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Item Corrigendum: Uncovering Disease Mechanisms in a Novel Mouse Model Expressing Humanized APOEε4 and Trem2*R47H(Frontiers Media, 2022-02-07) Kotredes, Kevin P.; Oblak, Adrian; Pandey, Ravi S.; Lin, Peter Bor-Chian; Garceau, Dylan; Williams, Harriet; Uyar, Asli; O’Rourke, Rita; O’Rourke, Sarah; Ingraham, Cynthia; Bednarczyk, Daria; Belanger, Melisa; Cope, Zackary; Foley, Kate E.; Logsdon, Benjamin A.; Mangravite, Lara M.; Sukoff Rizzo, Stacey J.; Territo, Paul R.; Carter, Gregory W.; Sasner, Michael; Lamb, Bruce T.; Howell, Gareth R.; Pharmacology and Toxicology, School of MedicineAn author name was incorrectly spelled as “Daria Bednarycek”. The correct spelling is “Daria Bednarczyk”. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.Item tTARGIT AAVs mediate the sensitive and flexible manipulation of intersectional neuronal populations in mice(eLife Sciences, 2021-03-11) Sabatini, Paul V.; Wang, Jine; Rupp, Alan C.; Affinati, Alison H.; Flak, Jonathan N.; Li, Chien; Olson, David P.; Myers, Martin G.; Pharmacology and Toxicology, School of MedicineWhile Cre-dependent viral systems permit the manipulation of many neuron types, some cell populations cannot be targeted by a single DNA recombinase. Although the combined use of Flp and Cre recombinases can overcome this limitation, insufficient recombinase activity can reduce the efficacy of existing Cre+Flp-dependent viral systems. We developed a sensitive dual recombinase-activated viral approach: tTA-driven Recombinase-Guided Intersectional Targeting (tTARGIT) adeno-associated viruses (AAVs). tTARGIT AAVs utilize a Flp-dependent tetracycline transactivator (tTA) 'Driver' AAV and a tetracycline response element-driven, Cre-dependent 'Payload' AAV to express the transgene of interest. We employed this system in Slc17a6FlpO;LeprCre mice to manipulate LepRb neurons of the ventromedial hypothalamus (VMH; LepRbVMH neurons) while omitting neighboring LepRb populations. We defined the circuitry of LepRbVMH neurons and roles for these cells in the control of food intake and energy expenditure. Thus, the tTARGIT system mediates robust recombinase-sensitive transgene expression, permitting the precise manipulation of previously intractable neural populations.Item Sex-dependent, lateralized engagement of anterior insular cortex inputs to the dorsolateral striatum in binge alcohol drinking(eLife Sciences, 2024-08-28) Haggerty, David L.; Atwood, Brady K.; Pharmacology and Toxicology, School of MedicineHow does alcohol consumption alter synaptic transmission across time, and do these alcohol-induced neuroadaptations occur similarly in both male and female mice? Previously we identified that anterior insular cortex (AIC) projections to the dorsolateral striatum (DLS) are uniquely sensitive to alcohol-induced neuroadaptations in male, but not female mice, and play a role in governing binge alcohol consumption in male mice (Haggerty et al., 2022). Here, by using high-resolution behavior data paired with in-vivo fiber photometry, we show how similar levels of alcohol intake are achieved via different behavioral strategies across sexes, and how inter-drinking session thirst states predict future alcohol intakes in females, but not males. Furthermore, we show how presynaptic calcium activity recorded from AIC synaptic inputs in the DLS across 3 weeks of water consumption followed by 3 weeks of binge alcohol consumption changes across, fluid, time, sex, and brain circuit lateralization. By time-locking presynaptic calcium activity from AIC inputs to the DLS to peri-initiation of drinking events we also show that AIC inputs into the left DLS robustly encode binge alcohol intake behaviors relative to water consumption. These findings suggest a fluid-, sex-, and lateralization-dependent role for the engagement of AIC inputs into the DLS that encode binge alcohol consumption behaviors and further contextualize alcohol-induced neuroadaptations at AIC inputs to the DLS.Item Disruption of Plasmodium falciparum kinetochore proteins destabilises the nexus between the centrosome equivalent and the mitotic apparatus(Springer Nature, 2024-07-10) Li, Jiahong; Shami, Gerald J.; Liffner, Benjamin; Cho, Ellie; Braet, Filip; Duraisingh, Manoj T.; Absalon, Sabrina; Dixon, Matthew W. A.; Tilley, Leann; Pharmacology and Toxicology, School of MedicinePlasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, PfNDC80 and PfNuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites.Item mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency(American Society for Microbiology, 2024) Holmes, Michael J.; Bastos, Matheus S.; Dey, Vishakha; Severo, Vanessa; Wek, Ronald C.