Upstate Medical University

NIH Research Projects · FY 2025 · 2021-09

SUMMARY Genetic risk factors for Alzheimer's Disease can predict the increased likelihood of acquiring the disease yet it is becoming more evident that external factors are involved in the neurodegenerative disease progression. Recently two herpesviruses have been implicated in this disorder as they have been found in diseased tissues of Alzheimer's patients. Human herpes simplex virus 1 (HSV-1) and Human Herpesvirus 6a (HHV6a) are neurotropic herpesviruses that establish both lytic and lifelong latent infections of the nervous system. How these viral pathogens alter the outcome of neurodegenerative disease remains uncharacterized. Alzheimer's Disease is marked by distinct alterations of host proteins including the misfolding of Tau. Using a novel human iPSC-derived 3D cortical model system, we observed altered splicing of the mRNA that encodes Tau. Additionally, we observe a viral infection dependent increase in the accumulation of phospho-Tau, a post translational modification indicating the misfolding of Tau. These preliminary findings support the hypothesis that viral infections directly impact the host cell microenvironment resulting in changes in Alzheimer's Disease markers. The overarching goal of these studies is to identify infectious etiological agents that impact either the severity or frequency of Alzheimer's Disease progression. The objective of this proposal is to define the biological impact of neurotropic herpesvirus infections on accumulation of known drivers of Alzheimer's disease within a physiologically relevant model system. Our first aim will identify and define viral dependent changes within multiple host proteins involved in neurodegeneration. This will for the first time, a direct link between neurotropic virus infections and pathogenic neurodegenerations in cells of clinical relevance. The second aim will use novel RNA labeling protocols to profile the transcriptome from only infected cells within the 3D organoids and will utilize single cell sequencing to characterize the tropism of these neurotropic viruses. The third aim will utilize 3D organoids that harbor known genetic risk factor mutations found in the onset of Alzheimer's Disease and will determine if herpesviruses amplify the onset of neurodegenerative progression. Successful completion will clearly define the viral factors that influence Alzheimer's disease and will provide insight in pathogenic mechanisms that extend beyond Alzheimer's Disease.

