Author: orandall

Neuroinflammation is a key aspect of Parkinson’s Disease (PD) pathology. Extracellular alpha-synuclein (aSyn) aggregates influence immune responses in both the central nervous system (CNS) and periphery. The preformed fibril (PFF) aSyn rodent model of PD effectively mimics many PD features, including dopaminergic cell loss, behavioral deficits, and widespread α-synuclein inclusions. In the PFF α-syn model, PFF α-syn seeds promote endogenous aSyn recruitment, leading to synuclein propagation and neurodegeneration. Neuroinflammation has been confirmed in rats and mice post-PFF α-syn injection, though conflicting reports raise questions about its presence in mice. The study aimed to identify variables affecting inflammatory phenotypes in PFF aSyn mice, including endotoxin (LPS) in aSyn preparation, species-specific aSyn characteristics, and animal facility conditions. In the original study, we found that inflammatory phenotypes were more pronounced in mice with endotoxin-removed PFF aSyn. Species-matched mouse PFF aSyn induced inflammatory phenotypes in non-transgenic wild-type mice. Consistency in inflammatory phenotypes was observed across different facilities, depending on previous results. This supplementary study aimed to further explore inflammatory responses and validate findings, including the role of peripheral immune cells in brain regions with p-aSyn pathology and gliosis. The study’s completion will establish quality control guidelines and profiles of inflammatory factors in PFF aSyn mice.

Funder: Michael J. Fox Foundation

Amount: $259,169

PI: Jae Kyung Lee, College of Veterinary Medicine

Brain tumors are the leading cause of cancer-related deaths among children. Recent studies have identified new subtypes of brain tumors, including high-grade glioma with G34R/V mutations in the histone variant H3.3 (HGG-G34). This subtype primarily affects the cerebral hemispheres of adolescents and young adults. In addition to the histone mutation, HGG-G34 often exhibits mutations in both ATRX and TP53. However, the precise pathogenic mechanism of HGG-G34 remains poorly understood. Currently, there are no established therapeutic approaches tailored to this subtype, and patients continue to face a dismal prognosis. Therefore, there is an urgent clinical need to elucidate the cellular and molecular mechanisms underlying the development of HGG-G34 and to identify new therapeutic targets. To address these knowledge gaps, we have developed a new human embryonic stem cell-based model that allows us to introduce various combinations of mutations into a defined cell population. Using this mode, we have demonstrated that the three core mutations (H3.3G34R, ATRX, and TP53) specifically transform interneuronal progenitors of the ventral forebrain, shedding light on the cellular origin of HGG-G34. We also discovered that H3.3G34R and ATRX mutations cooperatively enhance the expression of DMRTA2, a forebrain-specific transcription factor, which is crucial for the high proliferation of HGG-G34 cells. Additionally, our data indicate that the majority of HGG-G34 cells exhibits characteristics of radial glial (RG) cell, a type of neural/glial progenitor cell that only exists in the developing brain. These findings collectively indicate a dysregulation in developmental programs in HGG-G34. However, the precise molecular mechanisms underlying tumorigenesis, including the transcriptional targets of DMRTA2, the exact role of ATRX mutation, and the maintenance of RG-like state, are still unclear. Additionally, the clinical relevance of RG-like cells in malignant brain tumors has not been fully studied. In this proposal, we aim to unravel the transcriptional targets of DMRTA2 by employing ChIP-seq and assess their involvement in tumorigenesis by loss-of-function experiments (Aim 1). Additionally, we will investigate the interplay between H3.3G34R and ATRX mutations by examining the impact of ATRX mutation on the expression, function, and distribution of DMRTA2 (Aim 2). Furthermore, we will examine the maintenance mechanisms and clinical relevance of RG-like cells through mouse xenograft models and analysis of clinical samples obtained from human patients (Aim 3). Our innovative HGG-G34 model, scientific expertise, and strong local collaborations uniquely position us to achieve these aims. The outcomes of this project are expected to provide novel insights into the tumor biology of HGG-G34 and serve as a foundation for our long-term goal of developing personalized treatment and diagnostic approaches for patients suffering from this devastating disease.

