Oakland University

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY OF FUNDED R15 GRANT (R15CA254006-01A1) A major component of many solid tumors, including lung cancers, are bone marrow (BM)-derived immune cells that migrate to tumors and aid in their continued growth. While the activity of these cells including tumor associated macrophages (TAMs) has been the subject of intense investigation, we recently identified that BM- derived hematopoietic stem and progenitor cells (HSPCs) are also present in growing tumors and can be functionally maintained intratumorally for long periods of time. Interestingly, the numbers of HSPCs present in tumors directly correlates to the eventual regrowth rates of tumors following radiation therapy (RT). The data suggests that HSPCs represent another important cell population involved in tumor biology, however; their mechanism of action is still unclear. Filling this gap in knowledge will add to the ever-changing understanding of tumor biology. The objective of this proposal is to determine how HSPCs are maintained in tumors and how HSPCs promote tumor regrowth post-RT. Our preliminary data support the idea that HSPCs are maintained through interactions of the integrin CD49f and laminins present within the tumor extracellular matrix. In Specific Aim 1, we will show that this interaction is indeed responsible for HSPC maintenance using in vitro and in vivo strategies that block or enhance this interaction followed by analysis of their effects on HSPC functionality. We will also define the intracellular signaling pathways involved in this process with initial studies focusing on focal adhesion kinase (FAK) signaling. These studies will characterize for the first time a tumor specific niche capable of maintaining HSPCs outside of the BM. In Specific Aim 2, we will demonstrate that tumor treatment with RT exacerbates HSPC migration to tumors and concomitantly disrupts the interaction between CD49f and laminin. We will also show that RT produces tumor microenvironments that favor the differentiation of these ‘released’ HSPCs into tumor supportive macrophages (specifically M2 polarized) to aid in tumor recovery. We will also test the effects of blocking the activity of HSPCs on tumor growth and regrowth post-RT. By completing the proposed studies, our long-term goal is to use the knowledge gained to make a significant contribution towards the development of more robust treatment strategies for patients suffering with solid tumor based cancers.

NIH Research Projects · FY 2025 · 2022-05

Project Summary/Abstract Multiple sclerosis (MS) is a degenerative disease, which affects the central nervous system (CNS). While most neurodegenerative diseases affect older populations, the onset of MS generally occurs early in life. There is no cure for MS. Currently used drugs have severe side effects. Past attempts to develop cell therapies to treat MS have met with limited success. The major challenges in developing cell therapies include invasive isolation techniques, limited growth and differentiation potential, as well as genetic instability of adult mesenchymal stem cells (MSCs). We have isolated and differentiated highly proliferative and primitive (p) MSCs into neural stem cells (NSCs). In our preliminary studies, transplantation of NSCs significantly reversed the clinical symptoms when transplanted at an early stage of experimental autoimmune encephalomyelitis (EAE) in a mouse model. These findings are very promising and provide a strong “proof of concept” for cell-based treatment of MS. Since we saw substantial improvement in EAE disease with a single cell dose, we hypothesize that multiple doses of NSCs will be more effective in ameliorating chronic EAE disease symptoms and promoting functional recovery. We envision that this innovative approach using NSCs will enhanced the potency of cell therapy as proposed in this study. The specific aims are: 1. To determine the therapeutic effects of repeated doses of NSCs on chronic EAE in mice. We hypothesize that similar to repeated use of drugs, repeated cell therapy treatments will be more efficacious. This hypothesis will be tested by injecting GFP-labeled NSCs in 3 doses to counter chronic EAE induced by MOG immunization in mice. Changes in the disease symptoms and progression will be monitored by performing neurobehavioral, neurological motor function, mechanical threshold response and cold response analyses to assess the effect of cell therapy on the disease progression and remission. 2. To investigate CNS pathology at cellular and molecular levels in NSC transplanted EAE mice. We hypothesized that NSC treatments will reduce inflammation and restore CNS function. Histopathological analysis of the CNS will be performed to access immune cell infiltrates. Composition of cell infiltrates will be assessed by immunohistochemical analysis of CNS sections. Levels of pro- and anti-inflammatory cytokines will be carried to assess the immunomodulatory properties of transplanted NSCs. NSCs are also likely to help in mitigating the imbalance of immune regulatory cells, reduce astrogliosis, and improve myelination. This will be investigated using appropriate cellular and molecular techniques. NSCs also express high level of neurotrophic factors, their role in neuroprotection will be explored. The effect of NSCs on the global gene expression in the CNS of EAE mice will be examined by RNA-seq and validated by qRT-PCR analysis. RNA-seq analysis should help in determining the signaling pathways involved in potential functional recovery of damaged CNS in EAE mice. The results of this research will provide fundamental insights into EAE and also help in developing cell therapies not only for MS but also for other neurodegenerative diseases.

