Robert Hsiu-Ping Chow - US grants

The mechanism of impaired insulin secretion in type 2 diabetes (TTDM) is poorly understood. In health, approximately 75 percent of secreted insulin is released in discrete pulses with a periodicity of approximately 6 minutes, and modulation of the magnitude of these pulses serves to regulate the insulin secretion rate. In patients with TTDM, the rate of insulin secretion is impaired by a selective reduction of the pulse mass (amount of insulin released during a pulse), not pulse frequency. In addition, in TTDM first phase insulin secretion in response to a glucose bolus is impaired. These deficits are present even though there appears to be abundant stored insulin in the islets of patients with TTDM. The incretin hormone glucagon-like peptide-1 restores pulsatility and first phase secretion. Taken together these observations suggest that the number of insulin granules available for rapid discharge in a discrete insulin pulse or first phase secretion is deficient in TTDM. Our overall hypothesis for the present studies is that the mechanisms of impaired insulin secretion in TTDM is a decrease in the readily releasable pool of insulin granules. Three specific aims test this overall hypothesis: Aim 1: Test the hypothesis that impaired insulin secretion in TTDM is due to a reduction in the size of the readily releasable pool of insulin granules. Aim 2: Test the hypothesis that the mechanism leading to this deficit is insufficient docking of granules from the reserve pool. Aim 3: Test the hypothesis that the readily releasable pool and insulin secretion can be restored by agents that enhance granules docking and/or inhibit undocking. We will use the method of total internal reflection fluorescence microscopy (TIRFM), a method that enables the visualization of individual granules near the plasma membrane within living secretory cells. We are well positioned to address these hypotheses with the following resources: (1) A fully established apparatus for TIRFM. (2) Access to a number of rodent models of TTDM, including GK and ZDF rats and a transgenic rat model in which human IAPP is expressed. (3) Access to human islets. (4) The support of the USC Diabetes Research Center.

DESCRIPTION (provided by applicant): Regulated exocytosis is shared among many secretory cell types across the phylogenetic tree. Fusion of vesicle and plasma membranes is mediated by a highly conserved molecular machine comprising the SNARE proteins, whose function is fine- tuned through interaction with other proteins. One of these other proteins is complexin (CPX), a ~150-amino acid protein having four isoforms (I-IV), that binds stoichiometrically to the SNARE complex. The molecular mechanism of complexin action is controversial, with some evidence for a negative role - "clamping" the SNARE complex and inhibiting exocytosis - and other evidence for a positive role, leading to enhanced exocytosis. We propose a systematic study of the function of CPX in mouse adrenal chromaffin cells and neuromuscular junction. These two model systems, one a hormone-secreting cell and one a synaptic preparation, have been used extensively to study calcium-dependent exocytosis. We have previously presented data supporting the hypothesis that CPXII plays a positive regulatory role in exocytosis in adrenal chromaffin cells, serving to prime vesicles. We propose to build on our previous work by testing the following specific hypotheses in chromaffin cells derived from CPXII knockout (CPX KO) mice: 1) CPXII must bind to the SNARE complex in order to facilitate priming. 2) Spontaneous exocytosis is not increased in knockout cells 3) CPXII facilitates molecular coupling of vesicles and calcium channels. 4) CPXII does not affect the Ca sensitivity of exocytosis. 5) CPXII must be phosphorylated to facilitate priming. We have preliminary evidence that CPXI also plays a positive role in the neuromuscular junction (NMJ). We will test the following hypothesis in a mouse CPXI KO model: 6) CPXI regulates the readily releasable pool in mouse NMJ. Knockout mice have been provided by our collaborator Nils Brose (Max Planck Institute, Germany). The multidisciplinary team includes Dr. Robert Chow, Dr. Jeannie Chen, Dr. Ralf Langen, and Dr. Chien-Ping Ko. Understanding CPX function could lead to new strategies to alter secretion rates to combat disease states or to improve biotechnological production of secreted products. PUBLIC HEALTH RELEVANCE The protein complexin controls how much cells can secrete. Understanding how it does this could lead to new strategies to alter secretion rates to combat disease states or to improve biotechnological production of secreted products.