NIH Research Projects · FY 2025 · 2021-07

Project Summary The general purpose of this proposal is to provide the principle investigator (PI) with the experience and skills necessary to become a successful and independent vision researcher. Lowering intraocular pressure (IOP), the primary modifiable risk factor for glaucoma and the mainstay of treatment, does not halt the progression of glaucoma in many cases. This discrepancy highlights gaps in our understanding of how IOP- induced mechanical stressors cause damage to retinal ganglion cells (RGCs), the major cell type affected in glaucoma. Thus, the PI’s long-term goal is to develop an independent research program dedicated to investigating mechanisms by which RGCs and their cellular and structural support system respond to mechanical stressors. These studies will set the stage for the development of new treatments, with the potential to prevent RGC death regardless of IOP. The consensus in the field is that the initial site of RGC injury occurs at the optic nerve head (ONH). Of the cells of the ONH, astrocytes are the most likely sensors of these mechanical stressors. A prime candidate through which astrocytes sense and respond to mechanical stimuli is mechanosensitive channels. In Aim 1, we will test the hypothesis that astrocytes are key mechanosensors within the ONH, and that they translate changes in surrounding stiffness and mechanical strain into alterations in ECM integrity and tissue stiffening. In Aim 2, we will test the hypothesis that inhibition of ONH astrocyte Piezo mechanosensitive channel activity will prevent IOP-induced astrocyte reactivity and RGC death. We will use a combination of tissue engineering techniques, biomechanical tools, and in vivo glaucoma models to accomplish our specific aims. By analyzing ONH astrocyte mechanosensation in glaucoma through a mentored approach, and by acquiring a solid foundation in the fields of astrocyte/matrix biology, mechanobiology, tissue engineering, and glaucoma model systems, the PI will uniquely position herself to identify such novel drug targets. The success of the proposed research and career development plan is reinforced by the exceptional multi-tiered mentoring environment at the Center for Vision Research at SUNY UMU, participation in the Syracuse Biomaterials Institute at Syracuse University, state-of-the-art facilities, and a strongly collaborative research community.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Our laboratory has a long standing interest in understanding the catalytic and regulatory mechanism of the proton pumping vacuolar ATPase (V-ATPase, V1Vo-ATPase), a dynamic multisubunit membrane integral rotary motor enzyme found in all eukaryotic cells. The V-ATPase acidifies the lumen of organelles and, in professional acid secreting cells, the extracellular space. Enzyme function is required for fundamental cellular processes such as endocytosis, bone remodeling, protein trafficking, acid-base balance, sperm maturation, and neurotransmitter release. While complete loss of V-ATPase function is embryonic lethal, partial loss or hyperactivity is associated with numerous human diseases such as osteopetrosis, diabetes, male infertility, neurodegeneration, and cancer. Moreover, some viruses such as influenza rely on the acidic environment created by the V-ATPase for infection. Fighting these diseases on a molecular level will require a detailed understanding of the structure, catalytic mechanism and regulation of the eukaryotic V-ATPase. In cells, V- ATPase activity is regulated by a unique mechanism referred to as “reversible disassembly”, wherein the complex reversibly dissociates into V1-ATPase and Vo proton channel, with both sub-complexes becoming autoinhibited. Despite its important role in V-ATPase physiology, the molecular mechanism of reversible disassembly is poorly understood. This gap in knowledge is largely due to a lack of both high-resolution structural information and an in vitro model system to study the process under defined conditions, aspects that we are working to address. An interesting, and technically challenging feature of the mammalian V-ATPase is that most of its subunits are expressed as multiple isoforms. However, as such isoforms display differential tissue enrichment, they may provide opportunities for targeted therapeutics. Indeed, several diseases have been linked to malfunction or upregulation of specific isoform containing V-ATPase. However, how different isoform combinations determine tissue localization, and whether these isoform specific complexes have unique biochemical or regulatory properties, is currently unknown. We have started to develop a system to purify wild type and mutant forms of human V-ATPase in an isoform specific fashion for biochemical and structural analyses. Further, we are developing single-domain antibodies (Nanobodies) against specific subunit isoforms to serve as research tools, and to explore isoform specific modulation of V-ATPase activity in disease. Our research program employs the tools of structural biology, cell biology, biochemistry and biophysics to address broad questions of V-ATPase catalytic and regulatory mechanisms. For some fundamental aspects of V- ATPase structure and regulation, we study the enzyme from yeast, a well documented model system for the human V-ATPase. We use human tissue culture for questions that cannot be addressed in yeast, such as structure and biochemical properties of specific isoform containing enzymes. The long term goal of our research is to find ways to modulate the activity of disease causing V-ATPases in an isoform specific way.

NIH Research Projects · FY 2025 · 2021-04

SUMMARY/ABSTRACT Traumatic brain injury (TBI) represents a public health crisis in the United States. TBI is a very common injury in young adults and causes long-term disabilities in cognition, learning and memory, emotional control, and sensory and motor function. A severe TBI can lead to lifelong physical and psychological problems and increase the risk of developing neurodegenerative diseases. Severe TBI in young adults is a significant public health problem and national burden because their lifelong disabilities, permanent productivity loss, and long- term daily care dependence not only seriously affect the life of an individual and their family but also create a heavy financial burden in the United States. In the traditional view, TBI is an event that only needs acute management and a brief period of rehabilitation. Today’s notion is that TBI is the onset of a chronic health condition that requires therapies for improving recovery months and years after TBI. However, no such a treatment is available in the chronic phase. The chronic phase exists in a long period from 3 or 6 months after TBI and throughout an individual’s life span. The lack of treatment to improve severe TBI recovery in the chronic phase is a critical problem for the country. Using a severe TBI model in young adult mice, we have demonstrated significant improvements in functional recovery by a combination treatment of stem cell factor (SCF) and granulocyte colony-stimulating factor (G- CSF) (SCF+G-CSF) in the chronic phase. However, it remains unclear how SCF+G-CSF treatment in the chronic phase of severe TBI improves functional recovery. The objective of this application is to determine the underlying mechanisms of the SCF+G-CSF-enhanced recovery in chronic TBI. Based on preliminary studies, we hypothesize that SCF+G-CSF-improved severe TBI recovery in the chronic phase is mediated by the enhancement of cerebral remyelination and neurostructural regeneration. Using the approaches of molecular and cellular biology, pharmacology, Cre-LoxP technology, 2-photon live brain imaging and neurobehavioral evaluation, this hypothesis will be tested through two Specific Aims. Aim 1 will determine how SCF+G-CSF treatment in the chronic phase of severe TBI enhances remyelination in cerebral white matter, and Aim 2 will define how SCF+G-CSF treatment in the chronic phase of severe TBI enhances neural structure regeneration. Searching for treatments to improve severe TBI recovery in the chronic phase is a highly-important-but-not- investigated field and an urgent national demand to improve the health of young adults living with severe TBI. It is expected that the accomplishment of the proposed mechanistic studies will significantly move the TBI research field forward by identifying a unique pharmacological approach to repair a severe TBI-damaged brain in the chronic phase.

