Donald Sakaguchi, Ph.D. - US grants

9311054 Jacobson Cholecystokinin (CCK) is one of the most abundant peptides found in the mammalian central nervous system. It appears to modulate many centrally controlled functions, including respiration, thermoregulation, reproduction, nociception, anxiety, and memory. By far the best-known role of CCK, however, is its action as a feeding satiety factor. Recently, Dr. Jacobson found CCK receptor binding in the facial motor nucleus, a substrate that mediates facial movement or expression, including nursing. Therefore, Dr. Jacobson speculates that CCK may also have a central effect on motor systems involved in the control of feeding-related behaviors. Utilizing the methods of immunohistochemistry and receptor autoradiography, she will determine if the cholecystokininergic system is playing a role in nursing behavior. Dr. Jacobson will compare the distribution of CCK receptors in neurons located within the Vllm nucleus which innervates facial muscles in neonates of different species. She will then use a metabolic marker to determine how manipulations in feeding, and the exogenous administration of CCK or its antagonists, affect the activity of neurons in developing motor systems. The results from these studies will further our knowledge on the central mediation of feeding behavior, and the role of cholecystokininergic system in this process. Understanding this fundamental mechanism could in turn have beneficial effects on management of obesity and eating disorders. ***

9311198 Sakaguchi This project will investigate the biological significance of certain specific molecules during the development of the brain . This project will test the hypothesis that the growing tips of nerve cell processes, or growth cones, use receptors for extracellular matrix molecules and specific cell adhesion molecules to navigate through the brain to their appropriate targets. The role of these molecules will be characterized using function-blocking reagents in the developing visual system. In addition, a combination of biochemical , immunological and molecular techniques will be used to examine the spatial and temporal distribution of these molecules during development. The results of these studies will provide important new information on the cellular and molecular basis of growth cone guidance and pathway selection. The longer term goal is to better understand the cellular and molecular mechanisms that regulate the formation of specific nerve connections during development. *** 9311198 Sakaguchi This project will investigate the biological significance of certain specific molecules during the develop | ~ w | ~ ! !. !. F ~ ~ ( Times New Roman Symbol & Arial " h ? ? saka William Proctor, IBN William Proctor, IBN

In this proposal we will investigate the ability of neurotrophic factors delivered via genetically modified stem cell transplants into the eye to reduce the progression of damage and visual loss in an inducible rodent model of glaucoma (chronic ocular hypertension). Our preliminary studies in rodents with induced glaucoma and retinal ischemia demonstrated a therapeutic effect of neurotrophic factor treatment. We found that exogenous application of brain derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) delivered from biodegradable microspheres was temporally correlated with recovering visual function (pupil light reflex and electroretinogram) in rats affected with experimental glaucoma or retinal ischemia. The central hypothesis of this proposal is that transplanted, genetically modified bone marrow-derived stem cells can be used as effective neuroprotective agents to mediate preservation and rescue of visual function in the rat model of experimental glaucoma. We will test this hypothesis by delivering neurotrophic factors [brain derived neurotrophic factor (BDNF) and glia cell line-derived neurotrophic factor (GDNF)] via intraocular transplantation of genetically modified mesenchymal stem cells (MSCs) to eyes damaged by laserinduced chronic ocular hypertension and RGC injury. The long-term objective of this study is to develop effective methods to restore visual function in animal models of glaucoma, and to minimize or halt the process of neuronal death due to ischemic or pressure related insult. In this proposal we will use multiple, innovative approaches to advance toward these goals. First, we will use lentiviral vectors to genetically modify MSCs for production of neurotrophic factors. Second, we will employ cell transplantation of the MSCs as a therapy for neuroprotection in glaucoma. Third, we will turn to functional assays using computerized pupillometry and electroretinography (ERG) to determine the ability of transplanted MSCs and locally released neurotrophic factors to restore visual function after retinal injury. These noninvasive assays of visual function can be repeated over time to provide objective information about the status of the retina and optic nerve. We propose two Specific Aims of study: Specific Aim I: We will determine if transplanted MSCs, engineered to release neurotrophic factors, can provide neuroprotection to glaucomatous eyes. Specific Aim II: We will investigate a combination therapy by co-transplanting BDNF-MSCs and GDNF-MSCs to determine if they have greater neuroprotective activity than either by themselves. Engineered MSCs are especially attractive as neurotrophic factor delivery vehicles for several reasons. First, MSCs can be easily isolated, cultured, and propagated as a source of adult stem cells for autologous transplantation, inciting minimal host immune response. In addition, they can be easily engineered for production of exogenous proteins. Furthermore, these cells can survive in the environment of the eye and have the ability to integrate within the inner retina to serve as delivery vehicles for therapeutic proteins.

