Background

2.1.1 Microglial activation status in ALS

Microglial activation is a major pathology in the spinal cord of ALS patients and SOD1 mice, as discussed in Chapter 1. Microglial activation is commonly seen in the spinal cord of SOD1 animal models at disease onset, and increases with disease progression (Hall et al. 1998b; Almer et al. 1999; Chiu et al. 2008; Gowing et al. 2008; Beers et al. 2011b; Yang et al. 2011), although microglial activation has also been demonstrated to precede disease onset in some studies (Alexianu et al. 2001; Graber et al. 2010; Sanagi et al. 2010). Microglial activation may play an active role in disease progression, as replacing SOD1 microglia with WT microglia in SOD1 mice modulated the rate of disease progression (Boillee et al. 2006; Lee et al. 2012). SOD1 mice are thought to show a biphasic pattern of disease symptoms, with a brief plateau after onset where symptoms progress slowly, followed by a more rapid decline in functional ability (Beers et al. 2011a). This switch, from slowly-progressing to rapidly-progressing phases of disease, is thought to correspond to a switch between anti-inflammatory and pro- inflammatory phenotypes of microglial activation (Beers et al. 2011b; Liao et al. 2012). Microglia can show two phenotypes of activation, which are best characterised by protein expression and cytokine production. The M1-like, neurotoxic, pro-inflammatory phenotype of microglial activation is characterised by the elevated expression of pro- inflammatory cytokines and production of reactive oxygen species and nitric oxide (NO); whereas the M2-like, neuroprotective, anti-inflammatory phenotype of microglial activation is characterised by increased expression of anti-inflammatory cytokines and reduced production of free radicals (Colton 2009; Appel et al. 2011).

The microglial production of NO is a key factor in the toxicity of activated microglia to motor neurons (Zhao et al. 2004; Zhao et al. 2006; Thonhoff et al. 2012). Microglial production of NO is controlled by the inducible nitric oxide synthase enzyme (iNOS), which converts L-arginine to L-citrulline, producing NO in the process (Andrew & Mayer 1999). L-arginine, the iNOS substrate, is also metabolised by another enzyme, arginase-1 (Arg1), which converts L-arginine to L-ornithine and urea but does not produce NO (Ash 2004). The relative expression of these two L-arginine-metabolising enzymes, iNOS and Arg1, determines how much NO will be produced from a given cell (Gobert et al. 2000). Accordingly, high levels of Arg1 and low levels of iNOS are

______________________________________________________________________ 42 associated with the M2-like, neuroprotective microglial phenotype, while high levels of iNOS and low levels of Arg1 are associated with the M1-like, neurotoxic microglial phenotype (Colton 2009).

The reported switch between M2-like and M1-like microglial phenotypes in mouse models of ALS has mainly been investigated using cytokine mRNA expression levels (Beers et al. 2011a). It is therefore of interest to determine whether changes in iNOS and Arg1 protein expression also occur over time in the microglia of SOD1 mice, and how these changes are temporally related to the development of motor neuron pathology and functional deterioration, as any such protein changes may be reflective of a change in microglial phenotype. As oxidative damage from microglial-produced NO is thought to injure motor neurons, the expression of the antioxidant-response protein, metallothionein-1/2 (MT-1/2), should also be examined in the spinal cord of SOD1 mice to examine the evolution of oxidative stress over the disease course. The timing of oxidative stress response induction may relate to the production of NO by microglia, which in turn may be increased by any shift from an M2-type (Arg1-expressing) microglial phenotype to an M1-type (iNOS-expressing) microglial phenotype.

2.1.2 Measuring disease progression in SOD1 mice

As the underlying aetiology of ALS is as yet unknown, current therapeutic strategies involve addressing the known pathologies which occur in ALS patients and SOD1 mouse models, such as oxidative stress and neuroinflammation. Addressing these pathologies may limit disease processes, and slow disease progression. It is therefore necessary to track the functional decline in SOD1 mice over time, in order to evaluate any beneficial effects of therapeutic compounds. The age of onset, rate of symptom progression, and survival times vary in SOD1 mice according to the level of mutant SOD1 protein expression, gender, and genetic background strain (Alexander et al. 2004; Heiman-Patterson et al. 2005; Acevedo-Arozena et al. 2011; Bame et al. 2012).

The SOD1 colony maintained at the University of Tasmania expresses the SOD1 transgene on a congenic C57BL/6 background. As this colony was to be used for evaluation of potential disease therapeutics (see Chapters 3, 4 and 5), it was necessary to establish baseline measurements of functional decline over time.

______________________________________________________________________ 43 2.1.3 Aims and hypothesis

This study had two aims. First, to characterise the spinal cord microglia changes in Arg1 and iNOS expression over time in SOD1 mice, and to examine these changes in the temporal context of the appearance of pathological changes in motor neurons, induction of oxidative stress responses, and onset of functional deficits in SOD1 mice. Second, to establish baseline measures of functional decline in SOD1 mice compared to their wild-type littermates which could be used in future studies to monitor disease onset and progression.

Hypothesis: Microglia will shift their expression of L-arginine metabolising enzymes with disease onset: prior to disease onset, the expression of Arg1 will be prevalent, while the expression of iNOS will be prevalent after disease onset, indicating a shift in the spectrum of microglial phenotypes from an M2- predominant to an M1-predominant phenotype in the lumbar spinal cord of SOD1 mice.

Two cohorts of SOD1 mice and their WT littermates were examined in this chapter. Spinal cord samples were obtained from the first cohort at various ages between 6 and 25 weeks of age, and these samples were immunostained for the glial markers Iba1, tomato lectin, and GFAP, for the M1/M2 markers Arg1 and iNOS, for the pathological markers ubiquitin, neurofilament, and protein from the human SOD1 transgene, and for the antioxidant protein MT-1/2. The second cohort of SOD1 and WT mice were monitored for body weight, stride pattern, wire hang duration ability, and neurological score between 6 and 25 weeks of age, in order to characterise functional aspects of disease progression.

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