Chenghua Gu - US grants

DESCRIPTION (provided by applicant): The normal functioning of the adult nervous system relies critically on the proper structure and function of the vascular system. Blood vessels provide neurons with oxygen and nutrients and protect them from toxins and pathogens. Nerves, in turn, control blood vessel dilation and contraction and also heart rate. Key to this functional interdependence is an extraordinarily tight physical association between nervous and vascular systems. In the periphery, nerves and vessels often run parallel to one another and in the central nervous system neural activity and vascular dynamics are tightly coupled. Indeed, emerging evidence shows that some neurodegenerative diseases, such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS), once thought to be caused by intrinsic neuronal defects, are in fact initiated and perpetuated by vascular abnormalities. Despite these important connections between the nervous and vascular systems, little is known about how the nervous system becomes so closely aligned with the vascular system during development. In this proposal, we have established a simple system using developing mouse somatosensory peripheral target innervation as a model to study this question. In Aim1, we will dissect the molecular mechanisms underlying the organization of the tight nerve/vessel association, particularly focusing on the role of a recently identified ligand-receptor pair, Sema3E-Pelxin-D1. We will apply both in vitro assays and in vivo mouse genetics approaches to address the function of Sema3E-Pelxin-D1 signaling in establishing the nerve/vessel association. In Aim 2, we will identify and characterize the intracellular signaling mechanisms downstream of Sema3E-Pelxin-D1 in neurons and endothelial cells. Using a novel image-based RNAi genome-wide screen, we have identified and validated several potential candidates mediate Sema3E-Plexin-D1 signaling. We will characterize their roles in Sema3E-Plexin-D1 signaling and compare whether similar signaling mechanisms are used in Sema3E -mediated axon guidance and endothelial cell migration. Together, these proposed experiments will uncover cellular and molecular mechanisms underlying neuro-vascular interactions. These results may also improve our ability to diagnose, treat, and prevent neurological disorders that affect both neurons and vessels, including: peripheral neuropathies, Alzheimer's Disease (AD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). PUBLIC HEALTH RELEVANCE: Understanding the interactions between vascular and nervous systems will advance the diagnosis, therapy, and prevention of several neurological diseases, including diabetic neuropathy and trigeminal neuralgia. Moreover, emerging evidence shows some neurodegenerative diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS), that once were thought to be caused primarily by intrinsic neuronal defects, actually may be related to vascular abnormalities. Finally, since mechanisms that control angiogenesis during development are likely to be essential for neovascularization in tumors, this study may have a direct impact on cancer treatment.

DESCRIPTION (provided by applicant): The central nervous system (CNS) requires a tightly controlled environment free of toxins and pathogens to provide the proper chemical conditions for synaptic transmission. This environment is maintained by the 'blood brain barrier' (BBB), which is composed of highly specialized blood vessels whose endothelium display specialized tight junctions and unusually low rates of transcellular vesicular transport (transcytosis). While BBB breakdown has recently been associated with various neurological disorders, an intact BBB also poses a major obstacle for drug delivery to the CNS. Pharmaceutical companies spend billions of dollars to develop drugs that can penetrate the BBB to treat disease. However, little progress has been made on manipulating the BBB due to a significant knowledge gap in understanding how BBB function is regulated and identifying the essential molecular constituents governing its processes. This limited understanding has also thwarted our ability to therapeutically manipulate the BBB. The major impediment to understanding the BBB is identifying its essential constituent and unraveling the mechanism by which these key regulators control BBB function. However, the current in vitro models rely on fully differentiated endothelial cells, which already contain unique properties that prevent their use in reconstitution studies. Similarly, the main technique to study the BBB has been EM, however its static snapshots do not provide information on active and dynamic vesicular transport, directionality, or their specifi routes to allow investigators to interrogate the key molecular mechanisms that regulate BBB integrity. Recently, with our research background in developmental co-wiring of nervous and vascular systems, we used the traditional developmental approach, to first mapped the precise timing of BBB formation and then identified neural cues that induce CNS endothelium to acquire BBB properties, and molecules with possible roles in

Project Summary/Abstract: Proper function of precisely wired neural circuits depends on a close physical and functional relationship with an equally complex and overlapping vascular network. Vascular and perivascular cells are heterogeneous both within and across brain regions, and this heterogeneity is thought to underlie the functional specialization that caters to local neuronal circuitry demands. Moreover, emerging evidence shows that vascular cells actively influence the activity of neuronal ensembles, and reciprocally, neuronal activity controls the function and patterning of vascular networks. Understanding the molecular and structural heterogeneity underlying these unique local interactions is fundamental to understanding how non-neuronal cells contribute to the emergence of neural activity patterns underlying cognition and behavior. However, an inventory of vascular cell types within a defined brain region or within functionally defined brain circuits does not exist, and methods to identify or manipulate intercellular connections at the neuro-glio-vascular interface to determine their influence on circuit function are lacking. To date, the creation of a vascular cell inventory with regional specificity has proven challenging because it is difficult to obtain adequate vascular and perivascular cell yields for analysis. This is because vascular cells represent only 5% of brain cells4 , and of course we cannot pool across regions without losing regional specificity. To overcome this challenge, we developed a dissociation protocol to enrich for vascular cell survival in mice, which allows us to study small, defined brain regions. As a proof of principle, we focused on the median eminence (ME) and a size-matched region of somatosensory cortex, regions selected for their distinct functions and small, defined spatial structures (~0.05 x 0.2 x 1.2 mm3). Our preliminary single- cell RNA sequencing (scRNA-seq) data revealed differences in cell composition and gene expression among vascular and perivascular cells. Moreover, we identified ligand-receptor interactions underlie different neuro-glio- vascular interactions. We will capitalize on this work to generate the first comprehensive inventory of vascular and perivascular cells in these regions and two additional regions, the subfornical organ (SFO) and the hippocampus. We will identify cell type- and regional-specific markers and generate mouse Cre lines to target these cells, and we will generate a neuro-glio-vascular connectome database from which other researchers can rapidly access the spatial arrangement of cells in a brain region, with morphology, cell-cell contacts, and ultrastructure for all cell types identified in our inventory. Our tools will provide an invaluable resource to identify the contribution of non-neuronal cells to structural and functional heterogeneity of neural circuits across brain regions. In addition, the cell type- and regional-specific markers are likely to be useful in other mammalian species for viral delivery of Cre recombinase. Finally, given the importance of non-neuronal cells in neurological diseases, this work will provide a vast source of understudied molecular and cellular targets, which could change how we think about brain disease and identify new modes of treatment.