Top-down micromanufacturing is the process for fabricating computer chips and microelectromechanical system sensing chips and is the basis for the current semiconductor and sensor industries. However, the emergence of various nanoscale structures and materials can significantly improve the performance of these chips. However, numerous challenges exist to integrate these nanoscale materials and structures on these chips seamlessly due to incompatibility of their fabrication process with standard microfabrication processes. This award supports fundamental research to develop a room-temperature microfabrication process to fabricate silicon nanotubes. The new process allows the manufacturing of silicon nanotubes with other functional elements or electronics on the same chip without thermal damage. Nanotubes and nanotube-enabled functional devices fabricated from a wide variety of materials such as semiconductors, compound semiconductors, and metals have great potential for applications in healthcare, biomedical, energy, aerospace, and chemical industries. Hence, the outcomes from this research benefits the U.S. economy and society. This research involves several disciplines including manufacturing, computation, neuroscience and material science, thereby helping broaden participation of women and underrepresented minority students in research and having a positive impact on engineering and science education.

The project seeks to develop a scalable, ambient temperature, top-down process to fabricate single crystal silicon nanotubes. The fabrication of the silicon nanotubes is realized by simply using a series of integrated circuit (IC)-compatible microfabrication processes. Specifically, polystyrene nanosphere (NS) beads are first self-assembled into a close-packed monolayer on a silicon wafer. These NS beads are then tailored by oxygen plasma reactive ion etching (RIE) to shrink their size. Using the NS beads as the mask, the silicon nanotubes are fabricated by inductively coupled plasma (ICP) Bosch process. This research fills the technical knowledge gap on how to realize the large-scale integration and arrangement of a single nanotube or an array of nanotubes in a controlled manner on a chip. The research team plans to perform sharp interface phase-field nanoscale modeling for fundamental understanding and control of the tolerance range of the processing parameters for fabricating robust silicon nanotubes. In addition, the research team plans to develop silicon nanotube-based patch-clamp nanoprobes for neuronal and cellular stimulation and recordings. Specifically, arrays of silicon nanotube-based patch-clamp nanoprobes embedded within microscale cell culture chambers are developed for recording electrophysiological activity in cultures of adult hippocampal progenitor cells that have differentiated into oligodendrocyte, astrocytes or neurons as well as mapping multiple individual synaptic connections between neurons.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

In the United States, about one in five Americans above the age of 18 suffer from diagnosable neurological disorders with no cure insight. As such, new, safe, non-invasive methods for the treatment of brain disorders are critically needed. Non-invasive techniques including repetitive transcranial magnetic stimulation (TMS) and transcranial direct current stimulation have had some success. However, progress has been limited due to poor understanding of the interactions of magnetic fields with nervous tissue. The molecular/cellular mechanisms of nervous tissue under TMS are still lacking. Hence, investigation of effects of transient magnetic fields on adult neurogenesis, cell differentiation and plasticity of nervous tissue (neurospheres) is essential in developing new treatment procedures and achieving the use of TMS as a neuromodulation tool for treating neurological disorders. The educational goal of this project is to effectively integrate research with educational activities and to train both undergraduate and graduate students in interdisciplinary studies to produce next-generation bioengineers. The PIs will develop a new Vertically Integrated Program (VIP) based on this research proposal entitled: Targeting Neurodegenerative Diseases Using Bioengineering Approaches. The VIP will unite undergraduate education and faculty research in a team-based context. The overall educational goal is to help next-generation workforce development by training students to carry out research with sound technical background and allowing them to gain hands-on laboratory skills for their advanced careers. The long-term goal is to design an automatic technical platform to synthesize a variety of in vitro central nervous system disease models to mimic in vivo conditions as closely as possible. This will facilitate the studies of TMS effects and drug screening assays for neurodegenerative disorders.

