Alejandro Caicedo - US grants

? DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how the islets of Langerhans function in vivo, either in the native environment in the pancreas or after transplantation to treat type 1 diabetes. Most studies on islet function have been performed in vitro, and as a consequence little is known about the role of innervation on islet hormone secretion. The long-term goal of our research is to understand the cell biology of islets of Langerhans within the regulatory networks that exist in the living organism. The objective of this particular application is to determine the role sensory innervation plays in islet biology using ne technological platforms allowing in situ and in vivo imaging of islets. For in vivo imaging, islets are transplanted into the anterior chamber of the eye, and their function is recorded locally and systemically after manipulation of the eye's neural input. The central hypothesis is that sensory axons detect molecules released during islet activity and in response release substances that modulate islet function. In the proposed mechanism, sensory axons are localized between endocrine cells, resident macrophages, and vascular cells and are therefore positioned optimally to screen the extracellular space for changes in the chemical composition. When activated by local signals, sensory axons release neuropeptides that dampen endocrine activity, alter vascular function, and keep resident macrophages quiescent, thus promoting tissue homeostasis. The rationale for the proposed research is that the results will contribute a missing, fundamental element to basic knowledge, without which islet biology cannot be understood. The proposed research is therefore relevant to the mission of the NIH that pertains to the pursuit of fundamental knowledge about the nature and behavior of living systems. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) The innervation patterns of sensory axons in mouse and human pancreatic islets; 2) The functional role of sensory axons innervating the pancreatic islet; and 3) The role of sensory axons in islet biology and glucose homeostasis in vivo. Under the first aim, the sensory innervation patterns of islets and the neurotransmitter receptor profiles of the innervated islet cell types will be systematically examined in mouse and human islets using immunohistochemistry and physiological recordings. Under the second aim, the response profiles of sensory axons innervating the islet and their effects on islet cells will be studied usng pancreas tissue slices from mice expressing functional reporters in sensory axons or islet cells. Under the third aim, local responses in the islet and regulation of glucose homeostasis by intraocular islet grafts will be challenged by activating, blocking or ablating sensory input. The proposed work is innovative because it capitalizes on new technological platforms that allows for the first time in situ and in vivo functional imaging of sensory axons innervating the pancreatic islet. The proposed research is significant because it is expected to advance and expand current models of the regulation of glucose homeostasis by pancreatic islets. Ultimately, such knowledge has the potential to impact the way diabetes is treated.

? DESCRIPTION (provided by applicant): Imaging mitochondrial signaling in beta cells (? cells) ectopically implanted in the eye. In the last 20 years, it became clear that defects in the mitochondrial energy producing system, either genetic or toxin-induced, cause many different phenotypes. Therefore, defects in one of the five mitochondrial oxidative phosphorylation (OXPHOS) complexes likely trigger distinct signaling pathways, which differentially affect specific cell types. This team will use mouse models with specific defects in complexes I, III or IV to explore enzyme-specific signaling events in mitochondrial disorders. A novel model will be used to test this hypothesis in a dynamic, real-time platform. The approach will involve implanting ? cells in the anterior chamber of the mouse eye, which allows for the use of imaging techniques to follow both cellular and mitochondrial function as well as its signaling patterns in vascularized environment akin to the in vivo situation. This is a multiple Principal Investigator application that will use the expertise of the group of Dr. Alejandro Caicedo (?-cell implant model, imaging physiological biomarkers) and the group of Dr. Carlos Moraes (mitochondrial physiology and mouse models of OXPHOS defects) to tease out signaling signatures associated with defects (genetic or toxin-induced) in specific OXPHOS complexes.

