Prf Ignacio Romero Romero

DISCOVERY AND CHARACTERISATION OF BLOOD-BRAIN BARRIER TARGETS AND TRANSPORT MECHANISMS FOR BRAIN DELIVERY OF THERAPEUTICS TO TREAT NEURODEGENERATIVE & METABOLIC DISEASES

Investigation into the molecular mechanisms underlying idiopathic intracranial hypertension Idiopathic intracranial hypertension (IIH) is a neurological condition characterized by increased intracranial pressure in the absence of a tumour or other diseases. IIH affects primarily pre-menopausal obese women but the contribution of reproductive hormones and/or obesity to the pathogenesis of this disease is unknown. Indeed, as its name implies, its aetiology remains elusive although a possible role for inflammatory mediators has been suggested. From a physiological point of view, raised intracranial pressure may be the result of increased cerebrospinal fluid (CSF) secretion, decreased CSF drainage or a combination of both factors. The objectives for this project are: Year 1. To determine transcriptomic and microRNA changes in choroid plexus of female control rats and rats fed a high-fat diet and in young and old female rats. Years 2 and 3. First, to establish an in vitro rat model of the blood-CSF barrier and, second, to select candidates from the transcriptomic/microRNA analysis that are differentially regulated and investigate their effects on the secretory and transport activity of rat and/or human choroid plexus epithelium (both cell lines and primary cultures)

Neuroendocrine prostate cancer (NEPC) is the most aggressive prostate cancer type. This disease is resistant to all available therapies, highly metastatic and rapidly fatal. We believe that the elucidation of NEPC-driving pathways could pave the way for the development of effective therapies. Long non-coding RNAs (lncRNAs) are a vast and mostly uncharted region of the human transcriptome. Despite their emerging role in cancer biology, no study has assessed the relevance of lncRNAs in NEPC development. Our collaborator Dr. Wang (BC Cancer Agency) has developed a unique collection of patient-derived prostate cancer models. This collection includes the first-in-field model of transdifferentiation from prostatic adenocarcinoma to NEPC. Aims. We propose to study the role of lncRNAs in NEPC development. The objectives of this study are to determine: 1) whether transcriptomic profiles of lncRNAs in PCa pre-clinical models can elucidate the mechanisms of NEPC development. 2) if selected lncRNAs are promising therapeutic targets for NEPC. Accordingly, our specific aims will be: AIM 1: Identification, functional and clinical characterization of NEPC-driving lncRNAs. AIM 2: Development of innovative therapeutic strategies to prevent NEPC development. At the end of this project, we hope to shed new light on an incurable disease, and to identify viable therapeutic targets, which will be investigated in future translational and clinical studies.

The general aim of the current proposal is to the function of the neurovascular unit across species and the impact of several conditions (development, inflammation, ageing, exposure to drugs)on the function of the blood-brain barriers. The aims are: 1. Prediciting brain barrier function using in vitro BBB models 2. Brain barriers signature in vertrebrates 3. Brain barriers as therapeutic and diagnostic target The OU will lead the project within the BtRAIN network focused on age-associated changes in gene and microRNA expression at the blood-brain barrier. See the network webpage http://www.btrain-2020.eu/

Intercellular communication between immune cells and tissue-resident cells is essential to coordinate an effective immune response and involves both cell-contact dependent and independent processes that ensure the transfer of information between bystander and distant cells. There is a rapidly growing body of evidence on the pivotal role of extracellular vesicles (EVs) in cell communication and these structures are emerging as important mediators for immune modulation upon delivery of their molecular cargo. In the last decade, EVs have been shown to be efficient carriers of genetic information, including microRNAs, that can be transferred between cells and regulate gene expression and function on the recipient cell. However, little is known about regulation of cellular function by EVs at the blood-brain barrier (BBB), the main route of entry of immune cells into the central nervous system (CNS), in pathological conditions. We have recently shown that EVs isolated from MS patients induce blood-brain barrier dysfunction, characterised by leakiness of the barrier, in a human culture model (1) and that the microRNA profile of brain endothelium undergoes profound changes in inflammation (2,3). Thus, the proposed project will investigate the role of microRNAs secreted in EVs in activation of brain endothelium and subsequent leukocyte migration. In year 1, the miroRNA profile of EVs released by cultured human brain endothelium in inflammatory conditions and in plasma of MS patients will be determined. In year 2, the effects of endothelial-derived and MS plasma-derived EVs on leukocyte adhesion, both leukocyte cell lines and peripheral blood mononuclear cells, under flow will be investigated. In year 3, specific microRNAs will be knocked-down in cultured human brain endothelium prior to EV collection and subsequent effects on leukocyte adhesion will be investigated

The project aims to identify specific nucleic acid aptamers that may be used as carriers for the delivery of drugs to the brain. The project has two clearly defined parts that will run in parallel with similar methodology but with different 'baits' for the selection of aptamers, one using human brain endothelial cells (the cells that form blood vessels) and the other proteins known to acts as cargoes across brain endothelium.

