The Microglial Component of Amyotrophic Lateral Sclerosis

Benjamin E. Clarke; Rickie Patani


Brain. 2020;143(12):3526-3539. 

In This Article

Microglial Molecular Changes in ALS

Changes in microglial gene expression in response to different stimuli have been described as M1 or M2 states, corresponding to whether they are deleterious or protective to neurons, respectively. However, this categorization of microglial states is regarded as an oversimplification as it is clear that microglia elicit graded and context-dependent responses when activated (Ransohoff, 2016; Ajami et al., 2018). It is more helpful to regard this as a reactive continuum, of which M1 and M2 are polar phenotypes. Nevertheless, specific gene expression signatures are likely to exist in ALS, across different subtypes of the disease and simultaneously involving both detrimental and protective factors (Chiu et al., 2013). The microglial expression profile has been shown to change during disease progression, as microglia from mSOD1 mice initially express an overall protective phenotype but undergo a transformation to an overall toxic state (Liao et al., 2012). This temporal transformation of microglial activation states has also been observed in other models of neurodegenerative conditions using single cell analysis, including models of Alzheimer's disease (Mathys et al., 2017), the EAE model of multiple sclerosis and a model of Huntington's disease (Ajami et al., 2018), with heterogeneity in activation states likely to be due to the chronicity and type of stimulus. Furthermore, microglia may elicit region-specific differences in response to either inflammatory cues or in disease states (Beers et al., 2011b; Nikodemova et al., 2014; Clarke et al., 2019). To add further complexity, certain factors may be protective or toxic in a stimulus-dependent manner. For example, the phosphoglycoprotein osteopontin has been shown to be detrimental in the EAE model but is protective in a model of spinal cord injury (Chabas et al., 2001; Hashimoto et al., 2007).

Factors Primarily Associated With Microglial Protection in ALS

Several factors including cytokines, secreted proteins, receptors and transcription factors are altered in ALS post-mortem tissue and mSOD1 models. It is important to note that the expression of these factors is not restricted to microglia and it is likely that many secreted proteins and cytokines released from astrocytes or infiltrating immune cells also play an important role in disease.

Several factors linked to microglial activation have been associated with protection in ALS (Figure 2). However, only a small number of microglial factors have been directly shown to confer neuroprotection in models of ALS. Several microglia-related cytokines associated with neuroprotection including IL-4, IL-10 and G-CSF have been reported to be altered in ALS patients and models (Jeyachandran et al., 2015; Lu et al., 2016; Moreno-Martinez et al., 2019a, b). Lentiviral delivery of IL-4 before the onset of disease in mSOD1 mice increased expression of other reportedly neuroprotective genes including Arg1, Retnla (Fizz1) and Chil3 (Ym1) and decreased genes associated with neurotoxicity including Tnfa, Ifng and Il1b (Rossi et al., 2018). IL-4 delivery also increased proliferation of microglia while delaying disease onset and improving motor function but did not affect motor neuron loss or lifespan of mSOD1 mice. Administration of IL-10 blocking antibodies increased microglial activation and accelerated onset of motor deficits in mSOD1 mice, whereas microglial-specific lentiviral upregulation of IL-10 delayed disease onset and extended survival of mSOD1 mice (Gravel et al., 2016). However, the effects of IL-10 on motor neuron death in mSOD1 mice were not investigated. Furthermore, G-CSF extended survival in mSOD1 mice, reduced microglial activation and was protective to motor neurons exposed to glutamate excitotoxicity in vitro, but did not rescue motor neuron death in mSOD1 mice (Pollari et al., 2011).

Figure 2.

Several factors have been either directly or indirectly implicated in affecting microglial activation and disease progression in ALS. Certain receptors including xCT and P2X7 have been linked to both neuroprotection and neurotoxicity depending on the stage of disease progression. Components of this figure were created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License.

Certain growth factors have been directly implicated in microglial neuroprotection. IGF-1 was reported to be decreased in neonatal mSOD1 microglial cultures (Xiao et al., 2007), but increased in later stages of disease in mSOD1 spinal cords (Chiu et al., 2013). Increasing levels of IGF-1 retrovirally reduced microglial activation associated with neurotoxicity, partially rescued motor neuron death, delayed disease progression and extended lifespan of mSOD1 mice (Kaspar et al., 2003; Dodge et al., 2008). Meanwhile, adeno-associated viral delivery of VEGF also partially rescued motor neuron death and motor function, delayed disease progression and was associated with a shift in microglial activation towards a neuroprotective phenotype (Wang et al., 2016). A neuroprotective role for the microglial enriched lectin, galectin-3, has also been proposed as deletion of Gal3 in mSOD1 mice accelerated disease progression and reduced survival (Lerman et al., 2012). Finally, microglial expression of the fractalkine receptor (CX3CR1) has been associated with neuroprotection in the mSOD1 mouse as mSOD1/CX3CR1 knockout mice displayed increased motor neuron loss and decreased lifespan (Cardona et al., 2006).

Factors Primarily Associated With Microglial Toxicity in ALS

Many factors that are either released from or detected by microglia have been correlated with neurotoxicity in ALS, but as with factors associated with neuroprotection, few have determined direct causality of motor neuron death in models of ALS. Eliminating the expression of one factor may lead to compensatory actions by others, making it difficult to delineate the contribution of individual factors to microglial neurotoxic responses in ALS.