The goal of this proposal is to develop a chip-based microfluidics platform that facilitates the rapid formation of three-dimensional in vitro cell culture models of the central nervous system, which will permit the investigation of mechanisms of organ development, cellular interactions, disease model progression under magnetic field stimulation and drug treatments within de?ned microenvironments. Specifically, the proposed efforts include (1) the development of a chip consisting of microchamber arrays so that neurospheres including diseased neurospheres such as Alzheimer?s disease (AD) neurospheres can be fabricated in an efficient manner; and (2) the studies of the behavior of healthy neurospheres and AD neurospheres under transient magnetic stimulation (MS) and drug treatment using this chip. Major innovations of this proposed project can be summarized as the following: (1) Using this type of microfluidic chip, large-scale neurospheres with tunable and quantitative compositions can be synthesized rapidly and inexpensively, facilitating studies of different types of neurospheres; (2) Using a concentration gradient generator at the upper stream of this chip, a series of AD models (AD neurospheres) with known concentrations of amyloid-? and/or phosphorylated-tau can be readily fabricated; and (3) developing this chip will thus facilitate studies of the effects of both MS and drug treatment on AD models.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Therapeutic nanocarriers have transformed the landscape of multiple diseases by enabling site-specific drug delivery. The localization of nanocarriers in biological cells is controlled by their physical and biochemical properties. The goal of this award is to design and manufacture hybrid liposomal nanocarriers, simultaneously tuning their mechanical and molecular properties to achieve high cellular uptake. This project demonstrates this goal in a brain model since nanocarrier transport through the blood brain barrier remains a critical challenge. Further, this work examines spatiotemporally controlled drug release from these nanocarriers using light to enable safe and targeted therapeutics. The research establishes mechanisms that show drug transport is controlled by novel physicochemical properties allowing the tailoring of a new class of nanocarriers targeted to study biological interactions. This class of nanocarriers advances drug delivery in difficult to treat disorders of the brain. The principles learnt can also be extended to achieve therapeutic response in other diseases, thus meeting national healthcare needs. This project seamlessly integrates research with education to transition this work through ‘lab-bench-to-classroom’ activities and by dissemination of ‘Fun with Color Capsules’ kits to K-12 students targeting underprivileged youths. By leveraging established and effective outreach programs, this work enables training of undergraduate and graduate students for the future workforce.<br/><br/>The physicochemical behavior of therapeutic liposomal nanocarriers drives their interaction with biological interfaces and controls endocytosis in cells. Yet which properties should be tuned to enable efficient nanocarrier transport through biological barriers remains paradoxical. Therefore, approaches that leverage unexplored properties of nanocarriers are imperative to enable a paradigm shift in spatiotemporally controlled drug delivery. The goal of this project is to design and manufacture unconventional nanocarriers via bottom-up, directed self-assembly approaches. The research involves fabricating hybrid liposomal nanocarriers (LNCs) that synergize the properties of soft (liposome) core and hard (gold) shell nanoparticles in a single manufacturing platform enabling tunability of the elastic modulus and surface ligands. The research hypothesis is that these properties are mutually dependent, and when simultaneously tuned, achieve cell- and phenotype-specific targeting and therapeutic function demonstrated in an in vitro blood brain barrier (BBB) model. Further, LNCs are functionalized with antibody fragments that specifically target cells in the BBB. Another aim is to track LNCs in both cells and neurospheroids and pursue combinatorial optimization of the ligand density with elastic modulus to determine the stiffness-ligand regime that impacts cell-specific targeting. Finally, the project aims to demonstrate that these properties of LNCs enable effective photothermally actuated drug transport across the BBB.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.