The neurotransmitter acetylcholine plays a major role in regulating insulin secretion. In several species parasympathetic innervation provides cholinergic input to beta cells, but recent studies show that the glucagon secreting alpha cell is a major source of acetylcholine in human islets. The existence of this novel source of acetylcholine in the islet implies that insulin secretion and glucose metabolism can be regulated by local cholinergic mechanisms that remain unknown. The long-term goal of this research program is to understand the contribution of cholinergic signaling to human islet biology in health and disease. The objective of this application is to determine how acetylcholine is secreted, how it is degraded, and how it impacts beta cell biology, using a combination of innovative in vitro and in vivo approaches. The central hypothesis is that paracrine cholinergic input from the alpha cell influences human beta cell function. In our model, acetylcholine is released independently of glucagon and activates beta cell muscarinic receptors, stimulating signaling cascades that promote insulin secretion and glucose homeostasis. Cholinesterases produced by beta cells shape the duration and magnitude of cholinergic signaling. The rationale for the proposed research is that the results will contribute a missing, fundamental element to basic knowledge, without which islet biology cannot be understood. The proposed research is therefore relevant to the mission of the NIH that pertains to the pursuit of fundamental knowledge about the nature and behavior of living systems. Guided by strong preliminary data, our central hypothesis will be tested by pursuing three specific aims: 1) Identify the mechanisms of acetylcholine release from human alpha cells; 2) Determine the location and role of acetylcholinesterase in alpha-beta cell communication; and 3) Determine the impact of cholinergic signaling on human beta cell function. Under the first aim, we will visualize secretion of acetylcholine and glucagon using optical indicators of exocytosis (pHluorins) and measure acetylcholine and glucagon release from human islets in real time using biosensor cells. Under the second aim, we will study the expression of cholinesterases in human islets using biochemical assays, RT-PCR, and immunohistochemistry, and test FDA-approved cholinesterase inhibitors for their ability to increase insulin secretion. Under the third aim, we will stimulate alpha cells and measure beta cell responses with novel probes for signaling molecules. To investigate long-term effects of islet cholinergic signaling in vivo we will use a humanized mouse model in which human islets are transplanted into the mouse eye. We will inhibit acetylcholine secretion, acetylcholine breakdown, and muscarinic receptors in intraocular human islet grafts and measure the effects on human insulin plasma levels and glycemia in the recipient mouse. The proposed research is significant because it is expected to make a strong and lasting impact on our understanding of the role of cholinergic signaling in human islet biology. Ultimately, such knowledge has the potential to impact the way diabetes is treated.

The three components of the peripheral autonomic nervous system, the parasympathetic, sympathetic and sensory nerves, work together to prevent life-threatening fluctuations in glucose homeostasis. They do so in part by regulating insulin secretion from the pancreatic islet. Stimulating autonomic nerves with electrodes has recently been recognized as a potential way to treat diseases (neuromodulation). Given its central role in glucose metabolism and diabetes, the pancreatic beta cell is considered a primary target for neuromodulation. To propose electrical stimulation of nerves to treat diabetes, however, it is essential to understand how islet nerves impact insulin secretion from the beta cell. The objective of this application is to determine the mechanisms nerves use to control insulin secretion. Recent anatomical studies show that sympathetic and sensory nerves innervate the human islet, but parasympathetic innervation is sparse. Importantly, these nerves do not contact beta cells directly but densely innervate the islet vasculature. We therefore hypothesize that autonomic nerves control blood flow and vascular permeability to adjust insulin release into the bloodstream. The rationale for the proposed research is that these mechanisms of nerve action could be intervention targets for neuromodulation in human beings, which is relevant to the mission of the NIH. Guided by preliminary data, our hypothesis will be tested by pursuing two specific aims: (1) determine the functional role of sympathetic innervation for insulin secretion, and (2) determine the functional role of sensory nerves for insulin secretion. Under the first aim, we will test that sympathetic nerves target vascular pericytes to change blood flow. We will transplant human and mouse islets into the eye of diabetic mice. In the eye, islet grafts restore normoglycemia and can be monitored non-invasively. Importantly, islet grafts are revascularized and reinnervated in patterns that resemble those of islets in the pancreas. We will stimulate, inhibit, and ablate sympathetic input to islet grafts and determine the effects on islet blood flow and simultaneously measure the effects on insulin plasma levels and glycemia. We will also test whether chronic activation of sympathetic nerves prevents the derangement of islet vasculature in mouse models of type 2 diabetes. Under the second aim, we will test that sensory nerves respond to local perturbations in the islet microenvironment to change the vascular permeability. To determine what activates sensory nerves in the islet, we will use mice that express functional indicators in sensory neurons and measure activity in nerve terminals in the islet in living pancreas slices and in vivo in neuronal cell bodies of the nodose ganglion after stimulating or injuring the islet. We will also activate sensory nerve chronically to test the impact on islet health in mouse models of type 2 diabetes. The proposed research is significant because the research plan takes into account the innervation pattern of the human islet and uses those aspects of mouse islet innervation that are representative of the human situation. Knowing how nerves control insulin secretion is crucial to propose neuromodulation as a therapeutic approach in diabetes.

