Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective demise of upper motor neurons in the motor cortex and lower motor neurons in the brainstem and spinal cord [1, 2]. Disease onset occurs in mid-life (50 to 60 years of age) and is followed by a rapid (2 to 5 years), progressive failure of the neuromuscular system and death. Although the aetiology of ALS is yet to be fully elucidated, several factors are likely to contribute to motor neuron injury, including excitotoxic and oxidative motor neuron damage, protein aggregation, impaired axonal transport, mitochondrial dysfunction, and non-cell autonomous damage mediated through glial cells - astrocytes and microglia [3, 4]. Most of the current insights into disease pathogenesis come from studies on animal models overexpressing mutant forms of Cu/Zn superoxide dismutase 1 (SOD1) . Autosomal dominant inheritance of mutant SOD1 accounts for 20 percent of familial ALS (FALS) cases, or 2 percent of all ALS cases [6, 7]. Overexpression of mutant forms of SOD1, including G93A, G37R and G85R mutant SOD1, in animal models faithfully replicates pathological features of the human disease [8–10]. Motor neurons expressing mutant SOD1 can escape disease if surrounded by a sufficient number of normal non-neuronal cells . Conversely, normal motor neurons surrounded by mutant SOD1-containing non-neuronal cells developed signs of cellular injury with the development of ubiquitinated protein deposits . Selectively reducing the levels of mutant SOD1 in motor neurons delayed early disease progression and extended lifespan by a mean of 22 percent (64 days). In contrast, reducing mutant SOD1 expression in microglia, the major immune cell of the CNS with a monocyte/macrophage phenotype, had no effect on onset and early disease but showed a large protective effect in late stage disease and ameliorated disease progression with a mean extension of survival of 99 days . Moreover, a significant slowing of disease progression was observed in double transgenic G93A-SOD1/PU.1-/- mice when the mice received wild type but not G93A-SOD1 bone marrow transplant .
While the mechanisms of microglial disease propagation remain to be fully elucidated, studies indicate that mutant SOD1-overexpressing microglia may acquire an exaggerated inflammatory phenotype and neurotoxic properties following sustained activation. Low levels of inflammatory mediators are present in the cerebrospinal fluid of ALS patients [14–16] and activated microglia are detected in the CNS  and in the neighbourhood of degenerating motor neurons in post-mortem studies of the human disease . SOD1 transgenic mouse and rat models of ALS also display signs of an inflammatory response in the CNS at all stages of the disease. Prior to the clinical signs of disease onset, microglia are in an early state of activation, and elevated levels of inflammatory mediators such as interleukin (IL)-6 can be detected [19, 20]. With the onset of symptoms and motor neuron cell death, fully activated (or reactive) microglia are present in the CNS and microglial production of the pro-inflammatory cytokine, tumour necrosis factor (TNF)-α has been demonstrated [21–24]. Elevated levels of TNF-α, monocyte chemoattractant protein (MCP)-1, macrophage-colony stimulating factor (M-CSF), interferon (IFN)-γ and transforming growth factor (TGF)-β [15, 23, 25, 26] and an increase in cyclooxygenase (COX)-2 activity and prostaglandin (PG) E2 levels [14, 15, 23, 25, 27] have been shown in mutant SOD1 transgenic mouse tissues and microglial cells. Administration of drugs such as minocycline, or inhibitors of COX-2 and peroxisome proliferator-activated receptor (PPAR), capable of reducing microglial activation, delayed both disease onset and progression in mutant SOD1 transgenic mice [28–31].
It is unknown whether microglia overexpressing the wild type form of human SOD1 can acquire altered functional properties. Overexpression of wild type SOD1 in animals did not reveal any overt pathology at four months of age  except signs of deficiency of muscle innervation and premature aging [33–36]. Thus, wild type SOD1 could also contribute to neuronal pathogenesis. For example, autopsy material from familial as well as sporadic ALS cases revealed Lewy body-like hyaline inclusions within motor neurons that immunoreacted with anti-SOD1 antibodies . Overexpression of wild type SOD1 in mutant SOD1 transgenic animals accelerated the disease course and shortened the lifespan of double transgenic animals . Additionally, wild type SOD1 acquired toxic properties similar to those of the mutant forms of SOD1 following oxidative damage . Therefore, it is possible that wild type SOD1-overexpressing microglia may also have altered cellular properties rendering the cells capable of propagating neuronal damage.
In healthy animals, microglia perform a surveillance function to maintain a physiologically healthy microenvironment . They accomplish this by sampling the surrounding tissue with numerous extruding and retracting processes . Alterations in the tissue microenvironment induce microglial migration to the site of damage, scavenging of extruded cellular or plasma proteins and clearance of damaged cell components through phagocytosis [41, 42]. These dynamic effector functions of microglia are dependent on the presence of diverse surface receptors, including cytokine, chemokine, immunoglobulin, and purinergic receptors [43, 44]. Efficient intracellular signalling, control of gene expression, and tightly regulated function of the actin cytoskeleton are also necessary for appropriate microglial effector responses [45, 46]. Interestingly, in vivo recordings of labelled microglia from SOD1 G93A-overexpressing mice revealed significantly increased microglial response towards laser-induced single axon transection at preclinical age (60 days) when compared to that in control mice, and a subsequent reduction in SOD1 G93A microglial response to the same injury with disease progression (90 and 120 days) .
The purpose of the current study was to investigate whether overexpression of the mutant SOD1 transgene (TG G93A) or the wild type SOD1 transgene (TG WT) in microglia could significantly alter their functional properties, potentially contributing to neurodegeneration and its propagation. Due to inherent differences between the two colonies of transgenic mice that we observed in our studies, we compared the differences between NTG and TG cells within colonies, and not between colonies. Specifically, we examined non-transgenic (NTG: NTG (G93A) and NTG (WT)) and SOD1-overexpressing transgenic (TG G93A and TG WT) microglial surface expression of integrin β-1 (a subunit of integrin cell adhesion molecules), the ability of microglia to spread on fibronectin-coated surfaces and to migrate over astrocytic monolayers, the ability to phagocytose apoptotic neuronal cell debris, and intracellular calcium changes in response to a pro-inflammatory stimulus, extracellular ATP. Mutant SOD1 caused the most marked changes in these functions but overexpression of wild type SOD1 also produced significant changes. Thus, it is essential to examine the effects of both mutant and wild-type SOD1 when investigating the role of microglial cells in ALS.