Funder: National Institutes of Health

Amount: $1,676,104

PI: Kosuke Funato, Franklin College of Arts and Sciences, Department of Biochemistry and Molecular Biology

The increasing rise in antibiotic resistance and the diminished discovery of new antimicrobials threatens global healthcare. Of particular concern are Gram-negative pathogens, as these organisms are intrinsically resistant to multiple classes of antibiotics and the discovery of novel drugs targeting these bacteria has remained challenging. The innate resistance of these organisms is provided primarily by their outer membrane (OM), a defining feature of Gram negatives that encapsulates their peptidoglycan layer. Unlike the inner membrane (IM) that is composed solely of glycerophospholipids (GPLs), the OM is asymmetrical with GPLs found in the inner leaflet and lipopolysaccharide (LPS) localized to the outer leaflet. This unique membrane organization affords protection from large polar molecules, as well as lipophilic compounds, creating an impervious barrier. Remarkably, the high-priority Gram-negative pathogen Acinetobacter baumannii can completely inactivate LPS biosynthesis as an alternative mechanism of resistance to the “last-resort” polymyxin antibiotics. The primary objective of this application is to investigate the mechanisms required for maintenance of the cell envelope of A. baumannii, regardless of LPS status. While the benefit of an asymmetric OM relative to a GPL bilayer is apparent due to the impermeable barrier it provides, the lack of LPS essentiality in A. baumannii can be used as a tool to explore novel mechanisms of OM stability in both the presence and absence of LPS. For Aim 1, we will investigate the role of surface lipoproteins that are induced during envelope stress and that are prominent during LPS-deficiency. We will also investigate how these proteins are transported across the OM. Aim 2 focuses on the characterization of two glycosyltransferases required for the tandem transfer of sugars during LPS synthesis, a unique mechanism in the assembly of a bacterial glycoconjugate. Finally, in Aim 3, we will characterize a novel OM cardiolipin synthase and how it impacts OM integrity. An OM cardiolipin synthase challenges current dogma that dictates all major GPLs are synthesized at the cytoplasmic face of the IM. Completion of the Aims will provide novel insights into cell envelope biogenesis and promote the development of novel therapeutics targeting Gram-negative pathogens.

Funder: National Institutes of Health

Amount: $2,696,610

PI: Michael Trent, College of Veterinary Medicine

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This project seeks to answer the question how much fresh (low salinity) water is carried from the Arctic Ocean along the East Greenland Coast into the North Atlantic Ocean and how much this transport may vary as more of the Greenland and Arctic ice sheets melt. For this purpose, an array of six moorings is to be deployed on the Northeast Greenland Shelf to make continuous measurements of temperature, salinity, and current velocities. An exciting new feature of this array includes a variable ballast buoy at the top of one of the moorings, the one closest to the coast, that allows measurements to be made all the way to the ocean surface when the region is ice free, but that prohibits collision of the instruments with sea-ice or icebergs in winter by keeping the mooring line below the ice then. The mooring observations are to be complemented by a modeling study that estimates how the East Greenland Coastal Current evolves over longer time scales. A collaboration with European partners who have a similar mooring array in deeper waters further offshore allows to examine the spatial extent of the current system. Together these efforts will fill a critical gap in our understanding of Arctic-Subarctic exchange, and results will be applicable to a range of scientific fields beyond physical oceanography including climate science, marine biogeochemistry, and fisheries management, among others. The oceanic circulation of the high-latitude North Atlantic is a critical component of our climate system and is potentially sensitive to the release of fresh, surface waters from the Greenland Ice Sheet and the Arctic Ocean. A large gap exists in our monitoring of this freshwater input on the Northeast Greenland Shelf (NEGS). This gap will be filled by measuring the southward-flowing East Greenland Coastal Current (EGCC) on the NEGS for the first time with continuous, direct measurements over an entire year. Based on existing data from summer shipboard sections and satellites, it is hypothesized that the freshwater transport in the EGCC is as strong as the freshwater transport of the better known East Greenland Current (EGC) further offshore at the shelf break. If true, the EGCC would be a major contributor to the total freshwater budget of the Arctic and a key player in Arctic-Subarctic exchange. In addition to the mooring array, it will be analyzed how these data fit into the larger scale NEGS circulation using model simulations, reanalysis products, and satellite data. The new ice-avoiding buoy technology that is to be developed as part of this project has the potential to be widely applicable to a range of environments and is significantly more cost-effective than other similar products. Results from this project will: (1) quantify the volume, heat, and freshwater transports of the EGCC on the NEGS, (2) compare these transports to those of the EGC measured by European partners, (3) identify the physical drivers of transport variability in the EGCC, and (4) assess the long-term variability of the EGCC and its role in the Arctic freshwater budget.