NIH Research Projects · FY 2025 · 2021-03

Human cytomegalovirus (HCMV) is a common pathogen that has infected a majority of the human population. While symptoms of HCMV are generally resolved in immunocompetent individuals, the virus remains for the lifetime within the host as a latent infection is established in the hematopoietic stem cells. HCMV has co- evolved with humans and as such, the virus as adapted to undermine host cell antiviral responses. How HCMV undermines these responses is not fully known. Our research has found that an understudied protein modification, S-nitrosylation, plays a role in how HCMV interacts with the antiviral response. We found that viral proteins critical for efficient lytic replication are post translationally modified with nitric oxide groups and that the resulting modifications alter the viral proteins ability to limit antiviral responses. We propose to mechanistically investigate how these modifications impact HCMV protein functions and to identify the impact of S-nitrosylation on HCMV replication. Completion of the aims above will contribute significantly to our knowledge the regulation of biological functions of critical viral factors as well as identify the mechanism by which viruses weaken host defenses against infection. This work will lay the foundation in identifying novel therapeutic targets for a pathogen that lacks a vaccine or a cure.

NIH Research Projects · FY 2025 · 2021-03

Project Summary Eukaryotic RNA polymerase I (Pol I) transcribes ribosomal RNA, a key component of ribosomes. Pol I transcription accounts for the majority of the total RNA in cells, and its upregulation in human cells is a hallmark of cancer while its downregulation is a hallmark of several developmental disorders. Pol I transcription is understudied compared to transcription by Pol II and even Pol III. Our preliminary work suggests fundamental differences between Pol I and Pols II and III that are the basis for this proposal. Our broad long-term objectives are to determine the molecular mechanism of Pol I transcription and how its dysregulation leads to cancer and developmental disorders. There are major gaps in our understanding of (1) the structural organization and architecture of Pol I transcription complexes; (2) the mechanism for how Pol I initiation factors interact with rDNA, which encodes ribosomal RNA; and (3) the molecular function of several key Pol I transcription factors in the activation process. The first rationale for this work is that determining the mechanism and regulation of Pol I transcription will form the molecular basis for understanding how Pol I defects lead to human disease. Our central hypothesis is that Pol I factors use a unique mechanism to carry out transcription and their structure and function is different from the mechanisms governing Pol II and III transcription. The second rationale is that understanding the Pol I transcription mechanism at the most basic and fundamental levels will translate to a better understanding of the connection between Pol I and cancer, leading to new cancer therapeutic strategies. Our proposed research will use a conceptually and technically innovative cross-organismal and interdisciplinary approach that employs a combination of bioinformatic, computational, molecular, biochemical, genetic, genomic, proteomic, and structural methods in the yeast and human cells. Guided by strong preliminary studies, we will test two specific aims: (1) Determine the unique “coactivator” role of TATA-binding protein (TBP) in Pol I transcription, and (2) Determine the mechanism of Pol I transcription activation. To accomplish these aims, we will use well-established and complementary approaches to identify and map novel Pol I interactions in their native context. We will complement these studies with structural modeling in combination with molecular, genetic, and biochemical functional assays to identify Pol I factor functions conserved from yeast to humans. The proposed research is significant because it will lead to a detailed description of the Pol I transcription mechanism and will provide a conceptual framework for understanding the link between Pol I and human disease. Ultimately, this work will illuminate new avenues for diagnosis, potential interventions, and the development of therapies targeting these novel protein-protein and protein-DNA interactions.