"Elucidating the role of a novel RARA-AEBP1 interaction in breast cancer" 

"Phenotypical and behavioral characterization of the Wac gene and its role in neurodevelopment"

“The effects of the food additive and Nrf2 activator, tBHQ on dendritic cell function”

“Breaking the barrier: Identification and Characterization of novel MmpL3 inhibitors against M. abscessus”

"Soluble epoxide hydrolase inhibition as a potential treatment for vascular contributions to cognitive impairment and dementia"

"Circuit-specific androgen receptor-dependent regulation of neuronal excitability"

ΔFOSB disulfide bond formation in vivo affects neuronal gene expression and behavior

Daniela Anderson 1, Brandon Hughes 2, Eric Nestler 2, AJ Robison 1

1 Neuroscience Program, Michigan State University, 2 Department of Neuroscience, Icahn School of Medicine at Mount Sinai

Epilepsy is a chronic neurological disorder diagnosed in about seventy million patients globally. It involves spontaneous recurrent seizures caused by an upsurge in hyperexcitability of neurons throughout the brain, often originating in the hippocampus. During seizures, the hippocampus dramatically upregulates expression of ΔFOSB, an immediate early gene transcription factor with a remarkable half-life of 8 days in the brain. Our research indicates that activity-dependent accumulation of ΔFOSB reduces excitability of glutamatergic hippocampal pyramidal cells, potentially serving as a mechanism to counteract hyperexcitability and seizures. In support of this, mice lacking the FosB gene have spontaneous seizures and malformation of the hippocampus, and thus the ΔFOSB AP1 complex emerges as a promising druggable target for potential therapeutic interventions in epilepsy treatment. Moreover, under redox stress ΔFOSB can form disulfide bonds at residue C172 with JunD, its obligate binding partner, and these bonds can alter the ability of ΔFOSB to bind DNA in vitro and in cultured cells. Our lab has developed a new transgenic mouse line harboring a C172S point mutation, as well as a separate full FosB KO mouse. These transgenic tools will allow us to determine the role of ΔFOSB and C172 disulfide bond formation in seizures as well as in other key behaviors regulated by ΔFOSB like drug responses, mood, and learning. We used an array of behavioral assays to measure anxiety-like behavior, anhedonia, locomotor activity, reward-seeking, drug responses, and learning and memory. We also characterized the susceptibility of these mice to pilocarpine-induced seizures. We then treated mice with potassium dichromate, an oxidative agent that causes redox stress in the brain. Western blots were conducted to measure levels of ΔFOSB levels in key brain regions, and CUT&RUN was used to measure binding of ΔFOSB to target genes. Identification mechanisms of ΔFOSB function under oxidative stress could be beneficial for treatment of epilepsy, addiction, among other brain disorders.

tBHQ inhibits dendritic cell-mediated CD4+ T cell activation in a Nrf2-dependent manner

Saamera Awali1,2,3 and  Cheryl E. Rockwell1,2,3,4

1Department of Pharmacology and Toxicology, 2Integrative Pharmacological Sciences Training Program, 3Institute for Integrative Toxicology, and 4Cell and Molecular Biology Program, Michigan State University, East Lansing, MI 48824

Dendritic cells (DCs) are professional antigen-presenting cells that bridge the innate and adaptive immune systems by activating naïve T cells through antigen presentation on MHC class II molecules. Previous work from our lab demonstrated that tert-butylhydroquinone (tBHQ)—a widely used food preservative and potent activator of nuclear factor erythroid 2–related factor 2 (Nrf2)—reduces DC expression of key functional markers, including MHC II, CD80, and CD86. We also found that tBHQ suppresses CD25 and CD69 expression and decreases IL-2 secretion in CD4+ T cells, suggesting impaired T cell activation. However, it remains unclear whether the effects of tBHQ on T cell activation are a direct consequence of its impact on dendritic cells, given the essential communication between these two cell types. Since DCs are critical for T cell priming and activation, we hypothesized that tBHQ impairs DC-mediated CD4+ T cell activation in a Nrf2-dependent manner. To test this, splenic DCs were isolated from female wild-type or Nrf2-knockout mice, pulsed with OVA, and treated with vehicle (0.005% ethanol) or tBHQ (0.5, 1, or 5 µM), then activated with LPS overnight. DCs were washed, counted, and co-cultured with splenic CD4+ T cells isolated from wild-type female mice for three days. T cell activation was assessed by measuring CD25 and CD69 expression via flow cytometry and IL-2 secretion via ELISA. tBHQ treatment significantly suppressed CD25 and CD69 expression and reduced IL-2 production by CD4+ T cells, but only when co-cultured with wild-type DCs. These effects were abolished in co-cultures using Nrf2-knockout DCs, indicating a Nrf2-dependent mechanism. Overall, our findings demonstrate that tBHQ impairs the ability of DCs to activate CD4+ T cells in a Nrf2-dependent manner, providing new insight into how this common dietary additive may alter adaptive immune responses.

Optogenetic activation of LHANts neurons alleviates pain in diet-induced obese mice

Katherine Black, Jariel Ramirez-Virella, Arnalda Zhao, Maria Faraj

Department of Physiology and the Neuroscience Program, Michigan State University

Obesity and chronic pain are often co-morbid and reduce quality and length of life. Because obesity and pain have been studied singly, however, there are no treatments to simultaneously address them. Separate studies demonstrate that Neurotensin (Nts) signaling via its GPCR receptors (NtsR1 and NtsR2) in the brain restrains feeding or promotes analgesia. Previous research from our lab has shown that chemogenetic activation of Nts-expressing neurons in the lateral hypothalamic area (LHANts neurons) both alleviates pain and reduces feeding, suggesting a common Nts system to address obesity-pain. However, LHANts neurons project to many brain regions and mediate a variety of physiological functions, so an LHANts projection that exclusively affects feeding and pain would be the ideal target for obesity-pain treatments. Since the ventral tegmental area (VTA) expresses both NtsR1 and NtsR2 and is involved in motivated behavior pathways, I hypothesized that activating all LHANts neurons or specifically the LHANts to VTA pathway could alleviate obesity-induced pain. To test this, an AAV to cre-dependently express channelrhodopsin was injected into the LHA of normal weight or diet-induced obese NtsCre mice, followed by implantation of an optical fiber into either the LHA or VTA. This allows for optogenetic activation of all LHANts neurons or the LHANts neurons that project to the VTA, respectively. We verified that obese mice have pain compared to normal-weight mice. Optogenetic stimulation during Von Frey pain testing revealed that activating all LHANts neurons, as well as selectively activating their projections to the VTA, increased pain withdrawal threshold. These data confirm the involvement of LHANts neurons in pain signaling in addition to laying groundwork for investigating other LHANts projection candidates using optogenetics and Von Frey pain testing.

P2X Antagonist PPADS Constricts Urinary Bladder Arterioles

Alexis Boron (Presenting)¹, Nathan R. Tykocki 2
1 Department of Physiology, Michigan State University; 2 Department of Pharmacology and Toxicology, Michigan State University

Bladder dysfunction affects millions of people, but few effective treatments are available. Changes in bladder function correlate to changes in blood flow, which
makes the vasculature a novel drug target for treatment of bladder dysfunction. Interestingly, bladder blood flow is not regulated by myogenic tone like in other organs, and the current regulatory mechanism for bladder blood flow is unknown. Many vasoactive compounds known to affect blood flow are released during bladder filling, including ATP. Thus, we tested the hypothesis that activating P2X receptors constricts urinary bladder arterioles. Urinary bladder arterioles from 10 to 16-week-old male C57Bl/6 mice were isolated and cannulated for pressure myography. In some experiments, the endothelium was denuded via air bubble through the lumen. The presence or absence of endothelium was verified by dilation to the muscarinic receptor agonist carbachol (1 μM). Constriction to the purinergic receptor agonist α,βMethylene-ATP (α,βMe-ATP; 100 nM) was measured before and after exposure to the nonselective purinergic receptor
antagonist PPADS (100 μM). α,βMe-ATP produced a rapid constriction followed by desensitization and dilation, which was blocked by PPADS. Interestingly, PPADS alone produced a constriction that was augmented in endothelium-denuded vessels. This was specific to bladder arterioles, as PPADS produced negligible constriction in mesenteric arterioles. Based on these data, activating P2X receptors constricts urinary arterioles, and intrinsic P2X activity may help maintain urinary arteriolar dilation. Overall, these data introduce purinergic signaling as a modulator of bladder blood flow, and future experiments will further explore these mechanisms.

