Sheppard, O. | bioRxiv Science

bioRxiv2019

Beta secretase 1-dependent amyloid precursor protein processing promotes excessive vascular sprouting through NOTCH3 signaling

Amyloid beta peptides (A{beta}) proteins play a key role in vascular pathology in Alzheimers Disease (AD) including impairment of the blood brain barrier and aberrant angiogenesis. Although previous work has demonstrated a pro-angiogenic role of A{beta}, the exact mechanisms by which amyloid precursor protein (APP) processing and endothelial angiogenic signalling cascades interact in AD remain a largely unsolved problem. Here, we report that increased endothelial sprouting in human-APP transgenic mouse (TgCRND8) tissue is dependent on {beta}-secretase (BACE1) processing of APP. Higher levels of A{beta} processing in TgCRND8 tissue coincides with decreased NOTCH3/JAG1 signalling, over-production of endothelial filopodia and increased numbers of vascular pericytes. Using a novel in vitro approach to study sprouting angiogenesis in TgCRND8 organotypic brain slice cultures (OBSCs), we find that BACE1 inhibition normalises excessive endothelial filopodia formation and restores NOTCH3 signalling. These data present the first evidence for the potential of BACE1 inhibition as an effective therapeutic target for aberrant angiogenesis in AD.\n\nSignificanceIn this study, we show that targeting amyloid beta processing provides an opportunity to selectively target tip cell filopodia-driven angiogenesis and develop therapeutic targets for vascular dysfunction related to aberrant angiogenesis in AD. Our data provide the first evidence for a safe level of BACE1 inhibition that can normalize excess angiogenesis in AD, without inducing vascular deficits in healthy tissue. Our findings may pave the way for the development of new angiogenesis dependent therapeutic strategies in Alzheimers Disease.

neuroscience↗

bioRxiv2018

Microglia-dependent presynaptic disruption in an organotypic hippocampal slice culture model of neuroinflammation

BackgroundSystemic inflammation, such as occurs during sepsis, bone fracture, infections or post-operative trauma, has been linked to synapse loss and cognitive decline in human patients and animal models. Organotypic hippocampal slice cultures (OHSCs) represent an underused tool in neuroinflammation; retaining much of the neuronal architecture, synaptic connections and diversity of cell types present in the hippocampus in vivo whilst providing convenient access to manipulate and sample the culture medium and observe cellular reactions as in other in vitro methods. Here we report the development of an OHSC model of synaptic disruption after aseptic inflammation and investigate the underlying mechanism.\n\nMethodsOHSCs were generated from P6-P9 C57BL/6, the APP transgenic TgCRND8 model, or wild-type littermate mice according to the interface method. Aseptic inflammation was induced via addition of lipopolysaccharide (LPS) and cultures were analysed for changes in synaptic proteins via western blot. qPCR and ELISA analysis of the slice tissue and culture medium respectively determined changes in gene expression and protein secretion. Microglia were selectively depleted using the toxin clodronate and the effect of IL1{beta} was assessed using a specific neutralising monoclonal antibody.\n\nResultsAddition of LPS caused a loss of the presynaptic protein synaptophysin via a mechanism dependent on microglia and involving IL1{beta}. Washout of LPS via medium exchange allows for partial recovery of synaptic protein levels after 2 weeks. TgCRND8 OHSCs do not show alterations in IL1{beta} expression at a timepoint where they exhibit spontaneous synaptophysin loss, and LPS does not alter levels of APP or A{beta} in wild-type OHSCs. This indicates that although synaptophysin loss is seen in both systems, there is likely to be distinct underlying pathogenic mechanisms between the neuroinflammatory and amyloid models.\n\nConclusionsWe report the development of an OHSC model of LPS-induced synaptophysin loss and demonstrate a key role for microglia and involvement of IL1{beta}. We propose that distinct molecular mechanisms lead to synaptophysin protein loss in LPS-exposed versus TgCRND8 OHSCs and provide a new experimental paradigm for assessing chronic changes in synaptic proteins, and synaptic plasticity, following acute inflammatory insults.

neuroscience↗