Funder: National Science Foundation

Amount: $3,376,389

PI: Nicholas Foukal, Franklin College of Arts and Sciences, Department of Marine Sciences

Bordetella pertussis (Bp), the causative agent of “whooping cough,” is the most important vaccine-preventable disease and an NIH and CDC high priority. The incidence rates of pertussis have increased over recent years, corresponding to the switch from the whole cell pertussis vaccine (wP) priming and acellular pertussis vaccine (aP) boosting vaccination in the early 2000s, to the current aP-only vaccination. Women given the safer but less effective aP vaccines are a rapidly increasing proportion of birthing mothers, creating a complex transition that we poorly understand and that may have particular risks to the most highly sensitive population, newborns. To protect neonates during the months before they can be fully vaccinated the CDC recommends a cocooning strategy, vaccinating all their potential contacts, creating local “herd immunity” to prevent exposure. This strategy is based on the dated and inaccurate belief that aP vaccines that protect against disease also prevent infection/transmission. In fact, there is growing evidence that aP vaccinated individuals, despite significant antiBp antibody titers, can still be colonized and can transmit Bp to close contacts, including their babies. Another approach, maternal vaccination where antibodies to Bp are transferred via placenta and colostrum/milk to the newborn, although the actual protective effect of antibody transfer is unknown. In fact, the efficacy of maternally transferred antibodies in protecting against colonization or disease has not been determined clinically. Such a clinical study would be challenging and costly and has not been attempted. The experimental evidence indicates that antibodies alone do little to prevent disease. Therefore, we are effectively in the middle of a nationwide test of the differential effects of wP-primed and aP-primed vaccination of mothers on the protection conferred to their offspring without a complete understanding of the likely impact. To address this urgent knowledge gap, we recently developed a novel mouse neonatal experimental infection system that more accurately models the unique features of the human neonatal immune system, enabling us to probe vaccine-induced protection against Bp transferred from mother to offspring. Our exciting preliminary data demonstrate that both the wP and aP maternal vaccinations confer substantial protection to the offspring’s lungs. These results demonstrate that we can measure the profound effects of maternal vaccination on the protection of pups. This approach allows us the specific means to examine the mechanisms of differentially conferred protection, as proposed below. Here we will utilize a combination of innovative immunological techniques to address the central hypothesis that transferred maternal antibodies contribute to protection in neonates via mechanisms we can distinguish using tools unique to the mouse model.