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY The molecular chaperone Heat Shock Protein-90 (Hsp90) is essential for the folding and activity of an array of `client' proteins involved in signal transduction pathways. They are also responsible for many maladies including cancer, neurodegenerative, autoimmune and inflammatory diseases. Hsp90 inhibition strategies are currently being explored in these diseases in pre-clinical studies and clinical trials, however the optimal use of Hsp90-targeted therapeutics remains unknown. This is partly due to our limited knowledge of Hsp90 regulation in cells. Unraveling the detailed regulatory mechanisms of Hsp90 function in cells can provide new strategies to increase the cellular potency of Hsp90 inhibitors. Hsp90 chaperone function is coupled to its ATPase activity, which is regulated by co-chaperones and posttranslational modifications (PTMs). However, it is unclear how these regulatory components work together to fine tune Hsp90 function and also contribute towards drug sensitivity. During the past five years we have made major contributions towards the understanding of Hsp90 regulation by co-chaperones and PTMs. i) New co-chaperones: We have identified three new co-chaperones, FNIP1, 2 (collectively FNIPs) and Tsc1, that decelerate the chaperone cycle and facilitate chaper- oning of both kinase and non-kinase clients. They are regulated by PTMs (phosphoryla- tion, O-GlcNAcylation, SUMOlyation and ubiquitination). Their expression also enhances Hsp90 binding to drugs and consequently sensitizes cells to Hsp90 inhibitors. ii) Post- translational modification of Hsp90: Our work during the past decade on Hsp90 PTMs has redefined the regulation of its chaperone activity and revealed the reciprocal regula- tory mechanisms between client proteins, co-chaperones, and Hsp90. We have recently shown that loss of TSC1 co-chaperone leads to hypoacetylation of Hsp90 and elevated its ATPase activity. It also subsequently decreased Hsp90 binding to its inhibitors. Our long-term goal is to unravel the molecular mechanism of Hsp90 chaperone regulation in cells and regulatory factors enhancing cellular potency of Hsp90 inhibitors. Our strategy is to use biochemical, biophysical and cell-based assays to decipher the interconnectivi- ty and compensatory mechanisms between the co-chaperones and PTMs. Our vision is to utilize this information to dissect the intricate network of regulatory signals involved in fine tuning Hsp90 function and their impact towards cellular sensitivity to Hsp90 inhibi- tors.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract Our overall goal in this project is to develop an in-depth understanding of the complex interplay between non- obstructive sleep apnea (non OSA) sleep disorders and recovery after stroke. Sleep is vital to overall health and quality of life. Abnormal or insufficient sleep is both a risk factor and consequence of stroke. Sleep also plays a critical role in motor learning, which is the foundation of rehabilitation strategies after stroke. Although there is a growing understanding of the interplay between sleep, stroke, and recovery in people with OSA these complex relationships in individuals post stroke with non OSA sleep disorders are not well understood. In order to develop targeted sleep interventions to support rehabilitation after stroke and promote optimal recovery, it is critical to gain a fuller understanding of the prevalence and impact of non OSA sleep disorders in people with stroke across the continuum of recovery. The specific objectives of this proposal will lay the necessary groundwork for this as we will characterize the proportion of people with stroke that have insomnia disorders, restless legs syndrome, and insufficient sleep; and evaluate the impact of these non OSA sleep disorders on recovery of activities of daily living, mobility/activity, and participation across the continuum of recovery after stroke. We will take an innovative approach to measuring sleep, mobility/activity, and participation using a combination of techniques across the measurement spectrum that will include self-report questionnaires, clinic-based measures of capacity, and body worn sensors. The body worn sensors will include actigraphy to measure sleep parameters, activity monitors to measure mobility/activity levels, and Global Positioning System (GPS) units to measure participation. Additionally, we will apply innovative, big data tools from topological data analysis for a data driven approach to discover complex, structural, non-linear interdependent relationships among stroke, sleep, and recovery of mobility/activity, and participation. Upon completion of this study we will understand the prevalence and impact of non-OSA sleep disorders on recovery of function, mobility/activity, and participation across the continuum of recovery post stroke. This is an important, necessary step to develop appropriate sleep-based interventions to complement targeted rehabilitation strategies to enhance the health and quality of life in people with stroke.