BAM! Investigating The Role Of Meningeal Macrophages And Receptor Activity-Modifying Protein 1 In Migraine Behavior And Etiology

Alex D. Chapman (Presenting)1, Jaewon Sim1, Hina Khan1, Greg Dussor2, Geoffroy Laumet1

1Department of Physiology, Michigan State University, 2School of Behavioral and Brain Science, University of Texas at Dallas

Migraine, a headache disorder characterized by throbbing head pain, nausea, and light/ sound sensitivity, affects about 1 billion people worldwide. It is suggested sensitization of trigeminal ganglion neurons that innervate the meninges, a release of calcitonin gene-related peptide (CGRP), and inflammation of the meninges are involved. Yet, the specific mechanisms and cell types through which CGRP and its receptor, Receptor Activity-Modifying Protein 1 (RAMP1), contribute to the cause of migraines are not fully understood. Recent studies highlight the importance of RAMP1-CGRP signaling of macrophages to elicit a pain response (Fattori et al., 2024). The purpose of our study was to investigate the role of macrophages in the context of migraine. Moreover, our cytokine array analysis showed a significant increase in the CX3CL1, a ligand that can activate macrophages, in mice of both sexes. We employed a well-established restraint stress-induced headache mouse model and found that stress induced facial allodynia, measured by von Frey, and higher meningeal CGRP release compared to controls. Preliminary flow cytometry data also suggests that RAMP1+ meningeal immune cells are disproportionately border associated macrophages (BAMs). BAM deletion (Pf4-DTR) prevented stress-induced facial allodynia. This data together suggests that meningeal macrophages play a vital role in migraine behavior and etiology. As a result, we have generated a Pf4-RAMP1 mouse line to further investigate the role of RAMP1 signaling on macrophages in migraine.

Identification of Distinct NtsR1-positive Neuronal Populations in the substantia nigra pars compacta.

Priscilla Coriano (presenting), Gina Leinninger
Molecular, Cellular and Integrative Physiology, Pharmachology and Toxicology, Michigan State University

Feeding behavior and energy balance are largely influenced by dopamine (DA) neuronal activity. The neurotensin (Nts) system is also largely linked to feeding and weight loss behaviors. Dopaminergic neurons in the substantia nigra pars compacta (SNc) are responsible for locomotor activity, motivation and habit forming. Modulating Nts and neurotensin receptors (NtsR) have been linked to reduce feeding and increase locomotor activity however, the mechanisms and brain regions responsible for these behaviors are yet to be parsed. In the SNc, I have found NtsR1 expressing neurons, though not all NtsR1 neurons in the SNc are dopaminergic. While SNc DA neurons are well studied in movement and locomotor function, their involvement in feeding regulation has not been characterized. Moreover, the specific role of SNc NtsR1-only neurons remains unexplored. This study aims to determine how chemogenetic activation of SNc NtsR1-DA and NtsR1-only neurons influences feeding, movement, and body weight Findings will clarify how distinct SNc NtsR1 neuronal populations contribute to feeding and movement control, offering insight into the neural basis of obesity-related behavioral changes. Understanding these pathways may inform novel targets for treating metabolic disorders without impairing motor function.

Nutrient Availability Regulates MYC-ecDNA Copy Number and Metabolic Phenotypes in Aggressive Cancers

Noah A. Dusseau (Presenting)1,2, Eunhee Yi1

1 Department of Physiology, Michigan State University, 2 Department of Pharmacology and Toxicology, Michigan State University

Extrachromosomal DNA (ecDNA) is a common driver of tumor evolution and therapy resistance in cancer. These circular DNA elements unevenly segregate during cell division, promoting genetic heterogeneity and aggressive tumor behaviors. The influence of ecDNA on cancer metabolism, however, remains poorly understood. This project investigated how nutrient availability affects ecDNA content and phenotypic states in HF2354 patient-derived glioblastoma neurospheres and PC3 C1 prostate cancer cells. Using fluorescent in situ hybridization and quantitative imaging, it was found that glucose and glutamine deprivation reduced ecDNA copy numbers and shifted cell populations toward low-ecDNA, survival phenotypes. HF2354 cells showed a reduction from 56.6±3.7 ecDNA per cell in nutrient-rich conditions to 43.1±4.6 in nutrient-poor conditions, while PC3 C1 cells decreased from 37.1±3.1 ecDNA per cell in nutrient-rich conditions to 22.5±3.2 in nutrient-poor conditions. These findings suggest that nutrient stress constrains ecDNA maintenance and may reprogram cancer metabolism. Linking ecDNA dynamics to metabolic adaptation could reveal vulnerabilities for pharmacologically targeting aggressive tumors.

Unzipping GILZ: Elucidating the Role of Glucocorticoid-Induced Leucine Zipper in TNBC

Devyn A. Hill (Presenting) 1, Jennifer B. Jacob 2,3  

1 Department of Molecular, Cellular and Integrative Physiology, Michigan State University; 2 Department of Biochemistry and Molecular Biology, Michigan State University; 3 Department of Pharmacology and Toxicology, Michigan State University

One in eight women have a lifetime risk of developing breast cancer, making it a leading cause of cancer related mortality in American women. Cancers that lack expression of hormone receptors (HR) or the HER2 receptor are known as Triple Negative Breast Cancer (TNBC) and are resistant to common treatments, making them very difficult to manage and leading to higher mortality. Evidence from our laboratory shows that the expression of the protein, Glucocorticoid-Induced Leucine Zipper (GILZ) encoded by the TSC22D3 gene, is strongly correlated with increased survival in patients presenting with TNBC. Using single cell RNA sequencing, we know that GILZ expression is highest in stromal fibroblasts and myeloid cells. GILZ expression is induced by glucocorticoids and directly inhibits inflammation by interacting with key enzymes, such as mitogen-activated protein kinases (MAPKs), and proteins like rat sarcoma protein (Ras) in inflammatory signaling. GILZ interactions with these biomolecules are primarily cytosolic, and are known to prevent downstream phosphorylation or translocation into the nucleus that supports their activation. This is hypothesized to result in reduced inflammation in women with TNBC, contributing to their prolonged survival with increased expression of GILZ. However, with what is currently known, the precise mechanism of GILZ action on down-stream inflammation remains unclear. This project aims to identify the transcriptional and post-translational regulation of GILZ to determine its potential as a target for therapeutic treatment. A precise understanding of GILZ activation and action will allow me to investigate how GILZ expression affects tumor growth, hypoxia-driven inflammation and ultimately its therapeutic potential. 

Pyruvate Kinase Activity Regulates Cystine Starvation-Induced Cell Death in Pancreatic Cancer

Tessa Jordan1,2*, Elliot Ensink1,3, Hyllana CD Medeiros1, Amir Roshanzadeh1,4, Thurston Galloway5, Anmol Pardal4, Lei Yu1, Lin Lin7,8, Costas Lyssiotis7,8, Sophia Y Lunt1,6

1 Department of Biochemistry and Molecular Biology, Michigan State University; 2 Department of Pharmacology & Toxicology, Michigan State University; 3 College of Osteopathic Medicine, Michigan State University; 4 Department of Cellular and Molecular Biology; 5 College of Human Medicine, Michigan State University; 6 Department of Chemical Engineering and Materials Science, Michigan State University; 7 Department of Molecular & Integrative Physiology, University of Michigan; 8 Rogel Cancer Center, University of Michigan

Pancreatic cancer is one of the deadliest cancers in the U.S. with the lowest 5-year survival rate at just 13%. Current treatments are limited to nonspecific cytotoxic therapies, which typically extend survival by an average of 4 months. Therefore, there is a critical need to develop new therapies to treat pancreatic cancer. Cancer cells rewire their metabolism to survive in the harsh microenvironment, to keep up with metabolic demands required for rapid growth and proliferation, to manage oxidative stress, and to evade cell death. One common metabolic alteration to most cancer cells is the switch in isoform expression of the glycolytic enzyme pyruvate kinase from muscle isoform 1 to muscle isoform 2 (PKM2). Unlike other pyruvate kinase isoforms, PKM2 can be allosterically regulated, enabling cancer cells to redirect metabolism in response to oxidative stress. This makes PKM2 a promising potential therapeutic target. While its role in supporting antioxidant defenses through pathways like the pentose phosphate pathway (PPP) is established, its involvement in other antioxidant processes, such as cystine metabolism, remains largely unexplored. Cystine is critical for redox homeostasis, primarily through its role in glutathione synthesis, which provides the cell’s major source of reducing power. Our lab has shown that PKM2 knockout (KO) pancreatic cancer cells are resistant to cystine starvation-induced cell death compared to PKM2-expressing cells. These PKM2KO cells also show elevated NADPH and reduced-glutathione (GSH) levels under cystine starvation. Furthermore, the combination of cystine starvation and PKM2 activation significantly reduced the viability of wild-type pancreatic cancer cells more than either treatment alone. Therefore, our hypothesis is that PKM2 regulates pancreatic cancer cell response to cystine starvation-induced cell death by altering metabolism and antioxidant defenses. Overall, this work will advance understanding of PKM2’s role in cancer metabolism and lead to targeting PKM2 as a novel treatment strategy for pancreatic cancer.