DESCRIPTION (provided by applicant): Human embryonic stem cells (hESCs) offer a potentially unlimited source of tissue for replacement therapy in type 1 diabetes. However, current differentiation protocols like the 5-stage Novocell protocol yield few insulin-producing cells, which in vitro are unresponsive to glucose. Only after in vivo transplantation do the cells acquire glucose responsiveness. The molecular switches that turn on glucose responsiveness remain a mystery. Recent studies highlight a central role of small noncoding RNAs called microRNA (miRNA) in the regulation of gene expression during development. The overarching goals of this proposal are to develop a rapid screen for stem cell- derived insulin-positive cells that respond to glucose and to characterize the genetic and epigenetic (miRNA-based) mechanisms important for acquisition of this important phenotype. As a starting point for this proposal, we have already conducted a miRNA microarray analysis on populations of stage-5-differentiated hESCs vs. human islet cells. Although both populations are known to be composed of diverse cell types, our preliminary analysis showed striking differences in the level of several miRNAs, including miR-375 - a miRNA previously shown by other groups to be important for maintaining pancreatic beta cell mass, and, thus, lending support to the idea that we may find miRNAs that regulate beta-cell-specific genes, including those regulating glucose responsiveness. We have also initiated development of a novel fluorescent reporter that identifies insulin-producing cells, as well as reports insulin secretion. Here we outline a systematic approach to test the role of miRNAs in regulating glucose responsiveness. Our specific aims are: 1. Develop a fluorescent reporter that both identifies insulin-positive cells and reports insulin secretion, as a tool to rapidly screen new protocols to activate glucose-response genes. 2. Identify miRNA candidates for regulation of glucose-response genes. We will express the fluorescent reporter in Stage-4 or -5 hESCs and use fluorescence-activated cell sorting (FACS) to isolate insulin-positive but glucose non-responsive Stage-5 hESC-derived cells. We will compare their miRNA profiles with human beta cells using a differential miRNA microarray approach. 3. Test the functional activity of candidate regulatory miRNAs using population and single-cell physiological and genetic analysis. PUBLIC HEALTH RELEVANCE: Type 1 diabetes treatment by human embryonic stem cells has been blocked so far, because, while scientists can coax the stem cells to turn into cells that make insulin, those cells don

DESCRIPTION (provided by applicant): Our overall aim is to assess the technical and biological noise in measured RNA levels in single cells in a number of human tissue types, and to develop analytical tools to address the complexity observed at the single-cell level. Understanding the sources and relative sizes of technical and biological noise has become essential, as the lower detection limit of RNA-Seq is now in the range of 10 picograms of total RNA -- i.e. the amount of RNA in single cells. Technical noise can come from several different sources that we will attempt to evaluate separately. These include: 1) sample procurement and RNA retrieval, 2) sequencing library preparation, 3) sequencing methodology, 4) batch effects in sequencing experiments, 5) bioinformatics approaches for data analysis, 6) gene-gene variability. Assessing the relative magnitude of technical noise from different sources will infor how to reduce that noise in future experiments, and thereby reduce interference with studies of meaningful biological variations or noise. Biological noise, or inter-cell differences arise from differences in cellular history or fate, stages of cell cycle, connections to neighboring cells, an true functional differences of ostensibly identical cells (e.g., different olfactory receptors amon olfactory neurons). We propose to study three different cellular systems that we expect to have different levels of inter-cell variation (biological noise): first, syncytiotrophoblast cells from placenta, which are expected to have relatively low inter-cell variation; second, olfactory neurons from nasal neuroepithelium, each of which is expected to express a different olfactory receptor, providing a positive control for differences in the RNA-Seq data; and third, individual Purkinje neurons from the cerebellum, which may have larger inter-cell variation. The method to extract cytoplasm from individual cells -- patch clamp pipette extraction -- does not require fully disrupting the tissue or dispersing the cells. We have already used patch clamp to determine the transcriptomes of multiple individual neurons in the mouse brain, using the cytoplasm extracted from single cells on which we had already performed patch-clamp electrophysiology recordings, followed by RNA-Seq. For each of the cell types chosen - syncytiotrophoblasts, olfactory neurons, Purkinje neurons, cortical neurons we will generate single-cell transcriptome datasets to evaluate heterogeneity among ostensibly similar cells, using patch clamp to extract cell contents and RNA-Seq; investigate sources of technical noise and apply a systematic approach to reduce technical noise. We will test whether neuronal plasticity is reflected as a change in the transcriptome. All analytical tools and the transcriptome database developed here will be shared openly on our website and all project data will be deposited into dbGAP and the Short Read Archive (or its replacement) 6 months after data QC.

