Richard Daneman - US grants

? DESCRIPTION (provided by applicant): The blood-brain barrier (BBB) is a term used to describe the unique properties of the blood vessels that vascularize the central nervous system (CNS). This barrier is crucial both to maintain brain homeostasis, which allows for proper neuronal function, and to protect the CNS from injury and disease. Many of the symptoms of stroke, multiple sclerosis, edema, and brain traumas are due to a breakdown of the BBB that accompanies the primary insult. Furthermore, the BBB provides an obstacle for the treatment of all neurological diseases as it greatly impedes drug delivery to the CNS. Therefore, understanding the mechanisms that regulate BBB function may identify novel targets to modulate the BBB, both to repair the BBB and to develop methods to bypass the BBB for drug delivery. Many of the properties of the BBB are manifested within the endothelial cells that make up the walls of the blood vessels, which allows them to restrict the movement of molecules, ions and cells between the blood and the brain. One important barrier property is that the CNS endothelial cells undergo transcytosis at extremely low rates, thus limiting the transcellular permeability. Although these are properties of the endothelial cells, studies have revealed that they are induced by interactions with the CNS microenvironment. In particular we have identified that CNS pericytes are important to induce BBB properties in endothelial cells during development, and they do so by inhibiting transcytosis through the endothelial cells. In preliminary experiments, we have identified that specific EHD family members are enriched in peripheral `leaky' vessels such as the liver and lung which undergo lots of transcytosis, compared to CNS vessels which undergo very low amounts of transcytosis. We have further demonstrated that genetic loss of pericytes, stroke and neuroinflammation all lead to up-regulation of EHD genes in CNS blood vessels that correlates with BBB leakage. We have used genetic mouse models to demonstrate that over-expression of EHD4 leads to leakage of the BBB in vivo, and conditional knockout of EHD1 and EHD4 in peripheral endothelial cells leads to less peripheral vascular leakage. Therefore we aim to test the hypothesis that: Pericytes regulate the BBB by inhibiting the expression of EHDs in CNS endothelial cells, and that up-regulation of EHDs is an important component of BBB dysfunction during neurological disease. We have developed transgenic mouse models to reversibly over-express EHD- GFP fusion proteins in endothelial cells, as well as conditional knockout models to delete EHDs from endothelial cells. Using these models we will address the following questions: How does up-regulation of EHDs lead to BBB dysfunction? Are EHDs required for BBB dysfunction during diseases? What leads to EHD upregulation at the BBB? Answering these questions will enable us to determine the role of EHDs in regulating the BBB and determine if they are a target to modulate the BBB to treat neurological diseases and aid drug delivery to the CNS.

ABSTRACT Alzheimer's disease (AD) is a debilitating, chronic neurodegenerative disease that is the most common form of dementia. The pathophysiology of AD includes the progressive loss of neurons and synapses throughout the cerebral cortex as well as subcortical regions, and is characterized by the buildup of amyloid-beta (A?) plaques and tau-containing neurofibrillary tangle pathologies throughout these regions. A? plaques have been reported to be associated with neuronal and glial cells (parenchymal plaques), or associated with blood vessels (vascular plaques termed cerebral amyloid angiopathy [CAA]). CAA is found in up to 90% of patients with AD, and is thought to lead to impaired blood flow, altered vascular morphology, inflammation, microbleeds and hemorrhage. Despite the importance of CAA, very little is known about how A? deposits around vessels. There are two main hypotheses: First, vascular plaques are generated through the aberrant secretion of A? by endothelial cells and/or mural cells. Second, vascular plaques are generated though dysfunction in clearance of A?. Here we test a novel hypothesis: perivascular fibroblasts secrete A? in the generation of CAA. Work in our lab has identified that perivascular fibroblasts are intimately associated with vascular A? plaques in AD postmortem tissue. We have also identified that that perivascular fibroblasts robustly express amyloid precursor protein (APP) and the enzymes that process amyloid (BACE1/2, PSEN1). Here, we will use a conditional knockout approach to determine whether fibroblasts are the key cell type that secretes A? in the generation of CAA in a well characterized mouse model of AD. We will also utilize single cell sequencing to examine the cellular heterogeneity and gene expression of the perivascular fibroblasts as a function of time in the mouse AD model. If we find that perivascular fibroblasts are key contributors to A? secretion in the buildup of CAA, then this analysis will give insights into to the mechanism that leads to this vascular A? accumulation. If we find that fibroblast secretion of A? is not necessary for the generation of CAA, this analysis will provide vital information about how the fibroblasts change during the formation of vascular A? plaques, with which they are intimately associated.