Evaluating the Disease-Modifying Potential of a Novel ROCK Inhibitor in Rodent Models of Synucleinopathy 

Michael Kubik1, Jeffrey P. MacKeigan2, Anna C. Stoll1, Joseph R. Patterson1, Christopher J. Kemp1, Jacob W. Howe1, Kathryn M. Miller1, Fredric P. Manfredsson1, Stephanie Celano2, Kelvin C. Luk3, and Caryl E. Sortwell1

1 Department of Translational Neuroscience, Michigan State University, Grand Rapids, MI USA,
2 Department of Pediatrics & Human Development, Michigan State University, Grand Rapids, MI USA
3  Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA USA

Rho-associated coiled-coil kinase (ROCK) activity in neurons, glia, and peripheral cell types has been implicated in Parkinson’s Disease (PD) pathology, including neuroinflammation, axonal degeneration, and a disruption of the blood-brain barrier (BBB) integrity. ROCK inhibitors have shown to be a promising therapeutic strategy in mitigating these pathological hallmarks, yet their effects on alpha-synuclein (α-syn) pathology and associated neuroinflammation remain unclear. This study evaluates the neuroprotective potential of a third-generation ROCK inhibitor, KL-00974, in the α-syn preformed fibril (PFF) model of PD at pathologically significant endpoints. In one study, we investigated phosphorylated α-syn (pSyn) pathology, microglial activation, following 2-months of KL-00974 administration. In another study we assessed nigral dopaminergic neuron survival. Our preliminary data suggest ROCK inhibition modulates neuroinflammatory responses and might help maintain BBB integrity. To investigate the mechanisms by which ROCK inhibition may grant neuroprotection, RNA sequencing will identify transcriptomic changes throughout the nigrostriatal system. Further, we quantified striatal dopamine content using high-performance liquid chromatography (HPLC). Our work investigating the effects of ROCK inhibition in the context of α-syn pathology and neuroinflammation highlights an important milestone in the search for disease-modifying therapies for PD. These results will guide future neuroprotective strategies targeting α-syn aggregation-induced neuroinflammation and neurodegeneration.

Impact of tissue transglutaminase (TG2) on fibrin(ogen) crosslinking and clot formation

Nana Kwame Kwabi Boateng (Presenting)¹, Riley Wimberley¹, James P. Luyendyk¹'²

¹Department of Pharmacology and Toxicology, Michigan State University, USA; ²Department of Pathobiology & Diagnostic Investigation, Michigan State University, USA

Cross-linking of polymerized fibrin by the transglutaminase coagulation factor FXIII plays a key role in the hemostatic and inflammatory functions of blood clots. Several studies suggest that in the context of necrotic tissue injury, another ubiquitously expressed transglutaminase, tissue transglutaminase (TG2), may also contribute to fibrin(ogen) cross-linking. TG2 imposes unique α-γ cross-links in fibrinogen both in the injured liver and in vitro. Although prior studies suggest potential effects of TG2 on fibrinogen hemostatic properties, this has not been examined in detail. We determined the impact of TG2 on thrombin-induced fibrin polymerization and cross-linking. Recombinant human TG2 imposed a concentration- and time-dependent increase in the formation of alpha-gamma crosslinks in human fibrinogen 2mg/ml (FIB 1, ERL). Thrombin treatment of human fibrinogen increased fibrin polymerization as determined by increased turbidity. Notably, whereas TG2 did not significantly increase turbidity at the concentrations tested, the addition of TG2 induced a concentration-dependent (0.92µg/ml, 2.8µg /ml, and 8.3µg/ml) inhibition of peak turbidity induced by thrombin addition. The results validate the capacity of TG2 to cross-link soluble fibrinogen independent of polymer formation and indicate that TG2 cross-linking of fibrinogen alters thrombin-induced turbidity in vitro. Ongoing studies are focused on the impacts of TG2 on fibrinopeptide release, fibrinogen solubility dynamics, and FXIII-directed cross-linking. The results suggest that TG2 may play a substantial role in defining the hemostatic properties of fibrin clots formed in the context of massive tissue injury.

Affects of Neurosteroids on Thalamic Neurons

Megan McGrath (presenting), Joe Beatty, Lee Cox

Department of Physiology, Michigan State University

Neurosteroids are steroid based molecules that are formed de novo in the brain and act as neuromodulators. Unlike systemic steroids which predominantly act via intracellular receptors to alter gene expression, neurosteroids are thought to exert mostly extra-genomic actions. The targets of neurosteroids are determined by the specific substituents and chirality, leading to varied activity profiles. Our studies utilizing whole cell patch clamp recordings in rat brain slices have found that neurosteroids can alter both the intrinsic and synaptic properties of neurons in the thalamic reticular nucleus, and thalamocortical relay neurons in the ventrobasal nucleus of the thalamus. Teasing out the conserved and unique mechanisms of different neurosteroids can help to elucidate the important mechanisms for each of their unique pharmacologic indications. 

Characterization of Sex Differences in Mouse Cocaine Self-Administration

Alice J. McQueney1, Melissa Rogers2, Daniela Anderson1, AJ Robison1,2

1Neuroscience Program, 2Department of Physiology, Michigan State University

Cocaine use disorder remains a significant healthcare crisis, with no FDA-approved pharmacological treatments currently available. Additionally, pronounced differences in the ways males and females respond to cocaine have been observed both clinically and in preclinical rodent models. It is therefore imperative that we further investigate the mechanisms of sex differences related to cocaine use disorder and potential pharmacological targets underlying these differences. Intravenous self-administration (IVSA) examines volitional intake of a drug and is the gold standard preclinical cocaine model, allowing investigation of motivation, escalation, and relapse to drug use. Although sex differences in cocaine self-administration have been examined in rats, there is currently a lack of research characterizing this difference in mice. This is a critical gap because mice provide easier genetic manipulation of the receptors and circuits driving the development of cocaine use disorder, and a higher throughput and reduced cost compared to other rodents. To establish sex differences in the mouse cocaine IVSA model, male and female mice were surgically implanted with an indwelling jugular catheter and trained in operant boxes to respond for intravenous infusions of cocaine. Dose-response curves will be generated to characterize the differences in the acquisition and maintenance of self-administration criteria and overall consumption of cocaine in both sexes at various doses of cocaine. The overarching hypothesis of this work is that male mice are less sensitive to lower doses of cocaine than female mice, and male mice will be more responsive to higher cocaine doses that may be aversive to female mice. This project will provide insight into the sex differences in cocaine motivation in mice and potentially lead to the identification of novel therapeutic interventions. 

Immunorepertoire characterization enables non-invasive classification of traumatic brain injury status in children

Annie Needs1,2**, Jillie Lynch1,2, Surender Rajasekaran MD MPH3,4, Jeremy Prokop3, Elora Hussain MD3,4, Daniel Woldring1,2

1 Michigan State University, Department of Chemical Engineering and Material Science

2 Michigan State University, Institute of Quantitative Health Science and Engineering (IQ)

3 Corewell Health

4 Corewell Health Helen Devos Children’s Hospital

Traumatic brain injuries (TBIs) in children result in over 500,000 emergency visits annually in the U.S., often leading to impaired cognitive development and increased risk of neurodegenerative diseases. Despite being a major public health concern, current diagnostic methods rely on subjective witness accounts and clinical observation. This absence of an objective method for assessing a pediatric patient's TBI status motivates the urgent need for the development of objective diagnostic tools. This research proposes leveraging the potent immune response to a TBI to address these gaps. Following a TBI, neuroproteins, typically found only in the immune-privileged brain, enter systemic circulation due to Blood-Brain Barrier damage, triggering anti-neuroprotein antibody production and a proinflammatory cytokine response. Our collaborators at Corewell Health’s Helen Devos Children's Hospital collected immunorepertoires of 17 pediatric severe TBI patients, revealing a sequence-level dynamic antibody signature in the pediatric TBI patients distinct from controls and other non-TBI trauma patients. Initial analysis of antibody heavy chain CDR3 sequences has demonstrated the potential for the antibody repertoire as a diagnostic biomarker. To evaluate this potential, classification models have been applied using k-mer representations of the heavy chain CDR3 sequences, showing early promise in distinguishing TBI patients from healthy controls. Ongoing investigation aims to identify key amino acid motifs within these sequences that may correspond with the immune system's specific response to a TBI. By taking advantage of the information-rich, patient-unique immunorepertoire, collected in a standard clinical blood draw, this research will establish a foundation for non-invasive, sequence-based diagnosis tool for pediatric traumatic brain injuries. 