NIH Research Projects · FY 2025 · 2020-09

Project Summary: Darwin Babino, PhD, a trained pharmacologist/electrophysiologist, has spent the last ten years working on several disciplines in the vision sciences. His proposal entitled “Assessment of murine retinal acuity ex vivo by machine learning of multielectrode array recordings” presents his overarching goal to improve vision restoration approaches by developing methods to test the potential of these techniques thereby accelerating the development of effective interventions. Dr. Babino and his primary mentor, Dr. Russell Van Gelder, have assembled a strong team of co-mentors at the University of Washington SOM and collaborators to guide him through the proposed training and research. His previous training will be supplemented with goals to help his development as an independent investigator: 1) Study design and practical learning in performing panretinal (MEA) biological experiments; 2) Fundamental and advanced techniques of the proposed optogenetic and stem-cell restoration techniques; 3) Application of advanced machine learning techniques; 4) Develop leadership and professional skills to establish an independent group. The ability to assess the function of panretinal circuitry will foster our understanding of the advantages and weaknesses of different restoration techniques (Aim 1). The work proposed here will improve an existing retinal acuity assessment tool which combines machine learning techniques on novel, high-density multielectrode array recordings of ganglion cell responses in several mouse models. The utility of this system will be demonstrated in assessing visual potential of the mouse retina in three different approaches to vision restoration that are challenging for in vivo assessment (Aim 2). In collaboration with Dr. Deepak A. Lamba at UCSF, we will apply our system to animals which have undergone stem-cell replacement of retinal cells including photoreceptor cells. An optogenetics approach will also be evaluated in collaboration with Dr. John Flannery at UC Berkeley whose group has developed vectors for expressing rhodopsin and cone opsins in ganglion and bipolar cells. Finally, differences between native and restored vison with small molecule photoswitches, light-activated inhibitors of voltage-gated potassium channels, which confer light-dependent firing on treated cells, will be assessed. The resulting advanced electrophysiology application will help elucidate fundamental questions about the functional retina, mechanisms that lead to retinal degeneration and the potential of several therapeutics for the treatment of retinal diseases. Furthermore, this career development award will facilitate Dr. Babino’s development into an independent investigator by priming an R01 grant application.

NIH Research Projects · FY 2025 · 2020-07

Project Summary Fetal Alcohol Spectrum Disorders (FASD) affects up to 5% of live births in the US each year and results in life- long physical, cognitive, and behavioral impairments. Alcohol exposure during neurulation, the formation and closure of the neural tube (~ 4th week of pregnancy in humans, gestational days 8-10 in mice), is associated with abnormal growth of midline structures, such as the cortex, septum, pituitary, and ventricles, and neurofunctional changes later in life. My preliminary work suggested that neurulation-stage alcohol causes cell cycle arrest or delayed cell cycle progression, resulting in disrupted proliferation and, ultimately, anomalous tissue and organ development. Specifically, we performed whole transcriptome profiling of the rostroventral neural tube 6 hr after alcohol exposure and found that many genes and gene networks related to cell cycle regulation and cell proliferation were altered by alcohol. In addition, neurulation-stage alcohol caused significant dysregulation of the sonic hedgehog (Shh) pathway and cell cycle genes. These changes in morphogenic signaling were concomitant with smaller rostral neural tube volumes and fewer actively dividing cells in alcohol-exposed embryos. In this proposal, we use a well-characterized mouse model of FASD to test the hypothesis that neurulation-stage alcohol exposure alters cell cycle regulation in the rostral neural tube through disruption of processes that regulate cell cycle progression. Aim 1 analyzes cell cycle arrest and G1- specific processes in the neural tube following prenatal alcohol. Preliminary data suggest dysregulation of molecular mechanisms that control the successful transition between cell cycle stages and the DNA damage response, possibly leading to impaired DNA integrity and replication errors. Aim 2 investigates pathways that control protein degradation and trafficking during the cell cycle, following up on previous work showing downregulation of genes encoding ubiquitylation enzymes by prenatal alcohol. Finally, Aim 3 examines epigenetic marks associated with chromatin that regulate cell cycle progression, as pathways related to chromatin modifications were found to altered by neurulation-stage alcohol in our preliminary studies. These experiments will provide evidence that mechanisms of cell cycle progression represent an under-studied pathway through which prenatal alcohol causes symptoms of FASD.