Funder: National Institutes of Health 

Amount: $393,345 

PI: Eric Harvill, College of Veterinary Medicine 

The objectives for this project will be: 1) measuring phenological shifts of these key pests and evaluating reliability of available monitoring tools and models; 2) refining IPM tools to ensure compatibility with evolving production practices, regulatory changes, and climate; 3) suppress pest populations across orchard agroecosystems with promising biocontrol agents and integrating enhanced IPM tactics; and 4) assessing the socioeconomic impact of enhanced IPM programs to promote grower adoption and market flexibility. Approach: The insect pests identified as of highest concern by Eastern growers include codling moth (Cydia pomonella), oriental fruit moth (Grapholita molesta), San Jose scale (Comstockaspis perniciosus), plum curculio (Conotrachelus nenuphar), invasive brown marmorated stink bug (Halyomorpha halys), and trunk-boring sesiid moth pests: peachtree borer (Synanthedon exitiosa), lesser peachtree borer (Synanthedon picitipes), and dogwood borer (Synanthedon scitula). For objective 1, we will use historical data sets and novel automated traps to improve decision support for codling moth and oriental fruit moth. Historical data on San Jose Scale activity and horticultural stress indicators will be integrated into novel prediction tools. Novel attractants for plum curculio and relationships between brown marorated stink bug and Samurai wasp captures will be evaluated in the field. Factors associated with grower perception of IPM tools that enable or create barriers to adoption will be quantified. For objective 2, we will conduct semi-field and field studies aimed at improving biocontrol agents for Lepidopteran pests, San Jose scale and brown marmorated stink bug. Novel management tools including scalable mating disruption for lepidopteran pests and San Jose scale will be developed in a series of field studies. Border-based management tools for plum curculio and brown marmorated stink bug including the use of deterrents and trap-based treatment thresholds will be developed using field studies. Grower perceptions of novel management tools will be captured. For objective 3, we will develop a risk-based decision matrix for pests of Eastern tree fruit based on risk levels quantified using field data. Integration of enhanced IPM tactics will be conducted in the field and compared with standard practices. In objective 4, our team will produce Extension-ready case studies highlighting the economics of enhanced IPM, hold workshops for growers and emerging farmer audiences, and mentor graduate students in Extension. Climate-smart recommendations will be delivered to Eastern tree fruit growers through field days, print, and digital media.

Funder: United States Department of Agriculture Agricultural Research Service 

Amount: $439,984 

PI: Brett Blaauw, College of Agricultural and Environmental Sciences 

Chronic environmental exposure to neurotoxic levels of the metal manganese (Mn) affects the basal ganglia system and has been linked to the etiology of Parkinsonism. Within the basal ganglia (BG), Mn primarily accumulates in the globus pallidus, resulting in dysregulation of the BG neural circuitry. As the most abundant glial cells in the CNS, astrocytes are critical for healthy brain function and are highly sensitive to Mn toxicity as the metal preferentially accumulates in astrocytes. For any neurotoxicological triggers, astrocytes are early responders and undergo a process known as “reactive astrogliosis”, characterized by morphological, molecular, and functional changes. In this context, increasing evidence suggests that Mn neurotoxicity elicits proinflammatory responses in astrocytes. However, the fundamental molecular neurotoxicological mechanisms regulating the transformation of an astrocyte’s homeostatic phenotype from quiescent to pro-inflammatory and the exact molecular regulators of astrocyte crosstalk with neurons and microglia in metal neurotoxicity remain enigmatic. In the emerging field of epitranscriptomics, the role of N6-methyladenosine (m6A) RNA modification, under the dynamic control of N6-methyltransferases, demethylases, and readers (m6A mRNA-binding proteins), in neurodegenerative diseases (NDs) is currently being explored. We recently obtained exciting new data showing that the m6A reader protein YTHDF2 is specifically downregulated by Mn in cultured human astrocyte cells and primary murine astrocytes as well as in animal models. Functional loss- and gain-of-function studies reveal an anti-inflammatory role for YTHDF2 in Mn-stimulated astrocytes, leading to a novel hypothesis that YTHDF2 may be a critical regulator impacting Mn neurotoxicity. Additional mechanistic studies identified SEK1 mRNA as a direct target of YTHDF2 and also discovered the ability of YTHDF2 to suppress the SEK1/JNK/cJun pro-inflammatory signaling axis and subsequent production and secretion of selected pro-inflammatory mediators in reactive astrocytic cultures. Very recently, we also observed a significant increase in the m6A readers YTHDF1 and YTHDF3, which can boost the translation of their respective targets. Thus, the following specific aims will be pursued to further expand our novel findings: (i) elucidate in animal models if chronic Mn exposure dysregulates YTHDF proteins in astrocytes, and whether this dysregulation contributes to Mn neurotoxicity by disrupting neuroimmune homeostasis; (ii) elucidate the pro-inflammatory signaling mechanisms underpinning YTHDF dysregulation in activated astrocytes during Mn neurotoxicity in animal models; and (iii) validate YTHDF proteins as key regulators of astrocyte-mediated immune and neuronal dysfunction in Mn neurotoxicity. Overall, we anticipate that our proposed studies will provide novel mechanistic insights into reactive astrocytic activation and neuroimmune interactions in metal neurotoxicology and will offer novel therapeutic targets to dampen the neuroinflammatory processes in environmental factor-related neurodegenerative diseases.