Exploring transcriptional changes in mouse ocular tissue exposed to phosgene oxime 

Ebenezar Okoyeocha (Presenting) 1, Natalie Vredevoogd 1, Shreya Paithankar 1, Bryan Masino 1, Poojya Anantharam 2, Dinesh Goswami 1, Charlotte Madigan 1, Megha Suresh 1, Dodd Sledge 4, Bin Chen 1,3 , and Neera Tewari-Singh 1

1 Department of Pharmacology and Toxicology, College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA.
2 MRIGlobal, Kansas City, Missouri, USA
3 Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, East Lansing, MI, USA
4 Michigan State University Veterinary Diagnostic Laboratory, Lansing, MI, USA

Phosgene oxime (CX), a toxic blistering and nettle agent, serves as an environmental toxicant. Injuries caused by CX extend beyond external organs, such as the eye, skin, and lungs, often impacting internal tissues due to its penetrative properties. Despite the eye’s vulnerability to toxic chemical agents, a research gap persists regarding changes in gene expression following ocular exposure to CX. Thus, this pilot study investigates the transcriptional changes underlying the toxicity of CX in mouse ocular tissue. The eyeballs of C57Bl6 mice (6–8 weeks old, male and female, n=3) were exposed to CX for 30 seconds or underwent sham treatment, followed by enucleation 24 hours after exposure. mRNA was extracted using the phenol-chloroform method and sequenced by NovoGene on the Illumina NovaSeq PE150 platform. Sample quality and mRNA concentration were evaluated using the Agilent 2100 system. Differential gene expression analysis was performed using DESeq2 with a standard cutoff of p-value ≤ 0.01 and |log2-Fold Change| ≥ 1. Functional pathway enrichment was conducted using EnrichR, and selectively differentially expressed genes (DEGs) were validated through qPCR. Our findings revealed 697 downregulated and 233 upregulated genes following CX exposure. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (p-value ≤ 0.01) showed significant upregulation of pathways related to the inflammatory response and cell death in ocular tissues. These results offer critical insights for studies aimed at finding effective treatments for phosgene oxime-induced ocular tissue injuries. Further analysis using single-cell transcriptomics and virtual therapeutic screening tools is necessary to identify biomarkers and clarify underlying molecular mechanisms.

Diet-Induced Metabolic Alteration Affects Chemoresponse in Ovarian Cancer

Adriana L. Ponton-Almodovar¹,², Mary P. Udumula³, Vrinda Khullar¹, Ramandeep Rattan³, Jamie J. Bernard²,⁴, Sachi Horibata¹,²,⁵

¹Precision Health Program, Michigan State University, East Lansing, MI; ²Department of Pharmacology and Toxicology, College of Human Medicine, Michigan State University, East Lansing, MI; ³Department of Gynecologic Oncology, Henry Ford Hospital, Henry Ford Cancer Institute, Detroit, MI; ⁴Department of Medicine, College of Human Medicine, Michigan State University, East Lansing, MI; ⁵Cell and Molecular Biology Program, College of Human Medicine, Michigan State University, East Lansing, MI

Ovarian cancer is the most lethal gynecologic malignancy with approximately 80% of patients developing resistance to chemotherapy. Obesity is associated with poorer survival among patients receiving platinum-based drugs; however, the role of obesity in chemoresistance is unclear. We have shown that high-fat diet (HFD)-induced obesity promotes ovarian cancer progression in tumor-bearing mice and alters metabolic balance in glutaminolysis activity when treated with carboplatin. Therefore, we hypothesize that factors secreted from adipose tissue metabolically rewire ovarian cancer and this is associated with glutaminolysis-driven proliferation and chemoresistance. We developed a novel model whereby factors secreted from human omental adipose tissue (OmFTF) stimulate the anchorage-independent 3D growth in soft agar of ovarian cancer cell lines. Our results show that OmFTF induces a significant increase in colony size, demonstrating that culture with adipocyte-secreted factors stimulates proliferation of OVCAR3 and OVCAR8 cells. Interestingly, these aggressively growing clones have a higher IC50 when exposed to cisplatin, and this is associated with elevated glutamate pyruvate transaminase 2 (GPT2), which serves as a pivot between glycolysis and glutaminolysis. To further explore the implications of the adipocyte-secreted factors ex vivo, we exposed OVCAR3 cells to ascites derived from HFD-fed tumor-bearing mice. We demonstrated that exposure to these HFD-derived ascites results in acquired chemoresistance and upregulation of GPT2. These data suggest that adipose tissue stimulates ovarian cancer chemoresistance through metabolic adaption to the microenvironment. To determine if GPT2 was associated with chemoresistance independent of OmFTF exposure, we characterized GPT2 gene expression in OVCAR3 and OVCAR8 control (CP0) and induced platinum-resistant (CP5) cell lines. GPT2 was only upregulated in resistant cells and knocking out GPT2 in resistant OVCAR8-CP5 resulted in a significant decrease in 3D growth. These studies implicate GPT2 as a novel metabolic link between excess adiposity and glutaminolysis-driven chemoresistance in ovarian cancer. 

VWF-platelet interactions drive hepatic injury in experimental acute liver failure

Caitlin Schneider1, Zimu Wei2, Renhao Li3, James Luyendyk2

1 Department of Pharmacology and Toxicology, Michigan State University;

2 Department of Pathobiology and Diagnostic Investigation, Michigan State University; 

3 First Affiliated Hospital, Zhejiang University School of Medicine

Background and Purpose: Acetaminophen (APAP) overdose is a leading cause of acute liver failure (ALF) in the United States. Altered hemostasis is one consequence of APAP-induced liver failure, with clinical and experimental evidence suggesting that thrombocytopenia is associated with poor outcomes. Experimental APAP overdose in mice drives colocalized hepatic accumulation of VWF and platelets in the injured liver, with prior studies suggesting that VWF may inhibit liver repair by promoting retention of hepatic platelet microthrombi. We tested the hypothesis that VWF-platelet interactions drive APAP-induced ALF.

Methods: VWF deficient mice were given a hepatotoxic dose of APAP (600mg/kg), an experimental setting of liver failure, with sample collection taking place 24 hours post-challenge. To determine the contribution of platelets in driving ALF, wild-type mice were given platelet depleting anti-GPIbα antibody (R300) prior to APAP challenge. To pharmacologically disrupt the formation of VWF-platelet microthrombi, we used a novel nanobody (Nd6) that reduces platelet GPIb engagement of the murine VWF A1 domain.

Results:  VWF deficient mice had reduced plasma alanine aminotransferase (ALT) activity, a marker of hepatocellular injury. Anti-GPIbα antibody-induced thrombocytopenia reduced plasma ALT activity, hepatocellular necrosis, hepatic congestion, and VWF deposition induced by APAP challenge. Administration of the Nd6-Fc fusion protein significantly reduced plasma ALT activity and hepatic congestion in mice challenged with 600 mg/kg APAP.

Conclusions: These results suggest that both VWF and platelets contribute to hepatocellular injury during APAP-induced ALF. Our findings support the hypothesis that pharmacologically targeting VWF-platelet interactions may be beneficial to reduce liver damage and prevent progression to ALF. While inhibiting procoagulant pathways in patients with ALF presents several challenges, treatment with Nd6-Fc could circumvent these issues by decreasing the pathologic activity of VWF while retaining the protein's hemostatic function.

Androgen receptor dependent regulation of projection-specific hippocampal neuronal excitability

Chiho Sugimoto1 (Presenting), Andrew L. Eagle2, Ryan Bastle3, Ian Maze3, AJ Robison1

1. Department of Physiology, Michigan State University 2. Department of Neuroscience, University of Texas at Dallas 3. Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai

Major depressive disorder (MDD) is highly prevalent and has a complex multifactorial etiology, with stress vulnerability emerging as a critical risk factor. MDD prevalence is almost double in women compared to men, but the biological basis of this sex difference is not understood. Additionally, roughly half of MDD patients do not respond to existing treatments, and thus there is an urgent need to understand MDD’s cellular and the molecular mechanisms to develop new therapeutic strategies. MDD is associated with abnormalities of the brain’s reward circuitry, and ventral hippocampal (vHPC) projections to the nucleus accumbens (NAc; vHPC-NAc) have been implicated in stress-induced susceptibility to anhedonia in male mice. However, despite the higher prevalence of MDD in females, studies that include both male and female subjects are lacking. Our lab used subchronic variable stress (SCVS), which induces an anhedonia-like phenotype in female mice but not males, to mechanistically investigate the sex differences in stress-induced anhedonia. We found that female mice have increased basal vHPC-NAc intrinsic excitability compared to males, and that this directly drives anhedonia-like behavior following SCVS. Moreover, vHPC-NAc circuit excitability is reduced by adult testosterone, but the mechanisms by which androgen receptors (AR) regulate this excitability remain unknown. We used ex vivo whole cell slice electrophysiology on WT- and conditional AR knockout (AR cKO)-L10GFP mice to examine the role of AR in vHPC-NAc excitability in male and female mice. We found that removing AR from vHPC-NAc increased excitability in males. Moreover, the increased circuit excitability in females compared to males was dependent on non-aromatized androgens, and conditionally knocking out ARs in the vHPC-NAc circuit reversed these effects. Action potential waveform analysis suggested that potassium and calcium channels may play a key role in this excitability change. Therefore, we used translating ribosome affinity purification sequencing (TRAP-seq) to examine downstream targets of AR and observed changes in expression of multiple ion channels and signaling pathways which drive sex-specific behaviors and vulnerability to stress. Furthermore, we investigated the AR signaling pathways necessary to drive changes in vHPC-NAc excitability by using pathway-specific AR knockouts. ARs can operate via the canonical pathway of binding DNA to alter gene expression or through non-canonical membrane-bound signaling in the cytosol, and we hypothesize ARs reduce excitability of vHPC-NAc neurons through directly changing gene expression in the nucleus.

