit of a

There is increasing evidence that implicates the involvement of the innate immune system in the progression of ALS. In particular, the inappropriate activation or dysregulation of the complement system may play a role in ALS pathology. Evidence for this includes elevated levels of complement activation fragments in the serum, CSF, spinal cord and motor cortex of ALS patients. This has also been supported with elevated levels of complement activation fragments in various animal models of ALS. Moreover, inhibition of the C5a receptor using a specific C5a receptor antagonist ameliorated disease symptoms in a rat model of ALS. Collectively, these studies suggest that complement activation may play a crucial role in the progression of ALS. Hence reducing complement-induced inflammation using inhibitors to

target complement factors could be an important therapeutic strategy to treat ALS.

and Medical Research Council of Australia (Project Grants 455856 and APP1004455).

No.2, (April 2010), pp.389-401, ISSN 1471-4159.

We acknowledge the funding support of the Motor Neuron Disease Research Institute of Australia (Charles & Shirley Graham MND Research Grant 2010), and the National Health

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complement inhibitor to treat the devastating and intractable nature of ALS.

et al., 2011).

**8. Conclusion** 

**9. Acknowledgments** 

680, ISSN 1529-2908.

ISSN 0741-5400.

**10. References** 

complement pathways (Figure 1). Hence inhibiting central components of the complement system, at the C3 and C5 level, may have benefits in slowing disease progression in ALS, as opposed to inhibiting an individual activation pathway. Specifically, our studies suggest that inhibiting the pro-inflammatory C5 activation fragment, C5a, which is central to, and generated by, all complement pathways, may be a novel therapeutic strategy to treat ALS.