Dysfunction of the BBB is a major hallmark of neuroinflammatory diseases that constitute a major public health concern such as multiple sclerosis (MS). BBB dysfunction in MS is manifested by a decreased expression of a number of proteins at the tight junctions(TJ) at the protein and/or mRNA level by capillaries. We have recently shown that treatment of human brain endothelial cells with cytokines either alone or in combination leads to downregulation of occludin, claudin-5 and ZO-1 mRNA levels. These observations led us to postulate that TJ mRNA expression by cerebral endothelium is regulated by a recently identified mechanism, the expression of micro-RNAs induced by neuroinflammatory mediators. Following funding on an innovative project, we have identified a group of microRNAs with a putative role in BBB dysfunction and the present study constitutes a proof-of-principle study. To date, no studies on the role of microRNAs on the pathogenesis of MS have been carried out. The results from this project have promising therapeutic applications to prevent BBB impairment in MS by using antisense molecular tecnhnology.

Relevance of the blood-brain interface in the disease mechanisms of Multiple Sclerosis and Alzheimer's disease

The student will examine the role of a family of proteins that protect cells from a form of death known as 'apoptosis'. In particular, the student will examine how the 'Bcl-2' affects cellular calcium transport. The working hypothesis is that Bcl-2 prevents the transfer of calcium between cellular compartments to avoid triggering apoptosis.

IIH is a neurological condition characterized by increased intracranial pressure in the absence of a tumor or other diseases but whose cause remains elusive although a possible role for leptin and inflammatory mediators such as CCL2 has been suggested. The objectives for this project are: Year 1 (to be based at KCL). To determine, first, the protein composition of CSF from IIH and control patients using proteomic analysis and, second, the effect of IIH CSF on the secretory and transport activity of cultures of porcine choroid plexus epithelium Year 2 (to be based at the OU with occasional trips to Sheffield). First, to establish an in vitro human model of the blood-CSF barrier and, second, to determine , using transcriptomic analysis, the effect of CSF from IIH and control patients on gene profile of cultures of human choroid plexus epithelium (both cell lines and primary cultures) Year 3 (based at the OU). To select candidates from the proteomic/transcriptomic analysis that are differentially regulated in IIH CSF/choroid plexus epithelium and investigate their effects on the secretory and transport activity of human choroid plexus epithelium in order to identify potential therapeutic molecular targets mediating these effects

Dysfunction of the blood-brain barrier (BBB) is a major hallmark of ageing of the central nervous system, an important economic and societal factor and major public health concern in view of the rapidly increasing proportion of the ageing population. BBB dysfunction in ageing is manifested by a decreased expression of a number of proteins at the tight junctions(TJ) and transporters at the protein and/or mRNA level by capillaries. Our preliminary data shows a decreased expression of transporters in cerebral blood vessels in aged mice in human brain endothelial cells, the cells that form the BBB, in combination with an increase of specific microRNAs. These observations led us to postulate that barrier-specific mRNA expression by cerebral endothelium is regulated by a recently identified mechanism, microRNAs, and that these regulation is altered during normal ageing. To date, no studies on the role of microRNAs on the ageing process of the BBB have been carried out. The results from this project have promising intervention applications to prevent BBB impairment in ageing by using antisense molecular tecnhnology.

IIH is a neurological condition characterized by increased intracranial pressure in the absence of a tumor or other diseases but whose cause remains elusive. A possible role for leptin and inflammatory mediators such as CCL2 has been suggested. The objectives for this project are: 1. To determine, the effect of these inflammatory mediators on cerebrospinal fluid (CSF) secretion and drainage 2. To determine the role of sex and high-fat diet on CSF secretion and drainage. 3. to establish an in vitro human model of the blood-CSF barrier and to determine , using transcriptomic analysis, the effect of inflammatory mediators on gene profile of cultures of human choroid plexus epithelium . These results may help identify potential therapeutic molecular targets for IIH