Clopidogrel Increases Blood-Brain Barrier Permeability In Angiotensin II-Induced Hypertensive Mice 

Afolashade Toritseju Onunkun, Megyn McCoy, Taylor Rabanus, Anne Dorrance PhD, Adam Lauver PhD

Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI

Arterial thrombosis is a leading cause of death globally. Hypertension is a significant risk factor for its development, and over 50% of patients with arterial thrombosis have hypertension. P2Y12 antagonists like clopidogrel are used for the management of thrombosis, but they carry a serious risk of bleeding. Although traditionally attributed to platelet inhibition, emerging evidence suggests that P2Y12-independent mechanisms contribute to clopidogrel’s bleeding. Endothelial dysfunction is a mechanistic risk factor for cerebral bleeding, and we have demonstrated that clopidogrel impairs endothelial function in cerebral arteries independently of P2Y12. To further investigate its endothelial effects, we assessed the blood-brain barrier (BBB), a function mediated by the endothelium. We hypothesized that clopidogrel would increase BBB permeability through a P2Y12-independent mechanism. Male C57BL/6 wildtype (WT) and P2Y12knockout (P2Y12KO) mice were implanted with Ang II-filled osmotic minipumps (800 ng/kg/min for 4 weeks). The mice received either 10 mg/kg clopidogrel or vehicle once daily. Systolic blood pressure, measured using tail cuff plethysmography, was significantly elevated in hypertensive mice compared to sham controls. BBB permeability, determined by 4 kDa FITC-dextran extravasation into the brain tissue, was increased in both clopidogrel-treated WT and P2Y12KO mice relative to vehicle-treated mice. These results suggest that clopidogrel increases BBB through a P2Y12-independent mechanism. Elucidating the cerebrovascular effects of clopidogrel is crucial for developing novel antiplatelet therapies devoid of such properties.

15-HETE: A Novel Lipid Mediator and Potential Modulator of Neurodegeneration in C. elegans Alzheimer’s Models

Fatemeh (Jana) Yousefsaber1*, Kin Sing Stephen Lee1,2, Morteza Sarparast1 , Jamie Alan2

1 Department of Chemistry, Michigan State University
2 Department of Department of Pharmacology and Toxicology, Michigan State University

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-β (Aβ) aggregation and tau hyperphosphorylation, yet the metabolic pathways linking these hallmarks to neuronal dysfunction remain poorly understood. Recent studies implicate oxidized lipid metabolites (oxylipins) as potent regulators of neuronal activity and survival. Several studies including ours, indicate dramatic oxylipin levels shift with disease progression, suggesting they may serve not only as biomarkers but also as active drivers of pathology. 15-hydroxyeicosatetraenoic acid (15-HETE), a downstream metabolite of arachidonic acid metabolism by 12/15-lipoxygenase (LO), has emerged as a compelling candidate. Clinical studies reveal elevated 15-HETE in the cerebrospinal fluid of patients with AD or mild cognitive impairment, and 12/15-LO expression is increased in affected cortical regions. 

To mechanistically test this link, we employed Caenorhabditis elegans AD models expressing human Aβ or tau, together with wild-type and aging cohorts. We show that expression of Aβ, tau, or aging significantly elevates 15(S)-HETE levels. Remarkably, supplementation of naïve worms with 15(S)-HETE was sufficient to induce neurodegenerative phenotypes, including impaired movement, and selective degeneration of dopaminergic ADE neurons. These effects were stereospecific, as neither 15(R)-HETE nor arachidonic acid produced comparable pathology. 

Together, these findings suggest that 15-HETE acts as a pathogenic lipid mediator in neurodegeneration and may represent a novel mechanistic link between lipid metabolism and AD-related neuronal dysfunction. Defining the downstream protein targets and signaling pathways of 15(S)-HETE will not only deepen understanding of disease pathogenesis but also open new therapeutic avenues that target lipid metabolism to mitigate neurodegeneration.

Alveolar Macrophage Ontogeny Drives Differential Inflammation in Response to Immune Activation

Reham A. Ammar1 (Presenting), Andrew J. Olive2

1Department of Pharmacology and Toxicology, 2Department of Microbiology, Genetics, and Immunology
College of Osteopathic medicine, Michigan State University, East Lansing, MI

Positioned at the interface between the external environment and internal milieu, the lungs require a fine-tuned immune response to maintain respiratory function. Macrophages derived from distinct progenitors are key regulators of immune responses in the lung. Fetal liver-derived tissue-resident alveolar macrophages (AMs) are first-line defenders that maintain lung homeostasis, while myeloid-derived macrophages are recruited during infections and inflammation. Although recruited macrophages differentiate into mature alveolar-like phenotype, they remain functionally distinct. Understanding these ontogeny-driven differences have remained challenging due to a lack of ex vivo models recapitulating the functions of these macrophage subsets. To address this, we recently developed fetal liver-derived alveolar-like macrophages (FLAMs) as a tractable model of AMs. Here, we introduce myeloid-derived alveolar-like macrophages (MAMs) as a new model of lung myeloid resident macrophages, that are phenotypically similar to AMs when grown in the lung cytokines GM-CSF and TGFβ. We hypothesize FLAMs and MAMs can be used to interrogate ontogeny-driven differences regulating inflammation. To test this, we are dissecting the inflammatory response of FLAMs and MAMs to the TLR4 activator lipopolysaccharide (LPS). Our data show that MAMs are more inflammatory than FLAMs, resulting in increased pro-inflammatory cytokine production. TGFβ drives lipid metabolism in FLAMs resulting in robust PPAR𝛾 expression.  As a transcription factor, PPAR𝛾 regulates peroxisomal function and fatty acid oxidation in AMs. We found MAMs have lower PPAR𝛾 expression and higher expression of CD14, a co-receptor of TLR4. Modulating PPAR𝛾 activity in MAMs resulted in reduced TLR4-mediated inflammation suggesting a key link between PPAR𝛾, lipid metabolism, CD14 expression, and inflammation. Transcriptome and metabolic flux analysis suggest MAMs upregulate glycolysis in response to LPS, in contrast to FLAMs which favor oxidative phosphorylation. In summary, our new models position us to understand ontogeny-driven differences regulating inflammation in the lungs and uncover pathways contributing to susceptibility to pulmonary infections and inflammatory diseases.

Presynaptic Suppression of Higher-Order Thalamocortical and Corticocortical Inputs to Mouse Somatosensory Cortex

Kelly E. Bonekamp1 (presenting), M. Lea Ratz1, Grant R. Gillie1, Mya K. Sebek1, Lingxi Xiong1, Shane R. Crandall1 

1Department of Physiology

Long-range, higher-order communication across the cortex is important for processes such as sensation, motor control, and cognition. In the cortex, these projections converge on GABAergic inhibitory circuits in the outermost cortical layer, layer 1 (L1). L1 inhibitory cells are thought to inhibit nearby dendrites of pyramidal cells in L2/3 and L5. However, they may also inhibit presynaptic terminals of the long-range inputs into L1. Presynaptic GABA receptors are metabotropic (GABABR), which are thought to be activated only by certain inhibitory cells. Potential candidates for this presynaptic inhibition include L1 neuron-derived neurotrophic factor (NDNF) cells or somatostatin (SOM)-expressing cells projecting to L1. If these inhibitory cells activate presynaptic GABABR on long-range afferents, they could suppress their own input from higher-order structures. Yet it is unknown if this occurs, or if presynaptic GABABR exist on cortical long-range afferents. Using the mouse whisker system as a model, we examined presynaptic inhibition of corticocortical (CC) and thalamocortical (TC) inputs into primary somatosensory cortex (vS1). We found that CC presynaptic terminals were inhibited more strongly than TC terminals by the GABABR agonist Baclofen. We also found that this presynaptic inhibition could be mediated by the recruitment of L1 NDNF cells and SOM cells. Finally, we found that a subset of NDNF cells known to activate GABABR are strongly recruited by TC inputs, suggesting TC inputs may recruit L1 NDNF cells to suppress incoming CC input. This may be a mechanism to tune down the gain of CC input to focus on incoming sensory input during states of increased arousal.

