Transformation from a neuroprotective to a neurotoxic microglial phenotype in a mouse model of ALS

Abstract

Neuroinflammation is a prominent pathological feature in the spinal cords of patients with amyotrophic lateral sclerosis (ALS), as well as in transgenic mouse models of inherited ALS, and is characterized by activated microglia. Earlier studies showed that activated microglia play important roles in both motoneuron protection and injury. More recent studies investigating the pathoprogression of disease in ALS mice have demonstrated that the in vivo activation states of microglia, including their anti- versus pro-inflammatory responses, are best characterized as a continuum between two extreme activation states which are represented as a neuroprotective M2 (alternatively-activated) phenotypic state or an injurious/toxic M1 (classically-activated) state; a more complete understanding and determination the temporal transformation of microglia activation states in the ALS disease pathoprogression is therefore warranted. In the current study, we demonstrated a phenotypic and functional transformation of adult ALS mice microglia that overexpress mutant superoxide dismutase (mSOD1). mSOD1 microglia isolated from ALS mice at disease onset expressed higher levels of Ym1, CD163 and BDNF (markers of M2) mRNA and lower levels of Nox2 (a marker of M1) mRNA compared with mSOD1 microglia isolated from ALS mice at end-stage disease. More importantly, when co-cultured with motoneurons, these mSOD1 M2 microglia were neuroprotective and enhanced motoneuron survival than similarly co-cultured mSOD1 M1 microglia; end-stage mSOD1 M1 microglia were toxic to motoneurons. Our study documents that adult microglia isolated from ALS mice at disease onset have an M2 phenotype and protect motoneurons whereas microglia isolated from end-stage disease ALS mice have adopted an M1 phenotype and are neurotoxic supporting the dual phenotypes of microglia and their transformation during disease pathoprogression in these mice. Thus, harnessing the neuroprotective potential of microglia may provide novel avenues for ALS therapies.

Fig. 1

End-stage mSOD1 and M1 phenotype are toxic to motoneurons. (A) End-stage mSOD1 microglia express decreased levels of Ym1 mRNA than 11 week old mSOD1 or WT microglia. (B) CD163 mRNA expression is decreased in end-stage mSOD1 microglia compared with 11 week old mSOD1 or WT microglia. (C) End-stage mSOD1 microglia express less BDNF mRNA than 11 week old mSOD1 or WT microglia. (D) End-stage mSOD1 microglia express 5-fold more NOX2 mRNA than 11 week old mSOD1 microglia. Both ages of mSOD1 microglia express more NOX2 than their age-matched WT counterparts. (E) The number of surviving WT motoneurons in co-cultures with end-stage mSOD1 microglia is decreased compared with either motoneurons co-cultured with WT microglia or 11 week old mSOD1 microglia. (F) When co-cultured with end-stage mSOD1 microglia, the average neurite number on the WT motoneurons was decreased compared with WT motoneuron co-cultured with WT microglia or 11 week old mSOD1 microglia. (G) Immunochemical evaluation with SMI-32 antibody showed motoneuron morphological alterations. When co-cultured with end-stage mSOD1 microglia, motoneurons were less healthy compared with motoneurons co-cultured with WT or 11 week mSOD1 microglia; there were fewer motoneurons and the remaining motoneurons had smaller somas and shorter neurites. (H) The numbers of surviving WT motoneurons in cultures with WT astroglia and 11 week old mSOD1 microglia were increased compared with similar cultures containing WT astroglia and WT microglia. (I) When cultured with 11 week old mSOD1 microglia and WT astroglia, WT motoneurons had longer neurites compared with cultures with WT microglia. ⁺p<0.05, end-stage WT MC vs. end-stage mSOD1 MC; ^#p<b0.05, 11 weeks mSOD1 MC vs. end-stage mSOD1 MC; ⁺⁺p<0.01, end-stage WT MC vs. end-stage mSOD1 MC; ^††p<0.01, 11 weeks WT MC vs. 11 weeks mSOD1 MC; *p<0.05, MN co-cultured with WT MC vs. MN co-cultured with end-stage mSOD1 MC; **_p<0.01, MN co-cultured with WT MC vs. MN co-cultured with end-stage mSOD1 MC; ^&p<0.05, MN co-cultured with 11 weeks mSOD1 MC vs. MN co-cultured with end-stage mSOD1 MC; ‡p<0.05, MN/AC co-cultured with WT MC vs. MN/AC co-cultured with 11 weeks mSOD1 MC; ^¶p<0.05, MN/AC co-culture with 11 weeks mSOD1 MC vs. MN/AC co-culture with end-stage mSOD1 MC. Scale bar on panel G=50 μm. MN = motoneurons, MC = microglia, and AC = astroglia.