Astrocyte–microglial association and matrix composition are common events in the natural history of primary familial brain calcification

Abstract

Primary familial brain calcification (PFBC) is an age-dependent and rare neurodegenerative disorder characterized by microvascular calcium phosphate deposits in the deep brain regions. Known genetic causes of PFBC include loss-of-function mutations in genes involved in either of three processes-platelet-derived growth factor (PDGF) signaling, phosphate homeostasis or protein glycosylation-with unclear molecular links. To provide insight into the pathogenesis of PFBC, we analyzed murine models of PFBC for the first two of these processes in Pdgfb ret/ret and Slc20a2 −/− mice with regard to the structure, molecular composition, development and distribution of perivascular calcified nodules. Analyses by transmission electron microscopy and immunofluorescence revealed that calcified nodules in both of these models have a multilayered ultrastructure and occur in direct contact with reactive astrocytes and microglia. However, whereas nodules in Pdgfb ret/ret mice were large, solitary and smooth surfaced, the nodules in Slc20a2 −/− mice were multi-lobulated and occurred in clusters. The regional distribution of nodules also differed between the two models. Proteomic analysis and immunofluorescence stainings revealed a common molecular composition of the nodules in the two models, involving proteins implicated in bone homeostasis, but also proteins not previously linked to tissue mineralization. While the brain vasculature of Pdgfb ret/ret mice has been reported to display reduced pericyte coverage and abnormal permeability, we found that Slc20a2 −/− mice have a normal pericyte coverage and no overtly increased permeability. Thus, lack of pericytes and increase in permeability of the blood-brain barrier are likely not the causal triggers for PFBC pathogenesis. Instead, gene expression and spatial correlations suggest that astrocytes are intimately linked to the calcification process in PFBC.

Microglia control small vessel calcification via TREM2

“…Calcifications in Pdgfb ret/ret ;Trem2 +/+ animals appeared as single nodules (Fig. 7A, upper panels) in accordance with the reported phenotype of calcifications in Pdgfb ret/ret mice 18, 22, 42 . In addition to the altered pattern, vascular calcification was markedly aggravated in Pdgfb ret/ret ; Trem2 -/- and Pdgfb ret/ret ; Trem2 +/- mice (Fig.…”

“…Since CAM resemble amyloid β plaque-associated microglia in AD 8 (Fig. 2A-D) with protein deposits such as amyloid beta precursor protein (APP) and amyloid precursor-like protein (APLP2) 42 , we next asked whether protein aggregation also occurs in brain calcifications. In order to investigate this possibility, we used Thioflavin T and Congo red, standard stains to detect amyloid fibrils in amyloid β plaques, and a recently developed amyloid interacting luminescent conjugated oligothiophene (LCO) - h -HTAA.…”

“…We previously reported that cerebral vascular calcifications are positive for bone matrix proteins, including osteocalcin 18 . Moreover, a recent study identified that several non-bone proteins, including APP, are enriched in vessel-associated calcifications 42 . A noticeable increase in vascular calcification (approx.…”

“…Vascular calcification in PFBC is sometimes described as ‘pearls on a string’ due to numerous, tiny spherical calcifications that encrust almost the entire abluminal side of capillaries 19, 98 . Interestingly, calcifications appear as single nodules 18, 20, 22, 24, 42 in mouse models of PFBC. Variants in a microglia-specific gene, TREM2 , have been associated with an increased risk for AD 99 .…”

“…Previously, we described that mouse platelet-derived growth factor B ( Pdgfb ) hypomorphs ( Pdgfb ret/ret ) develop brain vessel-associated calcifications similar to human PFBC 22 . Vascular calcification in human PFBC and mouse models of PFBC were conspicuously encircled by activated microglia 18, 19, 22, 41, 42 and demonstrated an osteogenic environment with the surrounding cells expressing osteoclast markers 18 . This observation raised the question of the cellular origin and function of osteoclast-like cells.…”