NIH Research Projects · FY 2025 · 2020-03

β-III-spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje neurons, and is required for dendritic arborization and signaling. Dominant mutations in the SPTBN2 gene encoding β-III-spectrin cause the neurodegenerative disorder spinocerebellar ataxia type 5 (SCA5). SCA5 causes degeneration of Purkinje cells, and an associated progressive gait and limb ataxia. There is no cure or therapy for SCA5. Many SCA5 missense mutations (including L253P) cluster within the β-III-spectrin actin- binding domain (ABD). Previously, we showed that increased actin binding is a shared molecular consequence of the ABD-localized mutations. Using novel Drosophila models, we demonstrated that a common cellular consequence of the ABD-localized mutations is reduced dendritic arborization. We further determined that truncation of the unique β-III-spectrin N-terminal domain (NTD), preceding the conserved ABD, rescues L253P-induced high-affinity actin binding and arbor defects. We also discovered that multiple phosphosites modulate the binding of wild-type β-III-spectrin to actin in vitro. However, it remains unclear how increased actin binding impairs the function of β-spectrin to support dendritic arborization. Moreover, the shared impact of SCA5 mutations to alter actin binding demands a better understanding of the mechanisms controlling β-spectrin-actin interactions. The objective of this application is to determine how SCA5-induced high-affinity actin binding leads to decreased dendritic arborization, and to elucidate mechanisms regulating spectrin-actin binding. In addition to the progress described above, using immunoprecipitation coupled to mass spectrometry, we identified numerous proteins in Drosophila neurons that physically associate with β- spectrin, including tropomyosin, myosins, kinases, phosphatases, and additional proteins. Intriguingly, tropomyosin, was recently shown to simultaneously bind actin and the unique β-spectrin NTD, suggesting tropomyosin is a key regulator of spectrin-actin binding. Further, tropomyosin loss-of-function alleles are known to increase dendritic arbor outgrowth. This suggests that modulation of tropomyosin, or other components of the interactome, may alleviate reduced arborization caused by SCA5 mutations. In this project we will define the Drosophila neuronal interactomes of the wild-type and L253P β-spectrin. We will use these interactomes and the well-developed Drosophila toolbox to guide a targeted genetic modifier screen for modulators of SCA5 arbor defects. Further, we will define in greater detail how the NTD, and its phosphorylation and binding to tropomyosin, regulates actin binding and SCA5 arbor defects. Numerous undergraduate students will participate in the execution of this project, providing important bench training for the next generation of problem solvers.