Spatial Domain Fragmentation and Loss of Spatial Gene Expression Organization in Liver Fibrosis

Derek E. Bowman,1,3 Jordi Pinero,5 Carlo Piermarocchi,4 Sudin Bhattacharya 1,2,3

1 Department of Pharmacology & Toxicology, 2 Department of Biomedical Engineering, 3 Institute for Quantitative Health Science and Engineering, 4 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, 5 ICREA-Complex Systems Lab, Universitat Pompeu Fabra, 08003 Barcelona, Spain

An outstanding question in biology is how individual cells assemble into interconnected units that give rise to tissue-level functions. An important first step is to understand the characteristic length scales over which cells influence each other in discrete “tissue domains” and to assess how the characteristic length scales of these domains change in tissue pathologies like liver fibrosis. Here, we introduce a method to quantify the characteristic length of tissue domains using a gene expression-based measure of cellular similarity. We apply this measure to published spatial transcriptomics data from human liver samples from healthy patients and those with liver fibrosis. Interestingly, while the healthy tissue demonstrated larger correlations in gene expression and larger domain size and a more gradual transition in correlations with length scale, the diseased tissue demonstrated lower overall correlation scores and an accelerated step-off in correlation that occurred at smaller radii. This suggests that cells in the healthy tissue are more similar over larger length scales, and their interactions are more ordered and extend over a greater distance. In contrast, the diseased tissue exhibited lower correlation scores and a steeper transition, suggesting that the cells tend to interact over shorter distances and form smaller, more isolated clusters. Quantifying the characteristic interaction lengths of cells enables us to better appreciate cellular organization and achieve deeper understanding of the mechanisms governing tissue-level biology.

Disruption of the Gas6-Axl-Matrix Metalloproteinase-12 Pathway Contributes to the Loss of Hepatic Vascular Integrity during Acute Liver Failure 

Cab Gomez, Kevin Conzemius, Maclain McAllister, Cheryl E. Rockwell, Bryan L. Copple

Department of Pharmacology and Toxicology, Institute for Integrative Toxicology, School of Human Medicine

Michigan State University, East Lansing, MI

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver injury (DILI) in the United States. Outcomes from APAP-induced acute liver injury (AALI) range from a full recovery to acute liver failure (ALF), a life-threatening condition that adversely impacts multiple organ systems. The transition from AALI to APAP-induced ALF (AALF) is marked by severe damage to the hepatic sinusoidal vasculature. The mechanism(s) responsible for this injury remains to be defined. Prior studies demonstrated that matrix metalloproteinase-12 (MMP12) protects the sinusoidal vasculature during AALI. Moreover, our studies revealed that MMP12 is upregulated during AALI by a mechanism requiring activation of the Gas6-phosphatidylserine receptor, Axl. Because of the importance of this signaling pathway to the maintenance of vascular integrity, we tested the hypothesis that the Axl-MMP12 pathway is disrupted during the transition to AALF, ultimately leading to damage to the sinusoidal vasculature. To test this hypothesis, we first identified the cell type that produces MMP12 in an Axl-dependent manner during AALI. By utilizing Cre/loxP technology, our studies demonstrate that deletion of Axl on Kupffer cells, the resident macrophage population of the liver, markedly reduced MMP12 levels during AALI. During the transition to AALF, MMP12 mRNA levels were lower in Kupffer cells when compared to mice with AALI. Although Axl levels on Kupffer cells were unaffected by the transition to AALF, levels of the endogenous Axl ligand, Gas6, were reduced which may account for the reduction of MMP12 during AALF. Consistent with this, either restoration of Gas6 levels or activation of Axl with an Axl activating antibody increased MMP12 levels during AALF.  Importantly, this was associated with a reduction in indices of sinusoidal vascular injury. Collectively, these studies indicate that deficient Axl receptor signaling contributes to the loss of vascular integrity during AALF and that this may be corrected by restoration of Gas6 levels. In future studies, we will investigate the impact of restoring Axl signaling on life-threatening complications that arise during ALF. 

Discovery Of Cardiovascular Mesothelium: A Foundational Study

Paul J. Chu (Presenting)¹, Lizbeth Lockwood¹, Janice M. Thompson¹, Emma Wabel¹, Leah Terrian¹, Stephanie W. Watts¹
¹Department of Pharmacology and Toxicology, Michigan State University

The mesothelium was previously thought to be a passive monolayer lining of cells that functions as structural support for our internal organs and cardiovascular system. Indeed, single-nucleus RNA-seq (snRNAseq) experiments from our lab identified a distinct mesothelial cell population in thoracic aorta perivascular adipose tissue (taPVAT).  Recent developments support that the mesothelium may be highly involved in a multitude of biological functions that are important to cardiovascular health. Due to the limited understanding of what the mesothelium is composed of, our experiments focused on characterization and identification of the cellular constituents of mesothelium, including biological markers, that surrounds various cardiovascular organs. The mesothelium of male Dahl SS rats was dissected from the kidney, mesentery, and thoracic aorta.  Histochemistry and polymerase chain reaction (PCR) were used to characterize these tissues.  Masson’s Trichrome staining visualized prominently red-stained cells within an extensive collagenous matrix. We tested a list of genes, supported by the literature, as potential markers for the mesothelium.  Podoplanin and Mesothelin as were identified as consistent gene markers of the mesothelium across these three tissue types. SnRNAseq additionally revealed that C4a gene expression was highly active and localized to mesothelial cells surrounding the taPVAT, confirmed through PCR. The abundance of C4a and the finding that Connexin 43 is expressed is evidence that the mesothelium is a potentially highly active tissue that is involved in the regulation and propagation of various biochemical and immunological signals within the body.

Structural Plasticity of ecDNA and Its Impact on Cancer Therapy

Emmanuel E. Korsah (Presenting)1, Noah A. Dusseau1,2, Eunhee Yi3 

1Department of Physiology, College of Natural Science, Michigan State University, East Lansing, Michigan
2Department of Pharmacology and Toxicology, College of Natural Science, Michigan State University, East Lansing, Michigan
3Department of Physiology, College of Human Medicine, Michigan State University, East Lansing, Michigan

Oncogene amplification is a well-known mechanism by which tumors evade treatment, occurring not only on linear chromosomes but also on extrachromosomal DNA (ecDNA). ecDNA is observed across various cancer types and is associated with intratumor heterogeneity, drug resistance, and poor clinical outcomes. Importantly, ecDNA exhibits high structural plasticity, as it can reversibly integrate into chromosomes as homogeneously staining regions (HSRs) under therapeutic stress, and re-emerge upon drug withdrawal, suggesting that integration may serve as a survival strategy. However, the precise mechanisms underlying integration remain unclear. Double-stranded breaks (DSBs) on ecDNA have been proposed to promote integration into chromosomes, which usually form ectopic HSRs. Treatment with the DNA-damaging agent doxorubicin increased the percentage of HSR-containing spreads in our commercially obtained ecDNA cancer lines. However, the presence of pre-existing HSR+ cells makes it unclear whether these HSRs arise from direct integration of ecDNA or from the drug-induced selective expansion of pre-existing HSR+ cells. To address this, we established pure ecDNA+ populations through single-cell cloning. In these lines, we detected rare spontaneous HSR formation after serial passages. While this confirms that ecDNA integration occurs de novo, the spontaneous nature of these events makes these cell lines a suboptimal model for mechanistic studies. Therefore, we are generating cell lines containing ecDNA using the CRISPR-C method. Targeted DSBs will then be introduced into ecDNA with Cas9, and the roles of specific priming and repair pathways in HSR formation will be tested by pharmacological inhibition or knockdown of repair proteins. Gene and protein expression profiles, together with drug sensitivity assays, will be compared across three groups: cells with ecDNA, cells with integrated ecDNA, and cells with pre-existing HSRs. Together, these studies will identify the molecular determinants that facilitate ecDNA integration and establish how this process alters therapeutic response.

Effects of Cocaine Intravenous Self-Administration on Brain White Matter

Z. FERNANDEZ 1,2 (Presenting), M. OSOWSKI 2, M. ROGERS 2, M. WILKERSON, I. TELUGUNTLA, M. HENEIN, S. PERET, A. LEE,  A. BENDER 3, N. SCHEEL 1,2, A.J. ROBISON 2*, C. QIAN 1,2*
Affiliations: 1 Dept. of Radiology, Michigan State Univ., East Lansing, MI; 2 Neuroscience Program, Michigan State Univ., East Lansing; 3 Cleveland Clinic Lou Ruvo Center for Brain Health, Las Vegas, NV

Addiction, affecting 48.5 million Americans, has been associated with changes in brain white matter (WM). Clinical diffusion tensor imaging (DTI) studies have shown reduced fractional anisotropy (FA) in the corpus callosum (CC) of individuals with cocaine use disorder (CUD), correlating with relapse, shorter lifespan, and poorer treatment outcomes. Rodent studies replicate these findings after experimenter-administered cocaine (4), but intravenous self-administration (IVSA)—a translational model where animals regulate intake—better reflects human drug use. While IVSA-DTI has been performed in rats, it has not yet been applied in mice, limiting genetic and mechanistic investigations. Here, we developed a mouse IVSA-DTI paradigm. Male C57BL/6 mice were implanted with jugular vascular access ports, trained for operant IVSA, and scanned across three timepoints using a 7T Bruker small-animal scanner. DTI data were processed and analyzed using a combination of MATLAB, MRtrix3, FSL, AFNI, and DSI Studio software. Cocaine-exposed mice showed FA and mean diffusivity (MD) changes in several addiction-related regions, including the pedunculopontine nucleus, amygdala, and hippocampus. Some alterations normalized with withdrawal, while others persisted or reversed, suggesting region-specific effects. Increased FA with decreased MD may indicate inflammation, whereas decreased FA with increased MD may reflect atrophy or cell death in these regions. FA changes were also detected in saline controls, potentially due to surgical effects, which will be addressed in future experiments. This study establishes IVSA-DTI in mice as a feasible and translational approach for investigating cocaine-induced WM adaptations. Leveraging transgenic models will enable future work to probe the circuit and molecular mechanisms underlying these structural changes.

