Short CommunicationTransformation from a neuroprotective to a neurotoxic microglial phenotype in a mouse model of ALS
Introduction
Amyotrophic lateral sclerosis (ALS) is a progressive and devastating neurodegenerative disease characterized by selective loss of upper and lower motoneurons in the cortex, brainstem, and spinal cord of the patient's central nervous system (CNS). Neuroinflammation is a prominent pathological feature in these CNS areas and characterized by the morphological activation of microglia (Appel et al., 2010). A similar set of neuroinflammatory responses, including microglia activation, occurs in mice overexpressing mutant Cu2 +/Zn2 + superoxide dismutase (mSOD1), a model of inherited ALS. However, until recently, there has been controversy as to whether the proliferation and activation of microglia in ALS promoted motoneuron survival or exacerbated the neurodegenerative process (Moisse and Strong, 2006). This controversy has begun to be resolved by the demonstration that wild-type (WT) microglia or microglia expressing less mSOD1 promoted neuroprotection and extended survival of ALS mice (Beers et al., 2006, Boillée et al., 2006). Furthermore, our in vitro studies utilizing primary neonatal microglia and embryonic motoneuron co-cultures provided evidence that WT microglia were less neurotoxic than mSOD1 microglia due to their enhanced release of neurotrophic factors, and attenuated release of free radicals and proinflammatory cytokines (Beers et al., 2006, Weydt et al., 2004, Xiao et al., 2007).
Microglia, as a component of the innate immune system, are of hematopoietic origin and colonize the CNS during early development (Beers et al., 2006, McKercher et al., 1996, Ransohoff and Perry, 2009). These immunocompetent cells display functional plasticity during activation involving changes in cell number, morphology, surface receptor expression, and production of growth factors and cytokines. Cumulative studies using diverse animal models of CNS injury have demonstrated that microglia have very distinct and different phenotypic states, and in line with other tissue macrophage populations, may exert either neurotoxic or neuroprotective responses depending on the physiologic conditions they encounter. Although an over simplification, microglial activation states can be divided into classically activated microglia (M1) and alternatively activated microglia (M2); the in vivo functions of microglia are probably better represented as a continuum between these two extreme activation states. M1 microglia are cytotoxic due to their secretion of reactive oxygen species (ROS) and proinflammatory cytokines, and increased level of NOX2 expression. In contrast, M2 microglia block proinflammatory responses, and produce high levels of anti-inflammatory cytokines and neurotrophic factors (Buechler et al., 2000, Gordon and Martinez, 2010, Tiemessen and Kuhn, 2007). Thus, the M1/M2 distinction should not necessarily invoke a concept of a cellular differentiation state, but as a spectrum of pro- versus anti-inflammatory responses. Therefore, a more complete understanding of how these distinct microglial phenotypes temporally influence the ALS disease pathoprogression process is warranted (Appel et al., 2009, Henkel et al., 2009).
Because the phenotypic and functional states of adult microglia during the pathoprogression of ALS have not been fully characterized, and because an earlier study using chimeric mice demonstrated that healthy WT motoneurons develop features of ALS pathology when surrounded by mSOD1-expressing glia, which reinforced the hypothesis that alterations of glial properties by the overexpression of mSOD1 contributes to the motoneuron injury (Clement et al., 2003), we characterized the adult mSOD1 microglial phenotypic states that exist over the course of disease in ALS mice when studied ex vivo using WT motoneurons with and without WT astroglia; we isolated and assessed the mSOD1 microglia for their cytokine and neurotrophic factor mRNA levels, and whether they were protective or toxic to WT motoneurons. The adult mSOD1 microglia were obtained from ALS mice that were either at the clinical onset of disease, during the initial slowly progressing phase, or near end-stage disease, during the rapidly progressing phase (Beers et al., 2008, Beers et al., 2011b). The results of this study indicate that early in the disease process, mSOD1 microglia expressed more M2 related mRNAs compared with near end-stage mSOD1 microglia; near end-stage microglia expressed more M1 marker. More importantly, when co-cultured with WT motoneurons, mSOD1 M2 microglia were neuroprotective and enhanced WT motoneuron survival than similarly co-cultured mSOD1 M1 microglia; end-stage mSOD1 M1 microglia were toxic to WT motoneurons. Although the addition of WT neonatal astroglia enhanced WT motoneuron survival when co-cultured with M2 microglia, they had only modest effects when included in cultures of end-stage disease M1 microglia and WT motoneurons. These data demonstrate the dual phenotypic and functional characteristics of mSOD1 expressing microglia over the course of disease, and again suggest that enhancing the M2 phenotypic state of microglia may have beneficial therapeutic effects.
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Experimental animals
mSOD1(G93A) mice on a C57Bl/6 genetic background were bred and maintained in The Methodist Hospital Research Institute's animal facility. All animals were housed in microisolator cages with access to food and water ad libitum, and all animals are specific pathogen free (SPF); sentinel mice are tested quarterly. All experimental procedures involving animals were approved by The Methodist Hospital Research Institute's Institutional Animal Care and Use Committee in compliance with National
End-stage adult mSOD1 microglia expressed diminished M2 mRNA levels
We previously demonstrated in vitro that in primary microglia/motoneuron co-cultures WT microglia were less neurotoxic than mSOD1 microglia due to their enhanced release of neurotrophic factors, and attenuated release of free radicals and proinflammatory cytokines (Beers et al., 2006, Xiao et al., 2007). However, these previous studies were conducted using neonatal microglia and were activated with lipopolysaccharide (LPS). Furthermore, we recently established in vivo that specific M2 markers
Discussion
Previous studies have shown that motoneuron death in ALS is non-cell-autonomous. An earlier report using chimeric mice demonstrated that healthy motoneurons develop features of ALS pathology when surrounded by mSOD1-expressing glia, reinforcing the hypothesis that alterations of glial properties by the overexpression of mSOD1 contribute to the motoneuron injury (Clement et al., 2003). Our previous reports, and that of others, have established that different microglial responses can mediate
Acknowledgments
We gratefully acknowledge A. Huang, J. Wang, X. Wang, S. Wen, M. Chen and D. Cridebring for their technical assistance. This work was supported by grants from the National Institutes of Health (NS70050 and NS067153) and the Muscular Dystrophy Association.
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Department of Neurology, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555, USA.