Alon Greenbaum

The brain critically relies on balanced production of neurons and glia during embryonic and early postnatal development. Recently developed clonal lineage analysis has revealed the behavior of neural stem cells (NSCs) giving rise to neurons in the cerebral cortex with unprecedented single-cell resolution. However, the clonal principles underlying the formation of glia by NSCs remains unclear and has yet to be systematically investigated using these new technologies. Gliogenesis is critical for proper neuronal functions and when disrupted, it can result in various neurological diseases. Reconstructing how glia are generated from individual NSCs and organized in the cortex during development is essential to understand the structure-function relationships and how they can be modulated by clone-specific factors. We have established a genetically-based single-cell lineage tracing technique utilizing MADM (Mosaic Analysis with Double Markers) mice to label NSCs in the developing cortex and begin to address this knowledge gap. Using this method we have found two distinct populations of glia that occupy different territories of the cortex and its related structure the hippocampal formation. The goal of the proposed research is to reconstruct, quantify, and mathematically model the behavior of individually labeled NSCs in vivo in neocortical and paleocortical areas. This effort requires the development of optimized imaging and analytical tools to ensure reliable and repeatable interpretation of quantitative data. To this end we are developing light sheet microscopy and AI-based automated quantification methods to facilitate unbiased and precise imaging and quantification of clonal data in the brain. Successful completion of our study will result in a comprehensive map of single NSCs and their glial progeny in various cortical regions. Our approach will also establish a platform for detailed quantitative and computational analysis of gliogenesis, glial diversity, and their potential for repair and regenerative approaches in the cortex in the context of various neurological disorders and brain injury.

The cerebral cortex critically relies on balanced production of neurons and glia during embryonic and early postnatal development. Recently developed clonal lineage analysis has revealed the behavior of neural stem cells (NSCs) giving rise to neurons in the cerebral cortex with unprecedented single-cell resolution. However, the formation of glia by NSCs remains unclear and has yet to be systematically investigated using these new technologies. Gliogenesis is critical for proper neuronal functions and when disrupted, it can result in various neurological diseases. Reconstructing how glia are generated from individual NSCs and organized in the cortex during development is essential to understand the structure-function relationships and how they can be modulated by clone-specific factors. We have established a genetically-based single-cell lineage tracing technique utilizing MADM (Mosaic Analysis with Double Markers) mice to label NSCs in the developing cortex and begin to address this knowledge gap. The goal of the proposed research is to reconstruct, quantify, and mathematically model the behavior of individually labeled NSCs in vivo. We will use the power of this labeling method to also screen for gene expression of glial clones at single cell resolution, which all together will help us decipher the general principles organizing glial clones in the cortex, and define how clonal siblings interact with each other. Using some of the identified genes that we have already identified, we will test their role in generation of glial clones in the cortex, which will further help define the biological system underlying clonal rules and principles of gliogenesis. Successful completion of our study will result in a comprehensive map of single NSCs and their glial progeny in various cortical regions. Our approach will also establish a platform for detailed quantitative and computational analysis of gliogenesis, glial diversity, and their potential for regenerative approaches in the cortex. Potential for Broader Impact Our approaches to understand how important constituents of the brain, the glial cells, develop have wide implications. Disruption of glial development is the root of a range of pathological conditions in the brain. Therefore, understanding the basic principles and cellular mechanisms that control gliogenesis is critical to appreciate not only how healthy development may be controlled by systematic production of glial cells, but also how abnormalities in gliogenesis may lead to devastating neurodevelopmental disorders.

