Microglia control small vessel calcification via TREM2

Abstract

23Microglia participate in CNS development and homeostasis and are often implicated in 24 modulating disease processes in the CNS. However, less is known about the role of microglia 25 in the biology of the neurovascular unit (NVU). In particular, data are scant on whether 26 microglia are involved in CNS vascular pathology. In this study, we use a mouse model of 27 primary familial brain calcification (PFBC) -Pdgfb ret/ret to investigate the role of microglia in 28 calcification of the NVU. We report that microglia enclosing vessel-calcifications, coined 29 calcification-associated microglia (CAM), display a distinct activation signature. 30Pharmacological ablation of microglia with the CSF1R inhibitor -PLX5622 leads to aggravated 31 vessel calcification. Additionally, depletion of microglia in wild-type and Pdgfb ret/ret mice 32 causes the development of bone protein (osteocalcin, osteopontin) containing axonal spheroids 33 in the white matter. Mechanistically, we show that microglia require functional TREM2 for 34 controlling vessel-associated calcification. In conclusion, our results demonstrate that 35 microglial activity in the setting of pathological vascular calcification is beneficial. In addition, 36we identify a new, previously unrecognized function of microglia in halting the expansion of 37 ectopic calcification. 38 39 2 injury results in microglial activation with the rapid development of processes that shield a 57 lesioned blood vessel section and phagocytose debris 2 . These findings support the important 58 role of microglia in vascular repair. 59Optimal functioning of the NVU, which mediates hyperaemia, is crucial for cerebral 60 perfusion 4 . Blood vessels play an integral role in brain development and provide a niche for 61 brain stem cells. In addition, cerebral vasculature senses the environment and communicates 62 changes to neural tissue, participates in glymphatic clearance, and controls immune quiescence 63 in the CNS [11][12][13][14][15] . Accordingly, dysfunction of the NVU accompanies or may even represent a 64 primary cause of many neurodegenerative diseases 4,16 . In the case of the primary familial brain 65 calcification (PFBC), bilateral basal ganglia calcification of blood vessels is a key diagnostic 66 criterion. The pathogenic mechanism points to a compromised NVU 17-20 . PFBC is a clinically 67 and genetically heterogenous disease caused by mutations in at least in five genes -MYORG, 68 PDGFB, PDGFRB, SLC20A2 and XPR1 21-25 . Of note, recent studies have estimated the 69 minimal prevalence of PFBC ranges from 4-6 p. 10,000, depending of the causative gene 70 mutation, thus suggesting that PFBC is not a rare disorder and is likely underdiagnosed 26,27 . In 71 addition, basal ganglia calcification is a common radiological finding, estimated in up to 20% 72 3 of patients undergoing CT imaging 28,29 . Although the effect of cerebral calcification on the 73 NVU and brain parenchyma is unknown, peripheral vascular calcification can lead to 74 cardiovascular morbidity and mortality...

Adult-induced genetic ablation distinguishes PDGFB roles in blood-brain barrier maintenance and development

“…Presence of osteopontin (SPP1) has been noticed in small non-calcified vessel-associated nodules in Pdgfb ret/ret brain thalamus and midbrain regions. 28 While we could identify rare small SPP1 positive nodules associated with the vasculature in the thalamus and midbrain regions, these were found both in control and Pdgfb iECKO brains (Suppl. Fig.…”

“…Finally, since mice hypomorphic for Pdgfb and humans haploinsufficient for PDGFB develop vessel-associated calcifications in the brain, 9 we asked if adult-onset deletion of Pdgfb triggers capillary calcification. We performed immunohistochemical analysis of vessel-associated calcifications by staining for three different proteins that selectively accumulate in calcifications, amyloid precursor protein (APP), amyloid precursor like protein-2 (APLP2) and osteopontin (SPP1) 13,, 28 in old control and Pdgfb iECKO mice. Four to six 75 µm-thick sagittal sections were analyzed per mouse, with sections chosen to include both outer cortex and deep brain regions in the analysis, the latter known to harbor abundant calcifications in Pdgfb ret/ret mice.…”

“…[4][5][6][7]20,21 Adult-viable constitutive Pdgfb loss-of-function mutants with strongly reduced pericyte coverage acquire abnormal brain vasculature including general microvessel dilation and rarefaction, skewed endothelial arterio-venous zonation, reduced expression of specific transporters, two models of BBB leakage, and regional microvascular calcification. 9,10,[13][14][15]28,29 In this study, using an adult-inducible model of Pdgfb ablation, we demonstrate that endothelial-derived PDGFB is also required for adult maintenance of pericyte coverage, but that the slowly progressing pericyte loss that follows upon adult-induced Pdgfb ablation causes some, but not all, of the abnormalities observed in constitutive Pdgfb mutants.…”

“…Presence of osteopontin (SPP1) has been noticed in small non-calcified vessel-associated nodules in Pdgfb ret/ret brain thalamus and midbrain regions. 28 While we could identify rare small SPP1 positive nodules associated with the vasculature in the thalamus (d) qPCR analysis on the endothelial cell genes Fgfbp1, Bmp6 and Angpt2 performed on freshly isolated brain microvascular fragments in young and old mice (for litter and n number see Supplementary Table 1). The genes of interest were normalized to endogenous Gapdh levels (DCq) and these are presented as arbitrary units (a.…”

Bi-allelic LoF NRROS Variants Impairing Active TGF-β1 Delivery Cause a Severe Infantile-Onset Neurodegenerative Condition with Intracranial Calcification

“…[39][40][41] Recently, a Pdgfb knockout mouse model of primary familial brain calcification has been used to investigate the hypothesized association of microglial dysregulation with ICC formation. 42 Microglia depletion in Pdgfb knockout mice using the CSF1R inhibitor PLX5622 resulted in aggravated intracranial vessel calcification, which suggests a protective role for microglia in the development of ICC.…”