Training strength modulates the temporal window of anisomycin induced disruption of contextual fear memory consolidation and reconsolidation:

J. D. Jaraczewski, H. Wang

¹Department of Physiology, Michigan State University

De novo protein synthesis is widely believed to be necessary for memory consolidation and reconsolidation, yet much of the evidence rests upon injection of protein-synthesis inhibitors such as anisomycin, which have many off-target effects. Recent calcium imaging and RNA quantification data from our lab indicates anisomycin increases neural activity, a process known to cause amnesia. Objective. To disentangle protein synthesis inhibition from its ancillary effects, we mapped the time course over which anisomycin disrupts contextual fear memories formed by either weak or strong training, during either consolidation or reconsolidation. Methods. A contextual fear conditioning protocol was used in which male 10-14 week old C57BL/6 mice received weak or strong context-shock pairings. For consolidation, animals were injected i.p. with saline or anisomycin at various time points post-conditioning. For reconsolidation, the same treatments followed a 3-min retrieval 24 h after training. Freezing, measured 24 hours after training and retrieval respectively, served as a measure of memory. Results. Consolidation: Weakly trained memories were disrupted when anisomycin was given up to 4 h but not 6 h  post-conditioning. Strongly trained fear memories were interfered with when anisomycin was administered immediately but not 1 h after conditioning. Reconsolidation: For weak training, anisomycin impaired memory at 1 h but not 3 h post-retrieval; strong memories were insensitive at all time points. Conclusions. Stronger conditioning shortens both consolidation and reconsolidation; reconsolidation is shorter than consolidation. Results align with previous time course data for chemogenetic excitation induced disruption providing evidence for an excitation based mechanism of disruption for PSIs. Future work will co-infuse anisomycin with chemogenetic silencing to determine if increased neuronal activity is required for PSI induced disruption. Clarifying this foundational question is essential for guiding translational strategies that aim to modify maladaptive memories in disorders such as PTSD.

Transient Receptor Potential Vanilloid Type 1 Channel mRNA is Expressed Throughout the Mouse Urinary Bladder

Marlene E. Masino,1,2 Emma Flood,2 Bhanuteja Madhu,2 Osvaldo J. Vega Rodriguez,2 Nathan R. Tykocki1,2

1Neuroscience Program, Michigan State University, East Lansing, MI USA; 2Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI USA

Transient receptor potential vanilloid type 1 (TRPV1) channels are implicated in nociception and may become sensitized during inflammation. In the urinary bladder, TRPV1 channels are distributed throughout afferent nerves and vascular smooth muscle cells, but their expression in other cell types is controversial. While germline reporter mice show TRPV1-expressing fibroblasts in the bladder wall, it is unknown if these cells can express TRPV1 channels after development. Additionally, sex differences in bladder TRPV1 channel expression is not thoroughly explored. We hypothesize that bladder fibroblasts from both male and female mice express Trpv1 mRNA after development.
Bladders from male and female C57Bl/6 mice were isolated, fixed, paraffin-embedded and sectioned onto glass slides. Thereafter, RNAscope was used to visualize mRNA expression for Trpv1 and the fibroblast marker Pdgfrα. Wheat germ agglutinin was also used to label cell membranes. Cells positive for Trpv1, Pdgfra, or both were then counted.
Trpv1 and Pdgfrα were co-expressed throughout the bladder of male and female mice. Trpv1 mRNA was also expressed throughout the bladder muscle and urothelium in cells where Pdgfrα mRNA was absent. As compared to females, male mice had significantly more cells that expressed both Trpv1 and Pdgfrα mRNA and fewer that expressed neither.
Together, these data suggest Trpv1 mRNA is expressed in multiple bladder cell types, including interstitial fibroblasts, past development. These data also reveal potential sex differences in bladder fibroblast populations that may account for differences in bladder sensation and remodeling in disease.
Funded by NIH R01-DK135696, R01-DK119615, and P01-HL152951.

Modulation of Fibroblast Growth Factor Receptor-1 Enhances Kynurenine Uptake in an In Vitro Model of Tamoxifen Resistant Estrogen Receptor Positive Breast Cancer

1,2Samantha Musso (Presenting), 1,2Romina Gonzalez Pons, 3Barbara Sankofi Mensah, 3Elizabeth Wellberg, and 1,4Jamie Bernard

1Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824
2Environmental and Integrative Toxicological Sciences Program, Michigan State University, East Lansing, MI 48824
3Department of Pathology, University of Oklahoma, Health Sciences Center, Oklahoma City, OK, 73104
4Department of Medicine, Michigan State University, East Lansing, MI 48824

Resistance to endocrine therapies like tamoxifen remains the major clinical factor contributing to mortality in estrogen receptor-positive breast cancer (BC).  An estimated 40% of women treated with tamoxifen will develop resistance and research indicates obesity can further reduce its effectiveness. We have identified fibroblast growth factor receptor-1 (FGFR-1) modulation to be critical mechanism connecting excess adiposity to aggressive tamoxifen-resistant BC.  Our untargeted metabolomics assay in a tamoxifen-resistant ER+ BC cell line (TAMR7) treated with FGF1 (subcutaneous adipose-secreted growth factor), significantly increased the concentration of only one metabolite: kynurenine (kyn).  An endogenous ligand of the aryl hydrocarbon receptor, kynurenine has been shown to modulate immune function.  Our published research indicating kyn is secreted from adipocytes and elevated in the serum of mice fed a high-fat diet is supported by epidemiological data illustrating circulating kyn levels are increased in women with obesity. Therefore, we hypothesize FGFR1 activation promotes growth in tamoxifen-resistance BC models through the enhanced uptake of kyn from the microenvironment. We treated MCF7 (sensitive) and TAMR7 cells with FGF1 and performed RNA-sequencing, flow cytometry, and soft agar colony formation assays. Via a novel flow cytometric monitoring system, kyn can be taken up from the microenvironment by MCF7 and TAMR7s.   Additionally, FGF1 increased the gene expression of SLC7A11, a kynurenine transporter, in TAMR7s. Low gene expression of the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase in MCF7s and TAMR7s suggests the increased intracellular kynurenine in the FGF1-treated TAMR7s was likely a result of increased uptake and not increased synthesis. Additionally, via soft agar assays, we demonstrated kyn increases colony size in a concentration-dependent manner in both MCF7s and TAMR7s. Uncovering the role of FGFR1-driven kyn uptake in models of tamoxifen resistance will provide a greater understanding of how diet and lifestyle may reduce the efficacy of endocrine therapies in ER+ breast cancer patients.

Ablations of the two subunits of the DNA-dependent protein kinase have distinct cellular impacts.

Giovanni Pascarella[1], Mariia Mikhova[2,3], Gargi Parkhi(Presenting)[4,5], Jared Godfrey[4], Joshua Heyza[3], Tomas Janovic[3], Andrew Olive[4], Piero Carninci[1,6], Jens Schmidt[3,7,8], and Katheryn Meek[4,5,8].

[1]RIKEN Center for Integrative Medical Sciences (IMS), Yokohama Japan; [2]Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, U.S.A; [3]Institute for Quantitative Health Sciences and Engineering, Michigan State University, East Lansing, MI, U.S.A; [4]Department of Microbiology, Genetics, & Immunology, Michigan State University, East Lansing, MI 48824, USA. [5]Department of Pathobiology & Diagnostic Investigation, Michigan State University, East Lansing, MI 48824, USA; [6]Human Technopole, Milan Italy; [7]Department of Obstetrics, Gynecology, and Reproductive Biology, Michigan State University, East Lansing MI, U.S.A; [8]correspondence.