In the US one out of eight people suffers from hearing loss. The common causes for hearing loss are age, frequent exposure to loud noise, genetic disorders and more. Hearing loss is often accompanied with other medical conditions such as a higher rate of depression, social isolation, and cognitive decline (e.g., dementia). Given that hearing loss is a non-lethal condition, most of our understanding about hearing loss in humans is provided by either postmortem examination, or animal models. Rodent models are commonly used to study normal and impaired hearing, and they vastly expanded our understanding of how sound is perceived at a molecular level. However, the rodent inner ear physiology, genetics and anatomy is different from the human, therefore, in some situations, this fact limits our understanding how mutations in deafness genes affect hearing. Moreover, the small size of the mice creates difficulties to assess and evaluate the translational value of emerging technologies to deliver and restore hearing via regeneration of hair cells or spiral ganglion neurons. To address these gaps, we would like to advocate for the use of the pig as a translational animal model to study the inner ear. Pigs are large animal models, and their size, anatomy, intelligence, and genetics is by far closer to humans than rodents. Consequently, the pig is becoming a popular animal model and it is commonly used in cardiovascular research, wound healing, organ transplantation, nutritional studies and more. In general, pigs are readily available for research as they are a popular form of livestock, and only in the US, over 100 million pigs are slaughtered annually. Here, to establish the pig as a large animal model, we will: (i) Characterize and compare the gene expression pattern of major deafness genes to those of mice and marmosets. (ii) Establish an image guided method to deliver therapeutics (e.g., viruses) to the middle and inner ear. (iii) Quantify the baseline expression of cochlear progenitors and hair cells across ages. Technically, we will utilize tissue clearing and labeling techniques together with advanced microscopy to image the whole porcine inner ear with subcellular resolution. This methodology facilitates the detection of subtle biochemical and morphological changes in the tissue. Overall, the success of these goals will open future avenues for testing new regenerative medicine approaches for hearing restoration, characterizing cellular and physiological changes during development, and studying hearing loss progression driven by genetic mutations that do not produce a deafness phenotype in mice.

My long-term goal is to correct hearing loss via regeneration of inner ear hair cells and repair of the neuronal innervations that support their function. Unfortunately, at this time there is no high throughput, non-destructive method of therapeutic delivery into the cochlea/inner ear. The objectives of this proposal, the next step toward the attainment of this long-term goal, are: i) to develop in-vitro model systems to track the transport of therapeutics through the round window membrane (RWM) or uptake into cells of the RWM, and ii) to create a method to enhance the efficiency of delivery of regenerative therapeutics into the RWM. The central hypothesis is that the utilization of exosomes to deliver cargo can enhance uptake of genetic modifiers by cells of the RWM and/or facilitate the passage of therapeutics through the RWM. The rationale for this proposal is that successful completion will open new opportunities to engineer the delivery of a pool of therapeutics into the inner ear through genetically induced de novo secretion from cells of the RWM or by nondestructive transport of therapeutics across the RWM. Also, the use of a large mammal model that is physiologically similar to humans will facilitate clinical translation. The following first two specific aims will be pursued during K99 and the rest during R00 phase: Aim 1) Create a realistic in-vitro RWM to test the passage of representative substances; Aim 2) Identify exosomes that can be transported through RWM or taken up into cells attached to the membrane; Aim 3) Demonstrate that exosomes can be used to transport therapeutic-like substances through the RWM or to introduce mRNA into cells associated with the RWM; Aim 4) Use exosomes in vitro and in vivo to deliver therapeutics that trigger hair cell regeneration and activate neurons for increased survival. Under the first aim a porcine RWM tissue explant or isolated cells from RWM will be used to create RWM models. For the second aim, RWM exosomes, mesenchymal stem cell (MSC) exosomes (gold standard), and liposomes (FDA approved) will be loaded with fluorescent protein or mRNA and compared for efficiency of transport through and uptake by the RWM. For the third aim, exosomes will be loaded with Atoh1 protein (known to trigger hair cell regeneration) or its mRNA, and proliferation/differentiation of hair cells progenitors exposed to such loaded exosomes will be evaluated. Finally, for the last aim, therapeutics for hair cells regeneration and neuronal repair will be loaded into exosomes for delivery across the RWM in-vitro and in-vivo porcine models. Upon completion of the K99, the expected outcomes are: 1. Availability of safe and translatable platforms to test transport of therapeutics into the inner ear, and 2. Data on the efficiency of exosomes for nonsurgical transport of therapeutics in the ear. During R00, we further expect to identify the regeneration and repair efficacy of hair cells and neurons in porcine model using exosomes. These results are expected to have a positive impact because they will enhance testing and delivering novel and regenerative therapeutics to treat hearing loss and improve the lives of people with communication disorders.

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen worldwide with an urgent need for a protective vaccine. C. trachomatis is closely related to the pig pathogen C. suis and both induce a cross-reactive immune response. The proposed research uses a novel C. suis pre-exposed outbred pig model to develop a C. trachomatis vaccine candidate; and it will provide an in-depth analysis of the protective immune response. This research will have three major benefits, it will: i) develop a novel C. trachomatis vaccine candidate, ii) further establish a valuable genetically diverse animal model for C. trachomatis research, vaccine development and testing; and iii) greatly improve our understanding of a protective immune response.