Proposal: 1404089
PI: Humayun, Mark S.
Title: Retinal Nanophotoswitch

Significance
The objective of the proposed research is to develop a novel neurophotonic molecular switch for light-activation of neurons. A visual prosthesis based on this nanophotoswitch (NPS) has the potential to improve the visual acuity for the millions of patients suffering from retinal degenerative diseases, such as retinitis pigmentosa and age-related macular degeneration. This proposal is very innovative. The biophysical mechanism is completely differentiated from electrical devices and other molecular photo-switch-based approaches. Beyond vision restoration, it is a generally useful approach for controlling excitable cells. If successful, it may have a great impact on patients who are underserved by current treatments.

The interdisciplinary research provides excellent educational opportunities for participating graduate and undergraduate students. The proposing team has an exceptional record on inclusion of women, under-represented minorities, and undergraduates in their research. They also have a good track record on outreach to the local community, and planned outreach activities include Research Experience for Teachers and activities for K-12 students.

Technical Description
Nanophotoswitch (NPS) offers a new tool to elicit electrical activity for basic science studies of neuronal function, both in vitro and also potentially in vivo. The hypothesis is based on the NPS design and results of pilot experiments, that light induces an electrical dipole in the NPS. Preliminary data indicate that an NPS based on ruthenium bipyridine (Rubpy) inserts into cell membranes and upon visible-wavelength illumination triggers action potentials in cultured excitable cells and in wholemount rat retina. When injected into the eye of blind photoceptor-degenerate rats, visual stimulation induces electrical activity in the superior colliculus. It was also demonstrated that NPS can both depolarize or hyperpolarize the membrane, depending on the environmental redox potential. This unique combination of bi-directional modulation of the membrane potential in one biophotonic switch affords the ability to both activate and inhibit the action potential firing of the illuminated cells with the same molecule, presenting largely increased flexibility in neuronal control. The NPS would be useful in studying any electrically excitable cell, including, for example, cardiomyocytes, smooth muscle cells, neuroendocrine cells, and certain glial and cancer cells. Since light-activated signaling unit is individual neurons, a visual prosthesis based on NPS system has the potential to provide higher visual acuity for the millions of patients with photoreceptor loss due to retinal degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Distinct from other nano-scale optical cellular modulating approaches using optogenetics or azobenzene-based photoswitches, this approach obviates the need for gene manipulation, toxic ultraviolet illumination or immunogenic molecules, due to the unique light-to-electrical signal transduction mechanism of the NPS.

This PFI: AIR Technology Translation project focuses on translating research on light-induced modulation of the activity of electrically excitable cells to fill the need for a treatment for the retinal degenerative diseases retinitis pigmentosa and dry age-related macular degeneration. The molecular artificial retina is important because over a million Americans suffer from advanced vision loss and this research has the potential to restore some level of vision in these patients, significantly improving quality of life.