NIH Research Projects · FY 2024 · 2020-07

Project Summary/Abstract The structure and dynamics of chromatin control the accessibility of DNA to regulatory factors during transcription, replication, recombination and DNA damage repair. PTMs of chromatin‐associated proteins and DNA methylation are complex epigenetic mechanisms that regulate gene expression and chromatin organization. The interplay between these mechanisms generate synergistic or antagonistic interactions that partition chromatin into (i) euchromatin: lightly packed chromatin, enriched in genes often under active transcription or (ii) heterochromatin: tightly packed and condensed chromatin, containing mostly silenced genes. The molecular mechanisms underlying this partition is not fully understood. Recent studies demonstrate that heterochromatin can assemble through liquid‐liquid phase separation (LLPS) driven by multivalent interactions between modified histone tails and the proteins that bind these epigenetic modifications. However, heterochromatin is also highly enriched in methylated DNA, which is reciprocally regulating, and regulated by the surrounding histone modification binding proteins and enzymes. How methylated DNA binding proteins and the patterns of DNA methylation co‐ordinate with the histone modification machinery in the LLPS‐mediated assembly of heterochromatin is not fully understood. This proposal will address these gaps in knowledge.

NIH Research Projects · FY 2025 · 2020-06

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology with mortality still approaching 10% in 5 years. The leading cause of death in SLE are infections due to the toxicity of immunosuppressant medications. Therefore, a significant unmet need exists for effective and non-toxic medications to treat SLE. Our central hypothesis has been formulated on the basis that effective treatment should target key checkpoints of pathogenesis, such as the depletion of reduced glutathione (GSH), which underlies the activation of the mechanistic target of rapamycin (mTOR) and inflammatory lineage specification of T cells, B-cell activation and antinuclear autoantibody production in SLE. The rationale for this study is supported by evidence that 1) GSH is depleted in peripheral blood lymphocytes (PBL) of SLE patients; 2) GSH depletion contributes to mTOR activation that drives T-cell dysfunction in SLE; 3) administration of N- acetylcysteine (NAC), which serves as a cell-permeable amino acid precursor of GSH, blocks the development of murine lupus; and 4) the preliminary studies suggest that NAC is safe, reverses GSH depletion and mTOR activation and improves disease activity in SLE patients. In our completed double-blind placebo-controlled pilot study, NAC was tolerated by 100% of patients on 1.2 g/day and 2.4 g/day dosages, while 33% of those receiving 4.8 g/day had reversible nausea. Placebo and 1.2 g/day NAC did not influence disease activity. Although both 2.4 g/day and 4.8 g/day NAC dosages showed preliminary evidence for clinical efficacy, 4.8 g/day NAC achieved greater drops in SLEDAI and BILAG scores. NAC raised GSH, blocked mTOR, and expanded T regs. The proposed phase II trial will employ the SLE Responder Index (SRI), as a clinically meaningful primary outcome measure that provides easily interpretable results and yields a feasible sample size that is associated with adequate power to detect therapeutic benefit over 1 year. To minimize potential intolerance of NAC and subject withdrawal, the study design includes an open-label dosage titration period. Patients who tolerate 2.4-4.8 g daily NAC for 3 months will be randomly assigned 1:1 to continue treatment on their tolerated dosage of NAC or matching placebo for 9 additional months. Beyond the premise of validating clinical efficacy and durability of this therapy in SLE, the proposed studies will test the hypothesis that depletion of GSH and cysteine and activation of mTOR predict immunobiological and clinical responsiveness to NAC. The proposed studies will significantly advance our understanding of immune-metabolic pathways that control T-cell lineage specification with translational relevance for the pathogenesis and treatment of lupus. The approach is innovative as it will employ a safe therapeutic intervention to define the role of cysteine depletion in redox-dependent mTOR activation and pro-inflammatory T-cell development in lupus patients in vivo. The results will bring new perspectives to our understanding of disease pathogenesis with broad translational relevance for clinical management of patients with SLE.