If the causes of type 1 diabetes are not known it is mainly because the human pancreatic islet and its interactions with the immune system have not been studied. The diabetes research community is now coming to terms with the lack of relevance to the human situation of results obtained in rodent models of diabetes. In response, there is a new concerted effort at obtaining and studying the relevant material, namely the human pancreas, in health and disease. The long-term goal of this research program is to understand the anatomical and physiological changes that occur in the human islet during the progression towards the diabetic state. The objective of this application is to determine how the endocrine, vascular and immune compartments mature and interact functionally during the postnatal development of the islet. We will focus on the juvenile maturation period because it is a stage during which early-arising autoimmunity is strongly correlated with the predisposition towards overt type 1 diabetes. The overarching hypothesis is that the onset of beta cell-directed autoimmunity is causally related to developmental alterations in the molecular phenotypes of islet cells and to changes in islet architecture. We propose that maturation processes make islets susceptible to inflammation and facilitate the development of autoimmunity. The rationale for the proposed research is that understanding what makes the islet vulnerable will not only help explain its downfall but also provide clues for intervention strategies. This project is thus relevant to the mission of the NIH and is responsive to the research objectives of the Funding Opportunity Announcement from the NIDDK entitled ?High-Resolution Exploration of the Human Islet Tissue Environment?. Guided by preliminary data, we will test our hypothesis by pursuing three specific aims: (1) determine the mechanisms of functional maturation of islet endocrine cells, (2) determine how endocrine control of vascular function is established, and (3) determine changes in the phenotype and behavior of islet resident macrophages. Under the first aim, we will study the massive structural and functional changes needed for beta and alpha cells to reach their full secretory potential. In all three aims, we will record cellular responses with functional imaging and measure hormone release in living pancreas slices from donors aged 0 to 10 years old. These studies will be complemented by scRNA-seq analyses of cells sorted from isolated islets. Under the second aim, we will determine how the endocrine cells establish control of the vascular pericyte, the major regulator of blood flow in the islet. Under the third aim, we will examine how the phenotype and function of the islet resident macrophages changes during the maturation of the islet. The proposed research is significant because the anticipated results could reveal developmental processes that diminish the islet?s natural defenses and trigger abnormal responses from local immune cells. Knowing these processes is crucial to propose intervention targets aimed at preventing the development of type 1 diabetes.

The three components of the peripheral autonomic nervous system, the parasympathetic, sympathetic and sensory nerves, work together to prevent life-threatening fluctuations in glucose homeostasis. They do so in part by regulating hormone secretion from the pancreatic islet. Stimulating autonomic nerves with electrodes has recently been recognized as a potential way to treat diseases (neuromodulation). Given its central role in glucose metabolism and diabetes, the pancreatic islet is considered a primary target for neuromodulation. To propose electrical stimulation of nerves to treat diabetes, however, it is essential to understand how islet nerves impact insulin secretion from the beta cell. The objective of this application is to determine the mechanisms nerves use to control hormone secretion from the pancreas. Recent anatomical studies show in detail how autonomic nerves innervate the islet, but how exactly autonomic nerves impact hormone secretion from the islet is not known. We hypothesize that parasympathetic, enteric, and sensory neural pathways act through intrapancreatic ganglia to modulate local cholinergic control of islet cell function. The rationale for the proposed research is that there is a need to understand the local mechanisms of autonomic nerve control of hormone secretion from the islet, which is relevant to the mission of the NIH. Guided by preliminary data, our hypothesis will be tested by pursuing two specific aims: (1) the role of pancreas sensory innervation in regulating islet function, and (2) the role of the intrapancreatic ganglion as a signaling hub controlling islet function. Under the first aim, we will test that the vagal sensory innervation of the islet participates in a vagovagal neuronal circuit regulating islet hormone secretion. We will selectively stimulate islet cells with a chemogenetic approach and gain genetic access to activated neurons with the Targeted Recombination in Active Populations (TRAP) system. We will combine TRAP with tools for labeling, tracing, recording, and manipulating neurons in the brainstem activated by islet cell stimulation. Under the second aim, we will test that intrapancreatic ganglia integrate signals from parasympathetic efferent nerves, enteric neurons, and sensory axons to compute an executive summary of gut and brain inputs to adjust the local cholinergic control they exert on islet cells. We will stimulate parasympathetic, enteric and sensory innervation of the ganglion ex vivo and in vivo with chemogenetic and optogenetic tools and measure the effects in intrapancreatic neurons. We will further study how manipulating intrapancreatic neuronal activity affects islet function and glucose metabolism (insulin plasma levels and glycemia). We expect that applying our novel approaches to measure and manipulate pancreas nerve activity will yield important information about how nerves affect islet biology. This contribution is significant because it will provide fundamental knowledge that will complete and revise models about the influence of autonomic nerves on endocrine pancreas function. Once a functional map of islet innervation is available, we will be able to propose neuromodulation to improve islet function in diabetes.