Role of microRNAs in ageing at the blood-brain barrier: integrated studies in human and mouse models

The aim of the project is to investigate how ageing changes the specialized functions of cells. We propose to study the function of blood vessels in the brain and spinal cord (also known as the central nervous system or CNS) since this a specialized biological system that is amenable to functional, cellular and molecular investigations. Blood vessel malfunction during ageing is manifested by the leakage of molecules from the blood circulation and this may result in damage to nerve cells and impaired cognitive and/or sensory functions. The key cells that control access of molecules into the brain and spinal cord are the 'endothelial cells', which line the blood vessels. Normally CNS endothelial cells have a key property, the presence of 'tight junctions' which present a barrier to the movement of molecules from the blood to the CNS. Endothelial cells also express another class of proteins termed transporters on their surface that selectively drive the transport of key molecules such as nutrients into the CNS and exclude noxious substances present in the blood. During ageing, endothelial cells lose the expression of tight junction and transporter proteins and the barrier becomes leaky but it is not known whether these changes occur progressively with age or the precise mechanisms leading to CNS blood vessel malfunction. This project investigates whether a newly-described mechanism controlling specialised properties of cells, applies to the control of the barrier tight junction and transporter function between the blood and the CNS in the context of ageing. Small regulatory molecules termed microRNAs can regulate and rapidly reduce the level of particular proteins in cells and we propose that this regulatory mechanism is altered in the cells that form the blood vessels of the CNS during normal ageing. The first objective of the project is to characterize the changes in CNS blood vessel function that occur during normal ageing in two model organisms with different life spans, humans and mice. We have chosen leakiness of blood-borne molecules as a physiological process maintained by blood vessels that is critical for the normal functioning of the CNS. It is not known at what point in the ageing process CNS blood vessels start to malfunction and a systematic study on this has never been reported. The second objective is to measure the extent and significance of changes in microRNA levels at the stage in which CNS blood vessel malfunction starts to occur in the two model organisms. Here, we will also analyze changes in the pattern of gene expression in CNS blood vessels and identify whether changes in specific genes linked to tight junction or transporter function are likely to be driven by alterations in microRNA levels. To do this, we will use state-of-the-art techniques to simultaneously determine millions of microRNA and target gene molecules in CNS blood vessels in young and old individuals. Finally, we will select specific microRNAs that are altered during ageing and manipulate their levels in CNS endothelial cells in laboratory settings. In doing so, we will determine whether this manipulation results in changes in expression of their gene targets and in blood vessel function. These experiments will confirm whether microRNAs are important regulators of specialized functions of cells during the ageing process. The main expected outcome of this project is knowledge of whether the ageing process of a biological system with specialized functions is driven by alterations in microRNAs. If this proves to be the case, alterations in microRNA levels may represent a novel mechanism of ageing in multiple cell types with different specialized functions. This knowledge then could benefit not only academics working within the field of ageing but also those investigating how specialized functions of cells are altered in disease states, in particular those affecting the brain.

The aim of the proposal is to test the hypothesis that age-induced changes in gene expression in cerebral endothelium leading to cerebrovascular dysfunction are mediated, at least partially, by changes in microRNA levels. MicroRNAs (miRs) are endogenous non-coding small RNAs that mediate gene silencing by translation inhibition and/or by mRNA decay of target genes. Evidence suggests a role for miRs in regulating the life span of model organisms but the mechanisms involved in this process are largely unknown. One of the first signs of ageing is the loss of specialized functions by cells. The objective of the work is to elucidate general mechanisms by which miRs may modulate normal ageing in a specialized biological system that is amenable to functional, cellular and molecular investigations, the blood-brain barrier (BBB), across two model organisms with differing life spans, humans and mice. First, we will characterize progressive changes in BBB function in humans and mice during normal ageing. Second, using microarray technology, age-deregulated miRs in the cerebral endothelium and age-associated shifts in the global pattern of target gene expression will be analysed so that changes predicted to be directed by alterations in miR levels at the BBB are identified. Third, specific age-deregulated miRs will be selected using their ability to modulate specialized BBB function and/or known pathways of cellular senescence in vitro. Finally, we will validate actions of specific miRs on putative gene targets and confirm age-induced changes in levels of selected miR/target gene pairs at the BBB. BBB dysfunction is recently emerging as an important factor leading to age-associated loss of homeostasis in the central nervous system and we envisage that age-induced phenotypic changes at the BBB mediated by miRs will be identified that may well be not only specific to cerebrovascular function but also constitute general pathways of ageing across biological systems.

The proposed research seeks to determine the roles of microRNAs in the endothelium of the blood-brain barrier (BBB) during normal brain ageing. It has two main aims: first to systematically characterise changes occurring in brain endothelial cells during ageing and, second, to identify the roles of microRNAs in these changes, and thus their possible involvement in age-induced BBB dysfunction. Comparison of microRNA changes with alterations in the general endothelial transcriptome will provide a powerful data set of altered endothelial gene expression in ageing. BBB dysfunction and endothelial pathology have been implicated in the development and progression of degenerative brain disease, such as Alzheimer's Disease. However, these processes, their interaction with brain parenchyma ageing and their roles in disease remain poorly defined. The results from the work will therefore have a major impact both on our knowledge base in the areas of microRNA functions, ageing and neuroscience and also in the future, for health and wellbeing, as the results should lead to knowledge that can be used in the development of strategies and therapies to prevent, delay or alleviate age-associated BBB dysfunction in neuropathological conditions. Such novel approaches are essential given the limitations of current strategies.
The immediate beneficiaries of the proposed research are academics, the public sector and third sectors. In the longer term, potential future beneficiaries include health professionals, the commercial private sector and general public.
The academic community will benefit from the proposed research because at present our knowledge of the roles of microRNAs is in its infancy. Evidence suggests that these molecules play a role in ageing and in endothelial cell biology, and the proposed work will provide new information in both these important areas.
Within the public and third sectors, the specific groups to whom this research is relevant are research organisations (including BBSRC, MRC), professional bodies (such as the British Society for Research on Ageing, the British Neuropathological Society and the British Neuroscience Association) and charities (e.g. Age UK, Alzheimer's Research Trust, Alzheimer's Society, Alzheimer's Association). These groups will benefit from the knowledge generated from the project, as it will inform decisions on the future research priorities and development activities in the areas of ageing and neuroscience, in particular neuropathology.
We do not anticipate any early developments that will warrant commercial exploitation, although there is potential for such exploitation in the future. We will ensure that any such developments are taken forward, with appropriate partners and IP procedures.
The health and wellbeing of the general public will benefit from this research in the longer term. For example, understanding how the process of normal ageing affects blood-brain barrier function, in particular that related to transporter activity which influences CNS availability of many commercial drugs, will impact on the development of therapeutic regimes targeted at the ageing population with neurological conditions. The public should also have the benefit of being informed about the work.