Project Summary/Abstract: The central nervous system (CNS) requires a tightly controlled environment free of various toxins and pathogens to provide the proper chemical composition for synaptic transmission. This environment is maintained by the `blood brain barrier' (BBB), which is composed of highly specialized blood vessels whose endothelial cells display specialized tight junctions and unusually low rates of transcellular vesicular transport (transcytosis). In concert with pericytes and astrocytes, this unique brain endothelial physiological barrier seals the CNS and controls substance influx and efflux. While BBB breakdown has recently been associated to initiation and perpetuation of various neurological disorders, an intact BBB is a major obstacle for drug delivery to the CNS. A limited understanding of the molecular mechanisms that control BBB formation has hampered our ability to manipulate the BBB in disease. Our recent discoveries changed our understanding of what makes the BBB impermeable. The BBB is formed by a single layer of endothelial cells that lines the walls of the brain's blood vessels. Historically, the restrictive feature of BBB has been attributed to the specialized tight junctions between adjacent endothelial cells. However, substances can also cross the endothelial layer by transcytosis, when material enters endocytic vesicles that are trafficked across the cell. We discovered that transcytosis is actively inhibited in brain endothelial cells to ensure BBB integrity. Our findings suggest that molecular pathways inhibiting transcytosis could be targeted to open the BBB for CNS therapeutics.We have also identified over 200 BBB candidate genes that are enriched in CNS endothelial cells compared to periphery endothelial cells. I propose to launch major new efforts leading to a major expansion in the scope of our work in the field of BBB. I will take the next eight years to bring my lab to the next level to (1) identify the full list of key BBB regulators in CNS endothelial cells, (2) understand what signals from non-endothelial cells maintain and regulate BBB permeability, and (3) determine how BBB permeability dynamically changes during different physiological and pathological conditions. We will also begin to work on translating findings from these studies to therapies. We will use a combination of mouse genetics, imaging, molecular, cell biology, and biochemical approaches. The experiments described here represent a major expansion in the scope of our work. Achieving the goals outlined here could have a major impact on neurology, enabling clinicians to open the BBB for transient delivery of drugs to the CNS, and conversely to close the BBB to slow the progression of neurodegenerative diseases. Given the transcriptome screens we have recently performed, the model systems we have devised, and the imaging tools we have recently developed, my lab is in a unique position to reveal the molecular and cellular mechanisms of the BBB.

Project Summary/abstract: There is no organ in the body that more heavily depends on a continuous supply of blood than the brain. The importance of the circulatory system to the brain is demonstrated by the fact that the brain makes up 2% of total body mass but it receives 20% of cardiac output. Furthermore, the brain does not contain local fuel reserves, as peripheral organs do. During behavior, brain regions are recruited for specific tasks and must be brought ?online? quickly. The brain regulates its own blood supply via a process called neurovascular coupling, in which neural activity rapidly increases local blood flow to meet moment-to-moment changes in regional brain energy demand. Impairments in neurovascular coupling contributes to neurodegeneration and vascular dementia. Neurovascular coupling is also the basis for functional brain imaging, which is currently the only way to infer brain-wide neuronal activation in humans. Despite the importance of understanding how the brain regulates its own blood supply, the molecular and cellular mechanisms underlying neurovascular coupling are still not clear. In this proposal, we have developed a two-photon in vivo imaging paradigm that can simultaneously measure neural activity (via a genetically encoded calcium indicator) and vascular dynamics (vessel diameter and blood flow) at single-vessel resolution in awake mice, enabling us to capture the neurovascular response to a natural stimulus (whisker stimulation) at high temporal and spatial resolution in vivo. We will combine this state-of-art in vivo imaging techniques with powerful molecular, electrophysiological, ultrastructural, and genetic approaches to elucidate the fundamental cellular mechanisms governing neurovascular coupling. Our preliminary data with in vivo live imaging and mouse genetics in the barrel cortex, suggest that endothelial cells (ECs) in the brain play active roles in neurovascular coupling. Traditionally, ECs were considered to be passively involved in neurovascular coupling, and to be a homogenous population. We found aECs and cECs display molecular and subcellular differences, and play distinct roles during neurovascular coupling. Our proposed work will identify the cellular, subcellular, and molecular mechanisms by which endothelial cells from different segments of the vascular tree initiate, transmit, and implement neurovascular coupling. Specifically, we will demonstrate how different type of ECs along the vascular tree mediate neurovascular coupling, thus connecting the dots between neural activity and local blood flow change. We expect that our ability to image CNS small blood vessels non-invasively in awake mice with high spatial and temporal resolution, along with other sophisticated techniques, will allow us to identify critical molecular and subcellular mechanisms that underlie neurovascular coupling in vivo. Moreover, our structural and molecular findings will provide tools that can be broadly used by the field to cerebrovascular biology. Finally, the data will provide critical information to accelerate our understanding and treatment of CNS diseases related to small vessels and vascular dementia.