The Microglial Component of Amyotrophic Lateral Sclerosis

Benjamin E. Clarke; Rickie Patani


Brain. 2020;143(12):3526-3539. 

In This Article

Lessons From Animal Models of ALS

Animal models have provided invaluable insights into microglial involvement in ALS. Mouse and human microglia are thought to share a large majority of genes, suggesting that animal models are a useful tool for the study of microglia (Galatro et al., 2017). However, it is important to note that differences in genes involved in immune function were found to exist between mouse and human microglia, which may be particularly relevant to neurodegenerative diseases such as ALS. Furthermore, much of what is known about the mechanisms of microglial involvement in ALS comes from studies of mouse models overexpressing mutant ALS proteins (Table 1). Although this caveat is partially addressed by using cells overexpressing wild-type protein as a control, the study of microglia expressing mutant genes at physiological levels is lacking.


Mutations in SOD1 were the first discovered to cause ALS (Rosen, 1993). Mouse models expressing mutant SOD1 (mSOD1) recapitulate key features of the disease including motor neuron loss, paralysis and premature death (Gurney et al., 1994). Elegant studies have concluded that neuron-specific expression of mSOD1 is not sufficient to recapitulate the full disease observed in mSOD1 mice and that non-neuronal cells play an important part in mSOD1 disease progression (Pramatarova et al., 2001; Clement et al., 2003; Yamanaka et al., 2008).

Microglial activation has been observed from the onset of disease in mSOD1 mouse models (Almer et al., 1999; Alexianu et al., 2001; Chiu et al., 2008). Removal of mSOD1 from CD11b expressing microglia using a Cre-lox system lengthened lifespan, but did not affect the onset of disease, suggesting that microglia are most important in the later phases of disease in mSOD1 mice (Boillee et al., 2006). Furthermore, pharmacological inhibition of CSFR1, a protein with roles in microglial proliferation, survival and maturation, reduced microglial activation and extended survival of mSOD1 mice (Martínez-Muriana et al., 2016). Proliferation of microglia during the symptomatic phase of disease may not play an important role in disease progression as ablation of part of the proliferating microglial cell population using mSOD1/CD11b-TKmt-30 mice treated with ganciclovir at symptomatic ages did not affect motor neuron survival (Gowing et al., 2008). However, as chronic systemic administration of ganciclovir was lethal, local delivery intrathecally into the spinal cord by osmotic pump was used instead in this study. This treatment left a significant number of proliferating microglia unaffected. Therefore, a reduced population of proliferating microglia may still be sufficient to effect the same level of damage in mSOD1 mice.

Expression of mSOD1 in microglia alone is not sufficient to cause motor neuron degeneration. Replenishment of microglia by transplantation of mSOD1 bone marrow at birth into PU1knockout microglia-deficient mice was not able to cause motor neuron loss even though transplanted mSOD1 microglia were morphologically more activated, with short ramified processes, than wild-type microglia (Beers et al., 2006). In terms of onset, survival and duration of disease, mSOD1/PU1knockout mice given mSOD1 bone marrow transplantation showed no differences to mSOD1 mice. However, mSOD1/PU1 knockout mice given wild-type bone marrow transplants survived for longer, had an extended disease duration and decreased motor neuron loss compared to mSOD1/PU1knockout mice given mSOD1 bone marrow transplants, suggesting that removal of mSOD1 from microglia is protective (Beers et al., 2006). While disease onset was reported not to be affected, there was a trend towards a delayed onset of symptoms in mSOD1/PU1 knockout mice given wild-type bone marrow transplants. However, it is important to note that this reduction in damage to mice given wild-type bone marrow transplants cannot be attributed solely to microglia as PU1 knockout affects all myeloid and lymphoid cells.

While early studies suggested that microglia only had a role in the late stages of disease, changes in microglia that occur before motor neuron loss may still be significant to disease pathomechanisms. Recent spatial transcriptomics of the mSOD1 mouse spinal cord showed that changes in microglial gene expression preceded changes in motor neurons (Maniatis et al., 2019). Furthermore, microglial numbers have been reported to be decreased before disease onset in mSOD1 mice (Gerber et al., 2012). Therefore, changes in microglia in the early disease phase, while more subtle, may still be relevant in ALS.