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of MedicineThe protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and latent cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5'-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogeneous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence. Importance: Toxoplasma gondii is an opportunistic pathogen important to global human and animal health. There are currently no chemotherapies targeting the encysted form of the parasite. Consequently, a better understanding of the mechanisms controlling encystation is required. Here we show that the mRNA cap-binding protein, eIF4E1, regulates the encystation process. Encysted parasites reduce eIF4E1 levels, and depletion of eIF4E1 decreases the translation of ribosome-associated machinery and drives Toxoplasma encystation. Together, these data reveal a new layer of mRNA translational control that regulates parasite encystation and latency.Item Sex-Dependent Synaptic Remodeling of the Somatosensory Cortex in Mice With Prenatal Methadone Exposure(Frontiers Media, 2022) Grecco, Gregory G.; Huang, Jui Yen; Muñoz, Braulio; Doud, Emma H.; Hines, Caliel D.; Gao, Yong; Rodriguez, Brooke; Mosley, Amber L.; Lu, Hui-Chen; Atwood, Brady K.; Pharmacology and Toxicology, School of MedicineRising opioid use among pregnant women has led to a growing population of neonates exposed to opioids during the prenatal period, but how opioids affect the developing brain remains to be fully understood. Animal models of prenatal opioid exposure have discovered deficits in somatosensory behavioral development that persist into adolescence suggesting opioid exposure induces long lasting neuroadaptations on somatosensory circuitry such as the primary somatosensory cortex (S1). Using a mouse model of prenatal methadone exposure (PME) that displays delays in somatosensory milestone development, we performed an un-biased multi-omics analysis and investigated synaptic functioning in the primary somatosensory cortex (S1), where touch and pain sensory inputs are received in the brain, of early adolescent PME offspring. PME was associated with numerous changes in protein and phosphopeptide abundances that differed considerably between sexes in the S1. Although prominent sex effects were discovered in the multi-omics assessment, functional enrichment analyses revealed the protein and phosphopeptide differences were associated with synapse-related cellular components and synaptic signaling-related biological processes, regardless of sex. Immunohistochemical analysis identified diminished GABAergic synapses in both layer 2/3 and 4 of PME offspring. These immunohistochemical and proteomic alterations were associated with functional consequences as layer 2/3 pyramidal neurons revealed reduced amplitudes and a lengthened decay constant of inhibitory postsynaptic currents. Lastly, in addition to reduced cortical thickness of the S1, cell-type marker analysis revealed reduced microglia density in the upper layer of the S1 that was primarily driven by PME females. Taken together, our studies show the lasting changes on synaptic function and microglia in S1 cortex caused by PME in a sex-dependent manner.Item Pharmacological Depletion of Fibrinogen Suppresses the Growth of Primary Tumors and Metastasis of Pancreatic Ductal Adenocarcinoma (PDAC)(2024-08) Chowdhury, Nayela Nabiha; Fishel, Melissa L.; Yeh, Elizabeth; Pollok, Karen; Manchanda, Naveen; Jerde, Travis J.PDAC is a highly metastatic disease often linked to the dysregulated activation of both coagulation and fibrinolytic systems. Clinically, patients show elevated plasma fibrinogen levels, particularly in those with distant metastasis. The presence of systemic fibrinogen plays a crucial role in shaping