NIH Research Projects · FY 2024 · 2019-09

PROJECT SUMMARY Throughout recorded history, humans have used natural products for medicinal purposes. With the onset of modern analytical and pharmaceutical methods, the active ingredients in some of these “natural remedies” were identified and became foundational chemicals in drug development efforts. Among the numerous examples, aspirin (bark of the willow tree), capsaicin (chili peppers), and opioids (opium poppy plant) are notable for their analgesic properties. Similar to these other natural products, Cannabis sativa has long been used for medicinal purposes, including pain relief. The cannabis plant (Cannabis sativa/indica) contains over 100 phytocannabinoids as well as over 500 other identified chemicals, including the terpenoids that give cannabis its distinctive flavoring and aroma. Beyond 9-THC and CBD, however, difficulties in identifying, isolating and purifying minor phytocannabinoids in quantities sufficient for in vivo evaluation have hampered adequate investigation of potential therapeutic uses of individual phytocannabinoids. Recently, we have secured access to purified minor cannabinoids synthesized via a patented biosynthetic process in quantities that allow for preclinical in vivo testing. The goal of the proposed project is to provide a comprehensive evaluation of these compounds alone, and in planned combinations, to determine their potential efficacy as analgesics. In addition, we will conduct a parallel investigation of selected terpenes that have been identified in the cannabis plant. Novel analgesic strategies are needed to combat over-reliance on opioids and the resulting secondary consequences of dependence and morbidity/mortality.

NIH Research Projects · FY 2025 · 2019-08

PROJECT SUMMARY/ABSTRACT The actin and microtubule cytoskeletons are filamentous polymers that regulate vital cell processes, including but not limited to division, morphogenesis, DNA synthesis and repair, phagocytosis, and motility. Exactly how the dynamics of individual actin filaments or microtubules are regulated and coordinated in these cell processes is not fully understood. The goal of this research is to understand actin and microtubule assembly and their coordination by 1) developing new tools to visualize these proteins and their regulators, and 2) examining the emerging convergence between these proteins and membrane-less biomolecular condensates. This project utilizes an advanced in vitro biomimetic microscopy system capable of simultaneously monitoring fluorescent actin filaments, microtubules, and other regulatory proteins to elucidate detailed molecular mechanisms at single- molecule resolution. To address these mechanisms in a more physiological setting, we plan to combine these probes with an opto-genetic system that mimics several neurodegenerative disorders by repositioning condensates from the nucleus to the cytoplasm. This innovative approach imitates the onset of most neurodegenerative disorders and allows us to quantify pre- and post-disease state changes at high-resolution in the same cell. Identifying the molecular mechanisms that underlie these intricate biological systems (e.g., the dynamics of the cytoskeleton and biomolecular condensates) will significantly contribute to our understanding of fundamental biological processes and the onset of most neurodegenerative conditions.

NIH Research Projects · FY 2025 · 2016-01

Project Summary Rod-mediated vision transitions seamlessly to cone-mediated vision as light levels rise through the mesopic visual range. Despite the fact that mesopic vision is the major mode of vision for people who spend most of their time indoors under artificial lighting, there is a glaring lack of knowledge of how rod and cone photoresponses shape the temporal, spatial, and spectral sensitivities of mesopic vision. For decades it has been accepted that rods subserve vision in dim scenes, detecting only slow variations in light levels (contrast), and cones subserve vision in bright scenes when contrast changes rapidly. Our recently published results show that at indoor (mesopic) light levels, rods drive the visual responses to fast — not slow — temporal variations. Our findings reveal, for the first time, that adaptation of both rod and cone-driven visual responses underlie the seamless transition between scotopic and photopic vision over the mesopic range, but we do not understand how this happens. Therefore, there is a critical need to understand the physiology and functional significance of rod responses at mesopic, indoor light levels. In this application our objectives are: (i) to determine the adaptation mechanisms that control the responses of rods to slow and fast light variations in mesopic lights, and (ii) to determine the behavioral significance of rod-driven visual responses to fast changing scenes in mesopic lights. Our model is that a hierarchical system of adaptation mechanisms is in place to differentially regulate rod sensitivity as lights rise through the mesopic range. To test this model we propose two related but independent aims that investigate the underlying cellular and perceptual mechanisms. These aims are to determine 1) how rods respond to slow and fast variations in mesopic lights, and 2) the behavioral significance of inner segment conductances to the adaptation of rod visual responses in mesopic lights. For the first aim we will combine the power of mouse genetics, electrophysiological recordings, quantitative modeling, and operant behavioral assays to determine how different phototransduction adaptation mechanisms, including background and bleaching adaptation, work together to differentially regulate the responses to slow and fast light variations and to overcome the crippling effects of response compression in mesopic illumination. For the second aim, we will use mice with rod-specific knockout of voltage-gated conductances that are typically activated at mesopic lights to probe the functional significance of inner segment conductances to rod-driven responses in mesopic lights. Our expected outcome is to provide evidence for mechanisms that the visual system uses to encode signal variations in mesopic lights (Advancement of Basic Knowledge). Knowledge of this mechanisms will open new strategies for understanding and treating retinal pathological conditions, with implications beyond this particular application (Innovation for Emerging Therapy).