Further evidence for early phenotypes of mSOD1 microglia has come from studies of primary cultures from neonatal mSOD1 mice. Neonatal wild-type microglial cultures were toxic to motor neurons in co-culture after exposure to exogenous mSOD1 (Zhao et al., 2010; Roberts et al., 2013). Furthermore, neonatal microglia overexpressing mSOD1 were toxic to motor neurons in co-culture under basal conditions and were more toxic than activated wild-type microglia after stimulation by the bacterial endotoxin, lipopolysaccharide (LPS) (Xiao et al., 2007). However, another study found no differences in motor neuron survival in co-cultures with neonatal mSOD1 microglia, but instead reported neurotoxicity when co-cultured with adult mSOD1 microglia (Frakes et al., 2014). One potential limitation of mSOD1 mouse models is that mutations within the SOD1 gene, along with mutations in FUS, fall within the overwhelming minority of cases that do not exhibit the pathological hallmark of TDP-43 nuclear-to-cytoplasmic mislocalization. It follows that other gene mutations may capture ALS pathomechanisms with greater fidelity and precision.


Mutations in TARDBP, encoding TDP-43, only account for ~5% of familial ALS cases, but mislocalization of wild-type TDP-43 is observed in >95% of all ALS cases (Neumann et al., 2006). TDP-43 is an RNA-binding protein involved in several roles in RNA regulation. Both wild-type and mutant TDP-43 have been reported to stimulate cytokine production in primary mouse microglia (Zhao et al., 2015). Furthermore, mice overexpressing wild-type or mutant TDP-43 displayed increased immunoreactivity of microglia at symptomatic ages (Swarup et al., 2011a). It has been suggested that TDP-43 plays an important role in microglial phagocytosis, since microglial-specific knockout of TARDBP enhanced amyloid clearance and synaptic loss in an Alzheimer's disease mouse model (Paolicelli et al., 2017). In a reversible mouse model of doxycycline suppressible neuronal human TDP-43 containing a defective nuclear localization signal, only small changes in microglial activation were observed preceding and during an aggressive disease course of 50% motor neuron loss and death within 10 weeks (Spiller et al., 2018). However, when TDP-43 expression was switched off during disease, mice recovered motor function and microglia became activated and cleared neuronal TDP-43. Microglia were shown to directly play a neuroprotective role in this process as pharmacological suppression of microglia negatively affected recovery of motor function (Spiller et al., 2018).


Mutations in another RNA binding protein, FUS, also account for close to 5% of familial ALS cases (Kwiatkowski et al., 2009; Vance et al., 2009). Interestingly, mislocalization of FUS has been recently found to occur in motor neurons of sporadic ALS cases, suggesting a role in the majority of ALS cases (Tyzack et al., 2019). However, the importance of microglial FUS in ALS is only beginning to be understood. Exacerbation of proinflammatory cytokine production occurred in primary microglial cultures after incubation with astrocyte conditioned media from astrocyte cultures overexpressing wild-type FUS (Ajmone-Cat et al., 2019). Mice overexpressing wild-type FUS also display microglial activation in the spinal cord (Mitchell et al., 2013). Furthermore, both pro- and anti-inflammatory microglial activation has been reported in mice expressing a truncated FUS protein lacking a nuclear localization signal, both before and after disease onset (Funikov et al., 2018), suggesting that mislocalization of FUS is sufficient to result in microglial activation.


An intronic hexanucleotide repeat expansion in C9orf72 is the most common cause of familial ALS cases (DeJesus-Hernandez et al., 2011; Renton et al., 2011). It is thought that both a gain-of-function mechanism through production of dipeptide repeats (DPRs) and a loss-of-function mechanism contribute to C9orf72-mediated pathology (Balendra and Isaacs, 2018). In mice, C9orf72 is highly expressed in microglia and while no neuronal death is observed in C9orf72−/− mice, microglial activation has been reported (O'Rourke et al., 2016). In a mutant C9orf72 mouse model expressing the neurotoxic DPR, poly-GA, immunization against poly-GA reduced microglial activation (Zhou et al., 2020). Furthermore, in a recent study, reduction of C9orf72 expression in a mouse model with a repeat expansion resulted in hippocampal microglial activation, implicating both gain and loss of C9orf72 function in modulating microglial activation (Zhu et al., 2020).