Abstract The blood-brain barrier is a term used to describe the unique properties of the blood vessels that vascularize the central nervous system. These blood vessels tightly regulate the movement of ions, molecules and cells between the blood and the brain, thus controlling the extracellular environment of the neural tissue. Despite the importance of the blood-brain barrier, very little is known about how this barrier regulates the neural environment to modulate complex behaviors. We have identified that the metabolic enzymes that generate and breakdown the monoamine neurotransmitters dopamine and serotonin are enriched in the endothelial cells of central nervous system blood vessels, suggesting that blood-brain barrier metabolism may regulate the levels of these monoamine neurotransmitters which are critical for many complex behaviors. In preliminary studies we have identified that deletion of these enzymes in endothelial cells leads to deficits in social interaction, and have further found evidence that these vascular enzymes act as a metabolic buffer to the transport of monoamine precursors. Interestingly, polymorphisms in the genes encoding each of these enzymes have been linked with autism risk and/or severity, however the site of action and mechanism by which they regulate autism-related behaviors is not known. Here we will test the hypothesis that blood-brain barrier metabolism regulates autism-related behaviors including social interaction. We will further examine the mechanism by which blood-brain barrier metabolism may regulate the levels of monoamine neurotransmitters within the central nervous system, and how this affects behavior. Our ultimate goal is to determine whether the blood-brain barrier is a therapeutic target to modulate behavior.

ABSTRACT Alzheimer's disease (AD) is a devastating chronic neurodegenerative disease and the leading cause of dementia in older adults. One of the hallmark pathologies of AD is accumulation of extracellular amyloid-beta (A?) ?plaques?. These plaques can form both in the parenchyma as well as in the walls of meningeal and cerebral blood vessels. Vascular A? plaques, termed cerebral amyloid angiopathy (CAA), are present in up to 98% of AD patients and can cause stroke, dementia, inflammation, cortical microbleeds, and hemorrhage. Despite the serious clinical ramifications of CAA, it remains unclear why plaques develop in vascular walls. With therapeutic prevention of CAA as a long-term goal, this proposal aims to investigate the role of microglia in regulating cholesterol homeostasis in endothelial cells (ECs)??the cells that make up the walls of blood vessels. There are several epidemiological links between cholesterol and AD, yet the specific mechanisms underlying this association are unknown. Our preliminary data show that microglial depletion causes upregulation of cholesterol synthesis enzymes in brain ECs. Because a higher cellular levels of esterified cholesterol have been shown to increase A? production, we hypothesize that microglial dysfunction in AD disrupts brain EC cholesterol metabolism, driving A? production and secretion, thereby potentiating CAA in AD. This proposal will test this novel hypothesis and will also determine how dietary cholesterol modulates brain EC cholesterol metabolism. Specifically, we will test how microglial depletion affects cholesterol synthesis in other neural cell types by quantifying expression of cholesterol synthesis machinery in astrocytes, neurons, and oligodendrocytes. As brain ECs lie at the interface between the brain and the blood, we will also investigate how increasing dietary cholesterol with a high fat diet modulates cholesterol metabolism in brain ECs. Finally, we will assess the separate and combined effects of microglial depletion and high fat diet on vascular pathology in a model of AD. Taken together, the experiments proposed here will advance our understanding of the association between cholesterol and AD, particualrly CAA. Furthermore, these data will identify whether therapeutic regulation of cholesterol synthesis or efflux specifically in brain ECs could be a successful clinical strategy for preventing CAA.

ABSTRACT Fibrosis, defined by the deposition of collagen I, is a devastating pathological event that occurs in many organs including the heart, kidney, liver and lung in response to injury and inflammation. This fibrotic response inhibits recovery inflammation and can even lead to organ failure. Despite the potential importance, very little is known about whether there is a fibrotic response in the central nervous system (CNS) following neuroinflammation that occurs in diseases such as multiple sclerosis, neuromyelitis optica, stroke and CNS infections, and how this response affects repair and recovery. Using experimental autoimmune encephalomyelitis (EAE), a mouse model of neuroinflammation, we have identified that a robust collagen I- based fibrotic scar forms covering the neuroinflammatory lesion and we hypothesize that this fibrotic scar inhibits the ability of reparative cells to enter the lesion. In preliminary studies using lineage tracing and single cell sequencing, we have identified that this fibrotic scar is formed by the activation and proliferation of fibroblasts. We have further generated methods to isolate and culture CNS fibroblasts providing an in vitro model to study the proliferation, migration and collagen 1 production from these cells. In this proposal we aim to determine whether the fibrotic scar is helpful or harmful for recovery following neuroinflammation and to further study the mechanisms that regulate fibrotic scar formation. We will first determine whether inhibition of fibrotic scar formation can lead to an increased recovery from EAE. We will then examine whether TGF? and PDGFR signaling pathways regulate fibrotic scar formation. We hypothesize that TGF? signaling drives the proliferation and collagen I production by the fibroblasts and that PDGFR signaling regulates the migration of the fibroblasts to the lesion. Our goal is to determine whether modulating the fibrotic scar is a potential therapeutic target to aid in recovery for patients with neuroinflammatory diseases.