Mean SEM; n=6 4-6-week old mice per group; n=7 12-16-week old mice per group; n=5 36-48-week old mice per group. Fibrinogen and fibrin fibrils initiated autophagy-dependent cell death in oligodendrocyte and pericyte cultures, whereas pharmacological and genetic manipulations of systemic fibrinogen levels in pericyte-deficient, but not control mice, influenced the degree of white matter fibrin(ogen) deposition, pericyte degeneration, vascular pathology and white matter changes. Thus, pericytes control white matter structure and function, which has implications for the pathogenesis and treatment of human white matter disease associated with small vessel disease. Introduction White matter is composed of myelinated axon tracts that maintain connections between individual neurons in different grey matter regions. Diffuse white matter disease is prevalent in the elderly, and is associated with small vessel disease1, which contributes to approximately 50% of all dementias worldwide including Alzheimer’s disease (AD)2C4 Individuals with AD develop early white matter changes5,6 with loss of oligodendrocytes and axons7 concomitant with cerebral vessel pathology, loss of vascular integrity, and blood flow reductions8C11. Despite the prevalence and clinical significance of age-related white matter disease associated with small vessel disease, the underlying biological mechanisms remain elusive. Here, we investigated whether brain capillary pericytes embedded in the wall of smallest brain vessels12C14 play a role in white matter health and disease. Pericytes control microvascular functions in neuron-dense grey matter regions including blood-brain barrier (BBB) permeability15C17 and cerebral blood flow18C22. They die in AD10,23C26 mild dementia27, stroke19,20 and cerebral autosomal dominant arteriopathy with subcortical infarcts (CADASIL), the most common genetic ischemic small vessel disease associated with cognitive impairment28. Nonetheless, the role of pericytes in the pathogenesis of these disorders, particularly the white matter lesions, is still poorly understood. It is also unclear if pericytes can control vascular integrity and blood flow in white matter axon tracts, which lack neuronal cell bodies. To address these questions, we studied microcirculatory changes in relation to white matter integrity in pericyte-deficient mice carrying 2′-Hydroxy-4′-methylacetophenone seven point mutations in platelet-derived growth factor receptor (PDGFR), which disrupts PDGFR signaling in vascular mural cells causing pericyte loss29. Adult mice are viable15,17, but develop early pericyte loss causing BBB breakdown and microvascular reductions15,17,29, without appreciable early involvement of vascular smooth muscle cells (VSMCs)30, making them a valuable model to study effects of pericyte loss on neurovascular and brain functions. Results Loss of white matter pericyte coverage and capillary integrity in AD Consistent with previous reports examining grey matter brain regions in post-mortem AD tissue23C26 here we observed a 50% loss of pericyte coverage and a 3-fold greater accumulation of blood-derived extravascular fibrin(ogen) deposits (indicative of capillary leakage and loss of vascular integrity) in the subcortical white matter of AD patients compared to controls (Fig 1a-c; Table S1). This has been shown by immunostaining for pericyte marker PDGFR14,17, fluorescent staining of endothelial-specific marker lectin17, and immunostaining of fibrin(ogen), with quantification analysis of pericyte coverage and fibrin(ogen) extravascular deposits. The microvascular pathology in AD white matter was associated with 50% loss of oligodendrocytes, as shown by immunostaining for oligodendrocyte lineage transcription factor 2 (Olig2)31, as well as loss of myelin, as indicated by immunostaining for myelin basic protein (MBP)31 (Fig. S1), consistent with previous findings in the white matter in AD7. Open in a separate window Figure 1 White matter microvascular changes in Alzheimer’s disease and pericyte-deficient mice(a) PDGFR-positive pericyte coverage (magenta), lectin-positive endothelial profiles (green), and extravascular fibrin(ogen) deposits (red) in the prefrontal subcortical white matter of an age-matched control (Braak I, upper) and AD case (Braak VCVI, lower) (bar = 20 m). (b, c) Quantification of pericyte coverage (b) and fibrin(ogen)-positive extravascular deposits (c) in the prefrontal subcortical white matter of controls (n=15) and AD cases (n=16). Mean .Animals also received TXA dissolved in drinking water at 2′-Hydroxy-4′-methylacetophenone 25 mg/mL, as previously reported41. circuits leading to white matter functional deficits before neuronal loss occurs. Fibrinogen and fibrin fibrils initiated autophagy-dependent cell death in oligodendrocyte and pericyte cultures, whereas pharmacological and genetic manipulations of systemic fibrinogen levels in pericyte-deficient, but not control mice, influenced the degree of white matter fibrin(ogen) deposition, pericyte degeneration, vascular pathology and white matter changes. Thus, pericytes control white matter structure and function, which has implications for the pathogenesis and