the complex tumor microenvironment characteristic of PDAC, evidenced by the excessive deposition of its active substrate, fibrin, in PDAC tumors. To investigate its contribution to disease progression, fibrinogen was significantly depleted from the TME in multiple PDAC patient-derived xenograft (PDX) models, and the impact on tumor growth and metastasis was followed. In an aggressive, metastatic orthotopic Pa03C model, Fib depletion using antisense oligonucleotide (ASO) treatment markedly decreased the size of primary pancreatic tumors and subsequent spontaneous metastasis to the liver. Following implantation of tumor chunks (PDX21) from a patient with high Fib staining, Fib ASO treatment significantly diminished growth of primary tumors. Using a third orthotopic model (PDX33) and lipid nanoparticle (LNP)-encapsulated-siRNA to deplete the fibrinogen--chain, tumor growth was significantly reduced compared to control. This confirms that Fib deposition within the tumor microenvironment (TME) is an important driver of disease progression. Mechanistically, global proteomics revealed a remarkable upregulation of matrisome and extracellular matrix (ECM)-associated proteins, indicating that the reduction in primary orthotopic Pa03C tumors was associated with fibrin-mediated TME remodeling. Loss of fibrin matrices led to enhanced collagen, laminin, fibronectin and emilin1 deposition, and increased recruitment of collagen-producing αSMA+ myofibroblasts. Notably, our data suggests that fibrinogen depletion altered the ECM composition, inducing TME remodeling which led to stiffer tumors that are less metastatic. To further interrogate the role of fibrinogen in mediating metastasis, we employed an experimental metastasis model to study the colonization of tumor cells in the liver in the absence of fibrinogen. In this model, fibrinogen depletion did not impede the colonization of Pa03C cells, suggesting that fibrinogen does not mediate the colonization of tumor cells in the cascade of events for liver metastasis and is likely involved in one of the other steps of metastasis. Collectively, our data showed that pharmacologically reducing systemic fibrinogen levels impeded tumor growth and metastasis by remodeling the TME.Item Aqueous VEGF-A Levels as a Liquid Biopsy Biomarker of Retinoblastoma Vitreous Seed Response to Therapy(Association for Research in Vision and Ophthalmology, 2024) Daniels, Anthony B.; Sishtla, Kamakshi L.; Bogan, Carley M.; Pierce, Janene M.; Chen, Sheau-Chiann; Xu, Liya; Berry, Jesse L.; Corson, Timothy W.; Pharmacology and Toxicology, School of MedicinePurpose: Regression of retinoblastoma vitreous seeds (VS) during intravitreal chemotherapy can be delayed, resulting in supernumerary injections. Similarly, VS relapse may not be clinically evident at first. A predictive biomarker of tumor regression and relapse could help guide real-time clinical decision making. Retinoblastoma is an oxygen-sensitive tumor; paradoxically, VS survive in the hypoxic vitreous. We hypothesized that VS elaborate pro-angiogenic cytokines. The purpose was to determine if pro-angiogenic cytokine signatures from aqueous humor could serve as a biomarker of VS response to treatment. Methods: Multiplex ELISA was performed on aqueous from rabbit eyes with human retinoblastoma VS xenografts to identify expressed proangiogenic cytokines and changes in aqueous cytokine levels during intravitreal treatment were determined. Confirmatory RNAscope in situ hybridization for VEGF-A was performed on human retinoblastoma tumor sections and VS xenografts from rabbits. For human eyes undergoing intravitreal chemotherapy, serial aqueous VEGF-A levels measured via VEGF-A-specific ELISA were compared to clinical response. Results: VEGF-A was highly expressed in human retinoblastoma VS in the xenograft model, and was the only proangiogenic cytokine that correlated with VS disease burden. In rabbits, aqueous VEGF-A levels decreased in response to therapy, consistent with quantitative VS reduction. In patients, aqueous VEGF-A levels associated with clinical changes in disease burden (regression, stability, or relapse), with changes in VEGF-A levels correlating with clinical response. Conclusions: Aqueous VEGF-A