Biology subjects

Sheppard, O.

Publications and source records attributed to Sheppard, O..

4 recordsLinked to original sources

bioRxiv2024

Lowering glucose enhances BACE1 activity and Aβ generation in mouse brain slice cultures

Numerous environmental risk factors are now recognised as contributors to the onset and progression of Alzheimers disease (AD). It is probable that, in most instances, AD arises from a combination of genetic predisposition and environmental influences. In particular, there is a strong correlation between vascular impairment and dementia, yet the specific mechanisms by which vascular impairment and AD are linked, remain unknown. Hypoglycaemia can occur both due to vascular impairment, and due to fluctuating glucose levels in the context of diabetes, another risk factor for AD, and could potentially be involved in disease pathogenesis. To assess whether low glucose could contribute to the build-up of brain amyloid-{beta} (A{beta}) seen in AD, we exposed wildtype mouse organotypic hippocampal slice cultures (OHSCs) to varying glucose concentrations. Lowering glucose levels leads to an elevation in both A{beta}1-42 and A{beta}1-40 secreted into the culture medium, accompanied by an increased accumulation of A{beta} within the slice tissue. This effect is replicated in OHSCs derived from the TgCRND8 mouse model of overexpressed, mutant APP and in human SH-SY5Y cells. The heightened A{beta} levels are likely attributed to an upregulation of BACE1 activity, which is also observed with lowered glucose levels. In contrast, OHSCs subject to hypoxia exhibited no alterations in A{beta} levels whether singularly, or in combination of hypoglycaemia. Finally, we found that alternative energy sources such as pyruvate, fructose 1,6-bisphosphate, and lactate can alleviate heightened A{beta} levels, when given in combination with lowered glucose. This study underscores the capacity to induce an increase in A{beta} in a wildtype ex vivo system by selectively decreasing glucose levels.

neuroscience↗

bioRxiv2021

Tau assemblies do not behave like independently acting prion-like particles in mouse neural tissue

A fundamental property of infectious agents is their particulate nature: infectivity arises from independently-acting particles rather than as a result of collective action. Assemblies of the protein tau can exhibit seeding behaviour, potentially underlying the apparent spread of tau aggregation in many neurodegenerative diseases. Here we ask whether tau assemblies share with classical pathogens the characteristic of particulate behaviour. We used organotypic hippocampal slice cultures from P301S tau transgenic mice in order to precisely control the concentration of extracellular tau assemblies. Whilst untreated slices displayed no overt signs of pathology, exposure to tau assemblies could result in the formation of intraneuronal, hyperphosphorylated tau structures. However, seeding ability of tau assemblies did not titrate in a one-hit manner in neural tissue. The results suggest that seeding behaviour of tau only arises at supra-physiological concentrations, with implications for the interpretation of high-dose intracranial challenge experiments and the possible contribution of seeded aggregation to human disease.

neuroscience↗

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↗