NIH Research Projects · FY 2025 · 2018-09

SUMMARY Current stroke research focuses more on understanding the brain’s self-protective and repair mechanisms. Detailed elucidation of these mechanisms is crucial as such knowledge could lead to development of therapeutic interventions which mimic or engage the brain’s self-protective/repair mechanisms and can lead to successful stroke therapy. With the proposed research we seek to develop potent and selective ‘drug-like’ small molecule activators of peptidase neurolysin (Nln) which will be used as research tools and lead chemical entities to address the critical unmet need for stroke treatments. Our recently published and pilot studies have identified Nln as a leading brain self-protective mechanism, functioning towards cerbroprotection and recovery after stroke. Functional significance of Nln in the post-stroke brain is based on its ability to inactivate several neurotoxic peptides and generate three cerebro-protective/regenerative peptides, which are known from numerous experimental and clinical studies to critically contribute to the outcome of stroke. Based on this evidence we view Nln as a central peptidase involved in protection of the brain following stroke. In this collaborative renewal application, we will leverage our expertise in multiple aspects of the drug discovery process to further optimize lead molecules of two distinct chemotypes, developed during the first cycle of this R01 grant, to improve their potency and ‘drug-like’ properties for selective activation of Nln as experimental therapeutic agents for cerebroprotection after ischemic stroke. This proposal has been formulated based on our compelling experimental data revealing the discovery of two diverse Nln activator chemotypes that bind to Nln and enhance its catalytic activity; extensive structure-activity relationship, hit-to-lead optimization and pharmacokinetic studies; and initial in vivo proof-of-concept efficacy studies, in two mouse models of ischemic stroke, demonstrating the cerebroprotective effects of a lead Nln activator. The goals of this proposal will be accomplished in three well-integrated aims: (1) conduct lead optimization to further refine activity and ‘drug-like’ properties of Nln activators; (2) perform biochemical and structural studies to characterize the activation mechanism that the identified Nln activators exploit; (3) determine the therapeutic potential of Nln activators in post-stroke cerebroprotection using two mouse models of ischemic stroke. This work is highly innovative because our multi-mechanism molecules are the first and only Nln activators described in the scientific and patent literature, and the therapeutic potential of such compounds has not been recognized before. The collaborative investigative team, comprising experts in medicinal chemistry and drug discovery, cryo-EM and structural biology, enzyme biochemistry and pharmacology, blood-brain barrier physiology and stroke pharmacology, is highly qualified to conduct the proposed studies. Our long-term goal is to translate the lead Nln activators from bench to bedside and develop an effective stroke therapy, which will transform the current treatment modalities for a vast number of stroke patients.

NIH Research Projects · FY 2025 · 2016-05

Project Summary / Abstract This project investigates the molecular and cellular mechanisms that govern vertebrate photoreceptor outer segment (OS) structure, a fundamental unsolved problem in photoreceptor cell biology. This basic science knowledge gap severely limits clinical understanding of (and treatments for) blinding diseases in humans and animals caused by mutations that disrupt OS structure. A broad variety of inherited retinal degenerations (IRDs) are caused by mutations that disrupt OS organelle structure. Because normal OS structure is required for healthy vision but is not yet understood, it is essential to advance knowledge of how the many hundreds of membranous disks required by each rod and cone are properly shaped and stabilized. The most longstanding and well-known examples of IRDs triggered by abnormal OS structure are caused by mutations in peripherin-2/rds (P/rds). This proposal builds upon our demonstration in the previous project period that P/rds directly generates membrane curvature, to precisely sculpt the hundreds of membranous disk rims present in rod and cone OSs. This advance provides a clear mechanistic explanation for the general effects of P/rds mutations on OS structure, but leaves open the critical and fundamental questions of how P/rds activity is regulated, and how inherited defects in P/rds generate a diversity of disease phenotypes. Our central hypothesis is that regulation of P/rds function by heterotypic interactions with rom1 and glutamic acid-rich proteins (GARPs) contributes to differences between rod and cone OS disk structures. The research strategy will therefore identify how P/rds works together with rom1 and GARPs to build and maintain normal OS structure, and will investigate the significance of these proteins for rod vs. cone disk architectures and how mutations can disrupt organelle structure to trigger particular retinal diseases. An integrated approach using contributions from human molecular genetics, in vitro biochemistry, in cellulo functional assays, and in situ analyses of engineered vertebrate animal models will be utilized to carry out the research. Specific Aim 1 will elucidate the significance of heterotypic (rom1 and GARP) interactions for P/rds structure, molecular function, and support of OS disk architecture. Specific Aim 2 will identify differential contributions of P/rds to rod and cone structures and viability. Overall, these studies are expected to clarify how normal rod and cone OS disk structures are generated and stabilized by photoreceptor-specific proteins that are essential for human retinal health and vision. This work will also provide an improved mechanistic basis for understanding how pathogenic mutations in P/rds can differentially impact rods and cones to produce a broad heterogeneity of IRDs.