Saturation of primate genomes with Alu repeat elements occurred at the prosimian/new-world monkey evolutionary juncture. Although Alu elements have clearly driven unique aspects of higher primate evolution, the presence of these elements also negatively impacts genomic stability. The appearance of high levels of Alu elements in higher primates precisely correlates with high, ubiquitous expression of the three polypeptides of the DNA-dependent protein kinase (Ku70 and Ku80 which as a heterodimer, function as the DNA-binding subunit for the catalytic subunit, DNA-PKcs). Here, we confirm that disruption of either of the Ku polypeptides in human cells results in rapid cell death; our analyses demonstrate that Ku ablation dramatically impacts RNA splicing (perhaps by binding intronic Alu elements). Cell death by Ku70 or Ku80 ablation can be substantially rescued by human Ku70 or Ku80, including Ku mutants that are defective in supporting non-homologous end-joining (NHEJ).  In contrast, cell death in Ku ablated cells is only partially rescued by prosimian Ku70 or Ku80 suggesting that Ku in higher primates has evolved distinctly from prosimians and non-primate mammals.  Finally, ablation of DNA-PKcs (which is not cell-lethal in cultured human cells) results in increased levels of Alu-non-allelic homologous recombination (NAHR). 

Comparing Models of Zone-Selective Steatosis Using Spatial Transcriptomics

Srijana Shrestha2, Jaide Mickel¹, Rance Nault¹

¹Department of Pharmacology and Toxicology, 2Genetics and Genome Sciences, Michigan State University

The liver is organized into lobules with zonated functional gradients along the periportal to centrilobular axis. Metabolic dysfunction-associated steatotic liver disease (MASLD), marked by the accumulation of lipid droplets in hepatocytes, is increasingly recognized as having many subtypes. In models of MASLD lipid accumulation shows zone selectivity, however, the mechanisms underlying zonated accumulation and its outcomes remain poorly understood.  We hypothesize zone-selective steatosis exhibits distinct patterns of spatially resolved gene expression which may be associated with MASLD subtypes.  To this end, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a model of periportal steatosis was compared to perfluorooctane sulfonate (PFOS) exposure and GAN (Gubra-Amylin NASH) diet centrilobular steatosis models. Spatial transcriptomics was performed on livers from male C57BL/6J mice fed a GAN or control diet, treated with PFOS (1 mg/kg/day) or 0.5% Tween-20 water and gavaged with TCDD (3 ug/kg) or sesame oil for 84 days. Spatial transcriptomics was performed on livers from male C57BL/6J mice fed a GAN or control diet, treated with PFOS (1 mg/kg/day) or normal water and gavaged with TCDD (3 ug/kg) or sesame oil for 84 days. An average of 576729 10 µm spots were detected per sample representing a total of 12558 genes. Cell deconvolution using RCTD found 11 expected cell types with periportal hepatocytes being the largest average proportion (~50.71%), followed by centrilobular hepatocytes (~19.62%) and Neutrophil (~6.72%). Spatially variable gene analysis (Moran’s I) confirmed localization of canonical markers of portal (Cyp2f2, Ass1, Arg1) and central (Glul, Cyp2e1, Oat) hepatocytes along with non-canonical genes including Saa1, Saa2, Gstm3, Serpina1e, and Cxcl9. Neighborhood clustering using STAGATE showed model specific patterns in spatial gene expression. These genes have the most distinct spatial distribution between models. The GAN diet (strongest) and PFOS showed a central zonation for canonical gene marker expression while TCDD was unclear. Together, these findings support the hypothesis that the initial site of fat accumulation may shape distinct molecular responses, which may underlie MASLD subtypes.

Meningeal resident macrophages cause migraine by disrupting meningeal lymphatic vessels

Jaewon Sim1 (presenter), Gregory Dussor2, Geoffroy Laumet1

1Department of Physiology, Michigan State University, 2School of Behavioral and Brain Science, University of Texas at Dallas

Migraine is a primary headache disorder and is ranked as one of the most common causes of neurological disease burden. The skull meninges are densely innervated with pain-sensing trigeminal nerves, making this tissue a key origin of headaches. However, we lack a clear understanding of the mechanisms that activate these nerves in the meninges and result in migraine. We used a well-established stress-induced headache model to elucidate the meningeal factors contributing to migraine. We observed persistent migraine-like behaviors in mice following repetitive restraint stress, such as facial mechanical hypersensitivity and photophobic grimacing. These behaviors were accompanied by cellular and structural changes in the dura mater. Dura mater from stressed mice exhibited an increased number of CD45low CD206high CD163high MHCIIlow border-associated macrophages (BAMs) and reduced diameters of meningeal lymphatic vessels (mLVs) in the transverse sinus. To study the mechanistic role of meningeal BAMs, we selectively and effectively depleted CD163⁺ BAMs from the dura mater using Pf4cre:DTR mice. Depleting these cells prior to stress exposure prevented migraine-like behaviors and restored mLV diameters. Meningeal BAMs are in close contact with lymphatic vessels, and vascular-associated macrophages are known to regulate vasculature by secreting matrix metalloproteinases (MMPs). In our study, treatment with an MMP inhibitor prevented stress-induced migraine-like symptoms and mLV degeneration. We are currently investigating whether BAMs upregulate MMPs in response to stress, thereby altering lymphatic vessels and contributing to migraine pathophysiology. The successful completion of this study will clarify the novel role of meningeal BAMs in driving migraine through dysregulation of mLVs and may lead to new therapeutic approaches.

CSF1R Inhibition as a Tool to Manipulate Microglia in Mouse Models of Aging and Amyloid-Enhanced Tauopathy

Lindsey N. Sime (1,2), Dylan Finneran, PhD (1); Brianna Jackman, MS (1), David Morgan, PhD (1); Marcia N. Gordon, PhD (1).

1 Department of Translational Neuroscience, Michigan State University
2 Neuroscience Graduate Program, Michigan State University

Microglia are implicated in both age-related neuroinflammation and the progression of Alzheimer’s disease (AD), but tools to manipulate their function in vivo remain limited. CSF1R inhibition using PLX5622 enables targeted depletion of microglia and has become a widely used method for probing their roles across diverse biological contexts. Here, we describe two distinct applications of this tool. In one study, we aimed to model microglial replicative senescence, a state increasingly linked to AD, but lacking a reliable in vivo model. Eight-month-old C57BL/6J mice undewent three or six cycles of PLX5622 and withdrawal. Microglia isolated after three cycles showed elevated Cdkn2a (p16Ink4a) expression, while microglia from six-cycle mice exhibited increased Cdkn2a and Cdkn1a (p21Cip1/Waf1) expression and shortened telomere length, consistent with a senescent-like state. In a separate study, we used chronic PLX5622 treatment to examine the role of microglia in amyloid-enhanced tauopathy. 11-month-old APP+PS1 mice received intravenous AAV-PHP.B.10-P301L-tau, and PLX5622 diet was initiated one month later and continued until study end. At 15 months, mice underwent behavioral testing, followed by sacrifice ad tissue collection. While histological and biochemical analyses are ongoing, preliminary data indicate that PLX5622 partially rescued tau-associated behavioral alterations in radial arm water maze and open field, despite no detectable changes in AT8-positive tau pathology or in soluble and insoluble Aβ and tau levels (ELISA). These findings suggest that microglial activity may contribute to cognitive outcomes through mechanisms beyond classical AD pathology, highlighting its relevance as a therapeutic target.

Distinct echinocandin responses of Candida albicans and Candida auris cell walls revealed by solid-state NMR

Kalpana Singh (Presenting)¹, Malitha C. Dickwella Widanage, Jizhou Li, Jayasubba Reddy Yarava, Faith J. Scott, Yifan Xu, Neil A. R. Gow, Frederic Mentink-Vigier, Ping Wang, Frederic Lamoth & Tuo Wang

1 Department of Chemistry, Michigan State University, East Lansing, MI, USA

2 Institute of Microbiology and Service of Infectious Diseases, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland

3 Infectious Diseases Service, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.

4 National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL, USA

5 Medical Research Council Centre for Medical Mycology at the University of Exeter, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter, EX4 4QD, UK

6 Departments of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA, USA

Invasive candidiasis affects 1.6 million people annually, with high mortality among immunocompromised and hospitalized patients. Echinocandins are frontline antifungals, but rising resistance limits their efficacy. Here, we show that Candida albicans and multidrug-resistant Candida auris share a conserved cell wall architecture yet differ markedly in their adaptive responses to echinocandins. Solid-state NMR reveals that both species possess a rigid inner layer of tightly associated chitin microfibrils and β-1,3-glucans, supported by a flexible matrix of β-1,6-glucans and additional β-1,3-glucans. Outer mannan fibrils rely on α−1,2-linked sidechains to maintain contact with the inner wall. In both species, caspofungin rigidifies β-1,6-glucans and mannan side chains, and reduces water permeability during β-1,3-glucan depletion. However, C. albicans undergoes wall thickening and alterations in chitin and glucan dynamics, whereas C. auris maintains integrity through β-1,6-glucan upregulation. Deletion of KRE6a, which encodes β−1,6-glucan synthase, reduces echinocandin susceptibility in C. auris, further highlighting β−1,6-glucan’s critical role in adaptive remodeling.