Funder: National Institutes of Health 

Amount: $441,529 

PI: Anumantha Kanthasamy, College of Veterinary Medicine 

Neuroinflammation is a driving force contributing to neurodegenerative diseases, including Parkinson’s disease (PD). Microglia are the primary immune cell of the brain and are among the first responders to infection, toxic insult and aggregated proteins and contribute significantly to neuroinflammation and neurodegeneration. The microglial inflammatory response, including inflammasome activation, has been demonstrated to be significantly associated with PD. Paraquat (PQ) is a commonly used herbicide that has been linked to increased risk for PD. PQ-induced neurodegeneration is tightly coupled to the activation of microglia and appears to require priming of the microglial response. Therefore, factors that modulate the microglial inflammatory response could lead to neuroprotection and slow the progression of neurodegenerative diseases. However, to date, no antiinflammatory drugs have proven successful in human clinical trials necessitating research on new targets. Hv1 (HVCN1) is a voltage-gated proton channel highly expressed on microglia in the brain and in other immune cells in the body. This proton channel regulates the activity of NADPH oxidase and production of reactive oxygen species in immune cells and especially microglia. Our preliminary data demonstrate that PQ directly increases Hv1 levels in microglia, possibly through an epigenetic mechanism involving histone acetylation. Further, our data demonstrate effects of PQ on the NLRP3 inflammasome that appear to be regulated by Hv1, providing a potential mechanism contributing to PQ-induced microglial priming. This proposal seeks to test the hypothesis that Hv1 regulates priming of microglia following PQ exposure through the NLRP3 inflammasome, leading to neuroinflammation and neurodegeneration. The Specific Aims of this project are to 1) Determine mechanisms of Hv1 regulation following paraquat exposure 2) Define the role of Hv1 in regulation of the NLRP3 inflammasome following paraquat exposure and 3) Determine the contribution of microglial Hv1 and the NLRP3 inflammasome in regulating neurodegeneration following paraquat exposure. Completion of these Aims will define regulatory mechanisms for Hv1 and determine the role of Hv1 in regulation of the NLRP3 inflammasome activation and their role in PQ-induced neuroinflammation and neurotoxicity. Together, these Aims will provide crucial information on the function of a novel regulator of neuroinflammation, Hv1, and determine whether targeting Hv1 may be a viable therapeutic strategy in toxicant-induced neurodegeneration. We will use the regional scale model as boundary conditions for two high resolution mine-specific models (Mission and Amelia mines). We will use these models to estimate impacts of mining on groundwater flows, water table elevations, and the dispersal of solutes under future climate scenarios.

If available, we will review the hydrologic model(s) prepared by Twin Pines’ consultants in order to understand key assumptions, model construction, and data sets and to possibly guide enhancements of our model.