treatment of human white matter disease associated with small vessel disease. Introduction White matter is composed of myelinated axon tracts that 2′-Hydroxy-4′-methylacetophenone maintain connections between individual neurons in different grey matter regions. Diffuse white matter disease is prevalent in the elderly, and is associated with small vessel disease1, which contributes to approximately 50% of all dementias worldwide including Alzheimer’s disease (AD)2C4 Individuals with AD develop early white matter changes5,6 with loss of oligodendrocytes and axons7 concomitant with cerebral vessel pathology, loss of vascular integrity, and blood flow reductions8C11. Despite the prevalence and clinical significance of age-related white matter disease associated with small vessel disease, the underlying biological mechanisms remain elusive. Here, we investigated whether brain capillary pericytes embedded in the wall of smallest brain vessels12C14 play a role in white matter health and disease. Pericytes control microvascular functions in neuron-dense grey matter regions including blood-brain barrier (BBB) permeability15C17 and cerebral blood flow18C22. They die in AD10,23C26 mild dementia27, stroke19,20 and cerebral autosomal dominant arteriopathy with subcortical infarcts (CADASIL), the most common genetic ischemic small vessel disease associated with cognitive impairment28. Nonetheless, the role of pericytes in the pathogenesis of these disorders, particularly the white matter lesions, is still poorly understood. It is also unclear if pericytes can control vascular integrity and blood flow in white matter axon tracts, which lack neuronal cell bodies. To address these questions, we studied microcirculatory changes in relation to white matter integrity in pericyte-deficient mice carrying seven point mutations in platelet-derived growth factor receptor (PDGFR), which disrupts PDGFR signaling in vascular mural cells causing pericyte loss29. Adult mice are viable15,17, but develop early pericyte loss causing BBB breakdown and microvascular reductions15,17,29, without appreciable early involvement of vascular smooth muscle cells (VSMCs)30, making them a valuable model to study effects of pericyte loss on neurovascular and brain functions. Results Loss of white matter pericyte coverage and capillary integrity in AD Consistent with previous reports examining grey matter brain regions in post-mortem AD tissue23C26 here we observed a 50% loss of pericyte coverage and a 3-fold greater accumulation of blood-derived extravascular fibrin(ogen) deposits (indicative of capillary leakage and loss of vascular integrity) in the subcortical white matter of AD patients compared to settings (Fig 1a-c; Table S1). This has been shown by immunostaining for pericyte marker PDGFR14,17, fluorescent staining 2′-Hydroxy-4′-methylacetophenone of endothelial-specific marker lectin17, and immunostaining of fibrin(ogen), with quantification analysis of pericyte protection and fibrin(ogen) extravascular deposits. The microvascular pathology in AD white matter was associated with Mouse monoclonal to GFAP 50% loss of oligodendrocytes, as demonstrated by immunostaining for oligodendrocyte lineage transcription element 2 (Olig2)31, as 2′-Hydroxy-4′-methylacetophenone well as loss of myelin, as indicated by immunostaining for myelin fundamental protein (MBP)31 (Fig. S1), consistent with earlier findings in the white matter in AD7. Open in a separate window Number 1 White colored matter microvascular changes in Alzheimer’s disease and pericyte-deficient mice(a) PDGFR-positive pericyte protection (magenta), lectin-positive endothelial profiles (green), and extravascular fibrin(ogen) deposits (reddish) in the prefrontal subcortical white matter of an age-matched control (Braak I, top) and AD case (Braak VCVI, lower) (pub = 20 m). (b, c) Quantification of pericyte protection (b) and fibrin(ogen)-positive extravascular deposits (c) in the prefrontal subcortical white matter of settings (n=15) and AD instances (n=16). Mean SEM. Observe Supplementary Table 1 for medical and neuropathological characteristics. (d) Representative blood-axon barrier permeability constant (and age-matched littermate control (+/+) mice generated from dynamic contrast-enhanced magnetic resonance imaging (MRI) scans. (e) The regional CC ideals in 4-6-, 12-16-, and 36-48-week older (green) and age-matched littermate control (+/+; blue) mice. Mean SEM; n=6 4-6-week older mice per group; n=7 12-16-week older mice per group; n=5 36-48-week older mice per group. (f, g) CD13-positive pericyte protection (magenta) and lectin-positive endothelial profiles (blue) in the CC of 12-week older and control (+/+) mice (f, pub = 40 m), and quantification of pericyte protection in the CC of 2-, 4-6-, 12-16-, and 36-48-week older (green) and control (+/+, blue) mice (g). Mean SEM; n=6 mice per group. (f, h) Fibrin(ogen)-positive extravascular deposits (green) and lectin-positive endothelial profiles (blue) in the CC of 12-week older and control (+/+) mice (f, pub = 40 m), and quantification of fibrin(ogen) deposits in the CC of 2-, 4-6-, 12-16-, and 36-48-week older (green) and control (+/+, blue).