181 White matter microglia heterogeneity P2RY12 ALSP ALSP TMEM119/CD45 NAWM - MS Preactive lesion - MS HLA-DR HLA-DR P2RY12 TMEM119 AC - MS HLA-DR MLD c f e d a b Figure 2. Microglia involvement in CNS diseases. Microglia are involved in CNS white matter diseases. Microglia in the normal appearing white matter (NAWM) in multiple sclerosis (MS) show ramified morphology similar to healthy controls (a). Clustering of microglia may represent preactive lesions in MS, but these cells still express homeostatic markers TMEM119 and P2RY12 (b). In active MS lesions microglia undergo transcriptional and morphological changes associated with phagocytosis and display an amoeboid morphology (c). In leukodystrophies, white matter in ALSP (in the frontal lobe) is almost devoid of microglia and only few cells express the homeostatic microglia markers TMEM119 and P2RY12 (d, e). Highly activated and amoeboid microglia are a key characteristic of the pathology of the leukodystrophy MLD (f). Scale bar=50 µm. ALSP = adult-onset leuko-encephalopathy with axonal spheroids and pigmented glia, MLD = metachromatic leukodystrophy. CD45 and CD11b, have been used to differentiate microglia from other macrophages using flow cytometry25. Unlike CD11b-high monocytes and macrophages, microglia do not require Myb but depend on factors such as Pu.1 and Irf8 for their development26,27. Recently, microglia specific markers were identified- transmembrane protein 119 (TMEM119) and P2Y purinoreceptor 12 (P2RY12) - which distinguish microglia from invading myeloid cells and CAMs28,29. However, both TMEM119 and P2RY12 are downregulated in neurodegenerative diseases including AD30 and active multiple sclerosis (MS)18. Conversely, Hexb, which encodes for the beta subunit of the lysosomal enzyme hexosaminidase B, is a recently discovered microglia-enriched gene which is stably expressed even under neuroinflammatory or demyelinating conditions. In addition, Hexb is only faintly detected in CAMs and circulating myeloid cells indicating that Hexb can be used for specific microglia detection in the CNS31. Approaches to determine heterogeneity To study the roles of microglia in health and disease, several reporter mouse models have been developed. While mouse models do not always represent the full complexity of the human CNS32,33, they do provide the opportunity to study specific microglia states in vivo and to target microglia genes in a temporally-controlled manner in development, homeostasis, ageing and following injury. One such model, the CX3CR1GFP mouse, tracks cells expressing CX3CR1- a chemokine receptor involved in chemotaxis and cell survival during homeostasis and inflammation34. CX3CR1 is abundantly expressed by homeostatic microglia35, and promoter-driven GFP expression allows tracking of microglia. However, this does not distinguish between microglia and CAMs and also detects other macrophages, dendritic cells and natural killer cells under pathological conditions36,37. Nevertheless, tamoxifen inducible
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