NIH Research Projects · FY 2026 · 2015-08

Project Summary: The ubiquitin pathway in corneal scarring Scarring in the cornea resulting from injury, trauma, or infection can lead to debilitating opacities and permanent vision loss. Acute scarring, similar to chronic fibrosis, is characterized by immune cell infiltration and the persistence of cells termed myofibroblasts. We have found that the deubiquitinase (DUB) USP10 is a key regulator of scarring pathways in the cornea. Knockdown of USP10 in vivo leads to a significant reduction in the development of myofibroblasts, cell apoptosis, the presence of CD45+ immune cells, and fibrotic extracellular matrix in a healing wound. We are proposing to test the central hypothesis that USP10 is a key regulator of myofibroblast persistence in a corneal scar via its multi- factorial role in wound healing. Specifically, since USP10 is a DUB for αv-integrins and p53, in Specific Aim 1 we will unravel the complex role of USP10 in integrin recycling and p53 dynamics in primary human corneal fibroblasts and in mice in vivo. The communication between fibroblasts and macrophages plays a critical role in scarring outcomes. In Specific Aim 2 we will develop novel 3D and 2.5D hydrogel systems with “tunable stiffness” that mimic a 3D environment close to the stiffness of the healing cornea. Using these hydrogels, mouse corneal fibroblasts will be cocultured with mouse bone marrow derived macrophages (M0/M1/M2) to elucidate the role of USP10 in macrophage-mediated myofibroblast development and contraction. In Specific Aim 3 using three separate mouse models we will assess the role of USP10 in immune cell infiltration into a corneal wound.

NIH Research Projects · FY 2025 · 2007-09

PROJECT SUMMARY/ABSTRACT The objectives of this proposal are to determine the mechanisms of photoreceptor protein compartmentalization. Retinal photoreceptors are polarized neurons whose major functions, include receiving and transmitting signals, are compartmentalized into discrete subcellular domains. Compartmentalization is critical for normal photoreceptor activity and reduced vision or blindness result from improper segregation of proteins. Despite their importance, the mechanisms underlying protein compartmentalization in photoreceptors, or any other neuron, remain poorly understood. Essential for understanding compartmentalization are the biophysical properties of the photoreceptor cytoplasm, the biophysical properties of the proteins that are destined to be compartmentalized and the forces that drive accumulation of proteins, against significant concentration gradients, into the specific compartments. We have uncovered a fundamental biophysical mechanism that may be central to protein transport and segregation in all electrically active cells: transport of charged proteins within the electrical field generated by the photoreceptor neuronal activity. We call this novel mechanism axial dynamic electrophoretic protein transport (ADEPT). We will use state of the art live cell fluorescence imaging tools developed in our lab, powerful transgenic and gene editing techniques in Xenopus, and sophisticated biochemical and cell biological approaches to address the following aims: Aim 1: Map the axial cytoplasmic electric field, Eax, in rod photoreceptors. Aim 2: Determine the impact of ADEPT on photoreceptor protein transport and compartmentalization in living photoreceptors. Aim 3: Determine the locations and influence of Arrestin interactions on their distributions and dynamics in living rods and cones.