Funder: National Institutes of Health 

Amount: $625,498 

PI: Jason Richardson, College of Veterinary Medicine 

Plasmodium vivax (Pv) is responsible for a significant portion of the malaria cases outside of Africa and causes significant socioeconomic burden worldwide. New therapies to treat Pv are needed, but a continuous, high yield culture system that enables the study of Pv’s biology has not been established. Without this tool, development of new therapies is stymied. The long-term goal of this research program is to study Pv biology to inform the development of new treatments for Pv. The objectives of this proposal are to (1) establish continuous, high yield culture systems for Pv’s asexual stages and (2) to use this culture system to test triggers of gametocytogenesis so gametocytes can be generated in vitro for study. This research is needed because it will yield a key tool to study Pv asexual and sexual stages, thereby, enabling mechanistic research to identify and develop new therapies. The major bottlenecks to establishing continuous cultures have been (1) Pv preferentially invades a specific subset of reticulocytes, or immature erythrocytes, in culture, and (2) low parasite numbers due to poor development even when reticulocytes are supplemented for invasion. In preliminary studies, our team has used P. cynomolgi (Pcy), a closely-related malaria parasite to Pv, to develop a strategy for addressing these obstacles. We have shown that Pcy strains vary in their ability to grow in culture with some strains growing in culture immediately whereas others adapt with time, suggesting that intrinsic variation within strains exists and can be selected for to establish continuous cultures. Using our Pcy strains that grow in culture, we have also shown that a bone marrow (BM) mimetic composed of secreted factors from BM mesenchymal stromal/stem cells (MSCs) significantly improves Pcy development in culture. Intriguingly, our data also indicate that BM reticulocytes may trigger the development of gametocytes. Based on these data and the prior literature, we propose to test the hypothesis that continuous cultures of Pv can be achieved by establishing Pv strains that invade multiple reticulocyte subsets in culture using conditions that mimic the BM where Pv thrives in vivo. This proposal will test our hypothesis by pursuing three specific aims. During the R61 phase, the experiments in Aim 1 will establish Pv strain(s) that robustly invade reticulocytes in culture while the experiments in Aim 2 will improve Pv development in vitro by mimicking the BM microenvironment. Together, these aims are expected to yield a continuous culture system for Pv asexual stages. After achieving this milestone, Aim 3 will use this culture system to test triggers of gametocytogenesis during the R33 phase. The expected outcomes of this research are a continuous culture system for Pv and new insight into Pv invasion, development and gametocytogenesis. This research will have an immediate impact on Pv research because it will provide essential tools that will enable mechanistic insight into Pv biology and provide new knowledge that may lead to new therapies.

Funder: National Institutes of Health 

Amount: $1,152,630 

PI: Chester Joyner, College of Veterinary Medicine 

Nematodes are among the top yield-robbers encountered by soybean farmers across the U.S. Almost all SCN resistant varieties in U.S. soybean production are derived from two genetic sources, imposing risk of genetic vulnerability and resistance breakdown. Soybean breeders and growers are limited in genetic sources for nematode resistance with competitive yield, which places U.S. soybean production at risk. Discovery of novel nematode resistance from genetically diverse sources is essential for sustainable soybean production. Deployment of resistant varieties will improve U.S. soybean production and protect yields and farmers’ income.

Our team has successfully developed a strong pipeline of soybean germplasm with resistance to SCN or RKN and competitive yield across all maturity groups. Based on previous discoveries, many genetic populations have been developed to confirm and deploy these genes for nematode resistance. In this new proposal, this team will use an integrated conventional and advanced genomic technologies to achieve the following objectives: 1) incorporate nematode resistance genes into elite high-yielding lines to develop SCN and/or RKN resistant soybean varieties in MG 0 through VIII; 2) identify novel sources of multiple nematode resistance from existing resistant sources or soybean germplasm from USDA Soybean Germplasm Collections; 3) map nematode resistance gene(s) to develop DNA markers for efficient breeding selection; and 4) incorporate SCN and/or RKN resistance into high-yielding lines with value-added seed composition or other key abiotic and biotic tolerance traits.

This work will benefit the entire value chain by providing new soybean varieties adapted to local growing conditions with resistance to multiple nematode species. This project will also provide new and improved materials to commercial and public breeders for use as parental stocks to develop high-yielding, nematode resistant varieties. DNA markers and QTL information generated will benefit soybean researchers, enabling marker-assisted selection and seeking understanding of the genetic mechanisms underlying nematode resistance.

Funder: U.S. Department of Energy

Amount: $333,489

PI: Zenglu Li, College of Agricultural and Environmental Sciences