Presenilin-1 regulates the constitutive turnover of the fibronectin matrix in endothelial cells

Abstract

BackgroundPresenilin-1 (PS1) is a transmembrane protein first discovered because of its association with familial Alzheimer’s disease. Mice with null mutations in PS1 die shortly after birth exhibiting multiple CNS and non-CNS abnormalities. One of the most prominent features in the brains of PS1−/− embryos is a vascular dysgenesis that leads to multiple intracerebral hemorrhages. The molecular and cellular basis for the vascular dysgenesis in PS1−/− mice remains incompletely understood. Because the extracellular matrix plays key roles in vascular development we hypothesized that an abnormal extracellular matrix might be present in endothelial cells lacking PS1 and examined whether the lack of PS1 affects expression of fibronectin a component of the extracellular matrix known to be essential for vascular development.ResultsWe report that primary as well as continuously passaged PS1−/− endothelial cells contain more fibronectin than wild type cells and that the excess fibronectin in PS1−/− endothelial cells is incorporated into a fibrillar network. Supporting the in vivo relevance of this observation fibronectin expression was increased in microvascular preparations isolated from E14.5 to E18.5 PS1−/− embryonic brain. Reintroduction of PS1 into PS1−/− endothelial cells led to a progressive decrease in fibronectin levels showing that the increased fibronectin in PS1−/− endothelial cells was due to loss of PS1. Increases in fibronectin protein in PS1−/− endothelial cells could not be explained by increased levels of fibronectin RNA nor based on metabolic labeling studies by increased protein synthesis. Rather we show based on the rate of turnover of exogenously added biotinylated fibronectin that increased fibronectin in PS1−/− endothelial cells results from a slower degradation of the fibronectin fibrillar matrix on the cell surface.ConclusionsThese studies show that PS1 regulates the constitutive turnover of the fibronectin matrix in endothelial cells. These studies provide molecular clues that may help to explain the origin of the vascular dysgenesis that develops in PS1−/− embryonic mice.

Age‐specific function of α5β1 integrin in microglial migration during early colonization of the developing mouse cortex

“…Though fibronectin was diffusely localized in the parenchyma, developing blood vessels marked by isolectin-GS-IB 4 (Figure 3e, e4) were highly immunoreactive for fibronectin (Figure 3e, yellow arrowheads in e1, e3 for zoom-in), as reported extensively in literature microglia were often found in close proximity with blood vessels (Figure 3e) (De Gasperi et al, 2012;Milner & Campbell, 2002b;Sheppard et al, 1991;Stewart & Pearlman, 1987). As previously described (Grossmann et al, 2002;Kierdorf et al, 2013;Pont-Lezica et al, 2011;Rigato et al, 2011).…”

“…Accordingly microglial migration speed was decreased in the newborn rabbit brain as a consequence of in utero inflammation and this was suspected to result from changes in adhesion molecule expression after inflammation (Zhang et al, 2016). 4.2 | Developmental decrease in fibronectin and microglial fibronectin receptor a5b1 integrin expression in the embryonic cortex All three parameters, microglial average migration speed, the cortical fibronectin deposition and microglial a5b1 integrin expression levels decreased from E13.5 to E17.5. Throughout development, fibronectin is highly expressed by blood vessels and along radial glial processes (De Gasperi et al, 2012;Milner & Campbell, 2002b;Sheppard et al, 1991;Stewart & Pearlman, 1987), which makes these structures ideal scaffolds to guide microglial migration. Throughout development, fibronectin is highly expressed by blood vessels and along radial glial processes (De Gasperi et al, 2012;Milner & Campbell, 2002b;Sheppard et al, 1991;Stewart & Pearlman, 1987), which makes these structures ideal scaffolds to guide microglial migration.…”

“…ECM proteins, such as fibronectin, are developmentally regulated (Sheppard et al, 1991). Nevertheless, the deposition pattern of fibronectin in the embryonic mouse brain remains controversial (De Gasperi et al, 2012;Sheppard et al, 1991;Stewart & Pearlman, 1987). To bring clarity on this pattern at the ages relevant for this study, fibronectin's deposition pattern in the embryonic cortex during the microglial colonization phase from E13.5 to E17.5 was analyzed using immunostaining ( Figure 3a) and cortical mean grey value quantification (Figure 3b).…”

“…Twenty-four different integrin heterodimers exist in vertebrates with varying ligand binding properties, cell and tissue distributions. Moreover, they can interact with blood vessels that are known to express fibronectin during development (De Gasperi, Gama Sosa, & Elder, 2012;Grossmann et al, 2002;Milner & Campbell, 2002b;Pont-Lezica, Bechade, Belarif-Cantaut, Pascual, & Bessis, 2011;Sheppard et al, 1991;Stewart & Pearlman, 1987;Tanzer, 2006). Our research group previously showed that microglial migration speed changes during cortex colonization in the mouse embryo (Swinnen et al, 2013) (Lau, Cua, Keough, Haylock-Jacobs, & Yong, 2013;Ruoslahti, 1996;Sheppard, Hamilton, & Pearlman, 1991) and is essential for normal embryonic development (Romberger, 1997) where it regulates cell differentiation and migration in general (Romberger, 1997;Tanzer, 2006).…”

“…Microglia in vitro express the corresponding major fibronectin receptor, a5b1 integrin (Milner, 2009;Milner & Campbell, 2003) and it has been shown in vitro that these cells can migrate along fibronectin matrix (Milner & Campbell, 2003;Nasu-Tada, Koizumi, & Inoue, 2005). Moreover, they can interact with blood vessels that are known to express fibronectin during development (De Gasperi, Gama Sosa, & Elder, 2012;Grossmann et al, 2002;Milner & Campbell, 2002b;Pont-Lezica, Bechade, Belarif-Cantaut, Pascual, & Bessis, 2011;Sheppard et al, 1991;Stewart & Pearlman, 1987;Tanzer, 2006).…”