Several cytokines are increased in ALS patients and models (Hensley et al., 2003; Moreno-Martinez et al., 2019a). However, few have been shown to directly alter pathology in models of ALS. While TNF-α levels are increased in both ALS patients (Poloni et al., 2000) and symptomatic mSOD1 mice (Hensley et al., 2003), deletion of TNF-α did not affect disease progression or microglial activation in mSOD1 mice (Gowing et al., 2006). Moreover, contradictory evidence has been reported over whether IL-1β affects mSOD1 disease progression. Deletion of Il1bextended lifespan and partially rescued motor neuron death of SOD1G93A mice (Meissner et al., 2010), but not SOD1G37R mice (Nguyen et al., 2001). Application of the cytokine M-CSF accelerated disease progression in mSOD1 mice, increasing microglial proliferation and proinflammatory signalling but did not significantly affect motor neuron loss (Gowing et al., 2009). In a recent study, inhibition of IFN-γ with blocking antibodies extended lifespan and improved motor function in mSOD1 mice and shifted microglial gene expression from a neurotoxic to a neuroprotective phenotype (Garofalo et al., 2020).

Upregulation of microglial receptors have also been observed in ALS. Increased expression of toll-like receptors TLR2 and TLR4 and co-receptor CD14 has been reported in post-mortem ALS spinal cord tissue (Henkel et al., 2004; Casula et al., 2011). Transfection of mSOD1 in microglial-like cells has been reported to result in activation of TLR2 (Liu et al., 2009), while deletion of TLR4 improved hindlimb strength and extended lifespan of mSOD1 mice (Lee et al., 2015). Furthermore, activation of proinflammatory signalling resulting from incubation of primary microglia with either mSOD1 (Zhao et al., 2010) or TDP-43 (Zhao et al., 2015) occurred via a CD14-dependent mechanism. The neuroregulin receptor 1 (NRGR1) has been associated with microglial toxicity in ALS as pharmacological inhibition reduced motor neuron death and microglial activation and delayed disease onset and progression in mSOD1 mice (Liu et al., 2018). Increased expression of several purinergic receptors has also been reported in mSOD1 microglia (D'Ambrosi et al., 2009; Cieślak et al., 2019). While P2X7 was implicated in motor neuron death as antagonism of microglial P2X7 rescued SH-SY5Y motor neuron like cells (D'Ambrosi et al., 2009), a more complex role for the P2X7 receptor has been proposed. While knockout of P2X7 accelerated disease in mSOD1 mice (Apolloni et al., 2013), antagonism immediately preceding the onset of symptoms partially ameliorated motor neuron loss and inflammation (Apolloni et al., 2014). Likewise, a dual role for the cystine/glutamate antiporter xCT, which is enriched in microglia, has been proposed as deletion of this receptor in mSOD1 mice initially hastened motor deficits but prolonged the later stages of disease (Mesci et al., 2015).

The transcription factor NF-κB is a master regulator of inflammatory responses that has been linked to microglial involvement in ALS models (Frakes et al., 2014). Several receptor complexes can cause the activation of NF-κB through phosphorylation of the p65 subunit, resulting in the translocation of NF-κB to the nucleus and the upregulation of several proinflammatory factors. Increased phosphorylated p65 has been shown to correlate with disease progression in mSOD1 mice and inhibition of microglial-specific NF-κB extended lifespan of mSOD1 mice (Frakes et al., 2014). Furthermore, inhibition of NF-κB rescued neurotoxicity of co-cultured mSOD1 microglia and reduced the levels of microglial proinflammatory cytokine release (Frakes et al., 2014). The mechanism of mSOD1 activation of NF-κB is not fully understood, but it has been suggested that the microRNA, miR-125b, promotes the activation of NF-κB in microglia by inhibiting the ubiquitin editing enzyme, A20 (Parisi et al., 2016). TDP-43 has also been shown to interact with and activate NF-κB (Swarup et al., 2011b). Inhibition of NF-κB was sufficient to improve motor neuron survival in a TDP-43 overexpressing mouse model (Swarup et al., 2011b). Furthermore, incubation of microglia with exogenous TDP-43 activated NF-κB and caused motor neuron death in co-culture (Zhao et al., 2015).

Inducible nitric oxide synthase (iNOS) is an enzyme that is upregulated in microglia following activation of NF-κB and is increased in the spinal cord of symptomatic mSOD1 mice (Almer et al., 1999). Inhibition of iNOS was shown to extend lifespan in mSOD1 mice, although the effect on motor neuron survival was not measured (Martin et al., 2007; Chen et al., 2010). iNOS converts the substrate L-arginine to nitric oxide (NO), a molecule that is able to cause motor neuron death in vitro (Zhao et al., 2004). Although NO has physiological roles in regulating vasodilation and synaptic transmission, NO can also react with superoxide species to produce the neurotoxic product, peroxynitrite (Calabrese et al., 2007). mSOD1 microglia have been found to produce more NO than wild-type microglia (Beers et al., 2006). NO production in mSOD1 microglia is thought to be NF-κB dependent, as inhibition of NF-κB signalling ablated NO production in mSOD1 microglia (Frakes et al., 2014).

Other sources of reactive oxide species (ROS) have been implicated in microglial-mediated motor neuron death in ALS. NOX2 is a ROS-producing catalytic subunit of the NADPH oxidase that has been implicated in neurotoxicity in ALS. Reduced NOX2 activity has been correlated with increased survival in ALS patients (Marrali et al., 2014) and NOX2 expression was reported to be increased in symptomatic mSOD1 microglia (Chiu et al., 2013). While earlier studies reported that inhibition of NOX2 with apocynin or deletion of NOX2 partially rescued motor neuron death and extended survival of mSOD1 mice (Wu et al., 2006; Marden et al., 2007; Harraz et al., 2008), these findings were not recapitulated by either genetic or pharmacological means (Seredenina et al., 2016). Together, few factors associated with microglial-mediated neurotoxicity have been directly shown to cause motor neuron death in ALS models. It is likely that a combination of factors act in concert to cause microglial-mediated motor neuron death in ALS.