levels correlate with extent of retinoblastoma VS, suggesting that aqueous VEGF-A may serve as a predictive molecular biomarker of treatment response.Item The impact of abstinence from chronic alcohol consumption on the mouse striatal proteome: sex and subregion-specific differences(Frontiers Media, 2024-06-03) Duffus, Brittnie-Lee M.; Haggerty, David L.; Doud, Emma H.; Mosley, Amber L.; Yamamoto, Bryan K.; Atwood, Brady K.; Pharmacology and Toxicology, School of MedicineAlcohol misuse is the third leading preventable cause of death in the world. The World Health Organization currently estimates that 1 in 20 deaths are directly alcohol related. One of the ways in which consuming excessive levels of alcohol can both directly and indirectly affect human mortality and morbidity, is through chronic inflammation. Recently, studies have suggested a link between increased alcohol use and the incidence of neuroinflammatory-related diseases. However, the mechanism in which alcohol potentially influences neuroinflammatory processes is still being uncovered. We implemented an unbiased proteomics exploration of alcohol-induced changes in the striatum, with a specific emphasis on proteins related to inflammation. The striatum is a brain region that is critically involved with the progression of alcohol use disorder. Using mass spectrometry following voluntary alcohol self-administration in mice, we show that distinct protein abundances and signaling pathways in different subregions of the striatum are disrupted by chronic exposure to alcohol compared to water drinking control mice. Further, in mice that were allowed to experience abstinence from alcohol compared to mice that were non-abstinent, the overall proteome and signaling pathways showed additional differences, suggesting that the responses evoked by chronic alcohol exposure are dependent on alcohol use history. To our surprise we did not find that chronic alcohol drinking or abstinence altered protein abundance or pathways associated with inflammation, but rather affected proteins and pathways associated with neurodegeneration and metabolic, cellular organization, protein translation, and molecular transport processes. These outcomes suggest that in this drinking model, alcohol-induced neuroinflammation in the striatum is not a primary outcome controlling altered neurobehavioral function, but these changes are rather mediated by altered striatal neuronal structure and cellular health.Item Histone-Modifying Complexes Regulate Gene Expression Pertinent to the Differentiation of the Protozoan Parasite Toxoplasma gondii(Taylor & Francis, 2005) Saksouk, Nehmé; Bhatti, Micah M.; Kieffer, Sylvie; Smith, Aaron T.; Musset, Karine; Garin, Jérôme; Sullivan, William J., Jr.; Cesbron-Delauw, Marie-France; Hakimi, Mohamed-Ali; Pharmacology and Toxicology, School of MedicinePathogenic apicomplexan parasites like Toxoplasma and Plasmodium (malaria) have complex life cycles consisting of multiple stages. The ability to differentiate from one stage to another requires dramatic transcriptional changes, yet there is a paucity of transcription factors in these protozoa. In contrast, we show here that Toxoplasma possesses extensive chromatin remodeling machinery that modulates gene expression relevant to differentiation. We find that, as in other eukaryotes, histone acetylation and arginine methylation are marks of gene activation in Toxoplasma. We have identified mediators of these histone modifications, as well as a histone deacetylase (HDAC), and correlate their presence at target promoters in a stage-specific manner. We purified the first HDAC complex from apicomplexans, which contains novel components in addition to others previously reported in eukaryotes. A Toxoplasma orthologue of the arginine methyltransferase CARM1 appears to work in concert with the acetylase TgGCN5, which exhibits an unusual bias for H3 [K18] in vitro. Inhibition of TgCARM1 induces differentiation, showing that the parasite life cycle can be manipulated by interfering with epigenetic machinery. This may lead to new approaches for therapy against protozoal diseases and highlights Toxoplasma as an informative model to study the evolution of epigenetics in eukaryotic cells.