The project will confirm efficacy of the lead molecules based on ruthenium nanophotoswitches, evaluate their toxicity and optimize the scale up of the top candidates for pre-clinical and clinical trials of the molecular artificial retina. The nanophotoswitches (NPS) have the following unique features: it is a small molecule that is easily delivered (through injectable solutions or implants) to the eye, where it embeds in the membrane and ambient light activates neurons. The mechanism is universally applicable, both for any type of retinal degenerative disease and for any patient suffering from retinal degeneration. This is important, because many retinal degenerative diseases have multiple genetic components, making gene therapy complex and difficult. The class of molecules currently being investigated for a molecular artificial retina may be catalytic, therefore requiring only small, infrequent doses, and inexpensive, due to the simplicity of the system. These features provide the following advantages restoring vision with infrequent injections when compared to AREDs, a daily dose of vitamins that potentially delays the progression of the disease, the only FDA approved treatment in this market space.

This project addresses the following technology gaps as it translates from research discovery toward commercial applications: Determine the biosafety of the lead NPS molecules and identify NPSs with low toxicity/ immunogenicity, evaluate therapeutic potential of the lead NPS, scale up the production of NPS to meet the market demand in quantity, and develop the polymer implant for sustained intraocular release of the NPS. In addition, personnel involved in this project, undergraduate students and postdoctoral scholars, will receive entrepreneurship and pharmaceutical/drug development experiences through customer discovery and collaborations with ophthalmologists conducting pre-clinical and clinical trials.

Our long-term objective is to understand the principles that orchestrate skin morphogenesis in development and wound regeneration. The understanding of biochemical signaling is well advanced. Yet, research into the roles of non-neural bioelectricity lags behind, although evidence for a role of bioelectricity in development, regeneration (McLaughlin and Levin 2018 16; Li et al., 2020 5) and wound healing (Zhao et al. 2012 32) is growing. Our research objective is to study the mechanisms underlying the development and regeneration of skin appendages. In two of our recent research papers, we were inspired to see bioelectricity in action in two tissue patterning processes. First, the orientation of elongating feather buds is regulated by synchronization of oscillating calcium channel activities in bud dermal cells, which is controlled by epidermal Shh signaling (Li et al., 2018 11). Second, the skin frequently shows pigment stripes along the body. The size and spacing of longitudinal pigmentation stripes in Japanese quail was recently shown to be controlled autonomously within melanocyte progenitor populations in a gap junction-dependent manner (Inaba et al., 2019 12). At the time these periodic black/yellow stripes form in embryos, the spacing is in millimeters, a large-scale patterning process that cannot be explained by the classical Turing reaction-diffusion mechanism (patterning in micrometer range). The results led us to think hard about how large-scale tissue architecture is built. While localized signaling centers involving morphogens (e.g., WNT, BMP, FGF) were shown to initiate periodic patterning of feather/hair buds, some unidentified mechanism capable of spanning large distances dynamically must work together to transduce the information over the long-distance scale (Inaba and Chuong, 2019 15). Bioelectricity work here provides a clue. Thus, we organized a multi-disciplinary team to analyze the mechanisms on how biochemical and bioelectric signals integrate to achieve the large-scale tissue patterning. We hypothesize, among other possibilities, transient bioelectrical signaling across gap-junction-coupled cell collectives may allow rapid, long-distance signaling with minimal decrement. Electropotential gradients are harnessed to propagate signals rapidly over the long distance (millimeters in milliseconds) to regulate intracellular messengers and pattern the much larger morphogenetic field. The developing avian skin explants provide an excellent model because of the quantifiable distinct patterns, planar topology for easier channel activity visualization, electric current perturbation and optogenetic gene activation ? not easy in the mouse model. Experimentally, we will first gauge the endogenous bioelectric landscape and evaluate the importance of bioelectricity in these two tissue patterning processes (Aim 1A, 2A). Then we will study how ion channels / gap junctions cross-talk with biochemical signals to achieve tissue patterns (Aim 1B, 2B). The work is likely to produce new findings and insights for future applications to use bioelectricity to benefit wound regeneration.