Browsing by Author "Perrin, Benjamin"

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    Analyses of the development and function of stem cell derived cells in neurodegenerative diseases
    (2022-12) Lavekar, Sailee Sham; Meyer, Jason; Canfield, Scott; Belecky-Adams, Teri; Mastracci, Teresa; Perrin, Benjamin
    Human pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some in vitro approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different in vitro systems that could help better mimic certain physiological systems that tend to not be mimicked by in vivo models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction. Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in in vivo models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using in vivo models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.
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    Biofilm and Virulence Regulation of the Cystic Fibrosis Associated Pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa
    (2020-05) Ramos-Hegazy, Layla; Anderson, Gregory; Perrin, Benjamin; Slayback-Barry, Denise
    Cystic fibrosis (CF) is a fatal, incurable genetic disease that affects over 30,000 people in the United States alone. People with this disease have a homozygous mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) which causes defects in chloride transport and leads to build up of mucus in the lungs and disruption of function in various organs. CF patients often suffer from chronic bacterial infections within the lungs, wherein the bacteria persist as a biofilm, leading to poor prognosis. Two of these pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, are often found in the lungs of patients with CF and are an increasing medical concerns due to their intrinsic antimicrobial resistance. Both species can readily form biofilms on biotic and abiotic surfaces such as intravascular devices, glass, plastic, and host tissue. Biofilm formation starts with bacterial attachment to a surface and/or adjacent cells, initiating the acute infection stage. Chronic, long-term infection involves subsequent or concurrent altered genetic regulation, including a downregulation of virulence factors, resulting in the bacteria committing to a sessile lifestyle, markedly different from the planktonic one. Many of these genetic switches from an acute to chronic lifestyle are due to pressures from the host immune system and lead to permanently mutated strains, most likely an adaptive strategy to evade host immune responses. Biofilms are extremely problematic in a clinical setting because they lead to nosocomial infections and persist inside the host causing long-term chronic infections due to their heightened tolerance to almost all antibiotics. Understanding the genetic networks governing biofilm initiation and maintenance would greatly reduce consequences for CF and other biofilm-related infections and could lead to the development of treatments and cures for affected patients. This study showed that in S. maltophilia, isogenic deletion of phosphoglycerate mutase (gpmA) and two chaperone-usher pilin subunits, S. maltophilia fimbrae-1 (smf-1) and cblA, lead to defects in attachment on abiotic surfaces and cystic fibrosis derived bronchial epithelial cells (CFBE). Furthermore, Δsmf-1 and ΔcblA showed defects in long-term biofilm formation, mimicking that of a chronic infection lifestyle, on abiotic surfaces and CFBE as well as stimulating less of an immune response through TNF-α production. This study also showed that in P. aeruginosa, the Type III secretion system (T3SS), an important virulence factor activated during the acute stage of infection, is downregulated when polB, a stress-induced alternate DNA polymerase, is overexpressed. This downregulation is due to post-transcriptional inhibition of the master regulatory protein, ExsA. Taken together, this project highlights important genes involved in the acute and chronic infection lifestyle and biofilm formation in S. maltophilia and genetic switches during the acute infection lifestyle in P. aeruginosa.
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    Characterization of TRPV4-mediated signaling pathways in an optimized human choroid plexus epithelial cell line
    (APS, 2022-12) Hulme, Louise; Hochstetler, Alexandra; Schwerk, Christian; Schroten, Horst; Ishikawa, Hiroshi; Tung, Chun-Yu; Perrin, Benjamin; Blazer-Yost, Bonnie; Biology, School of Science
    The objectives of these studies were twofold: 1) to characterize the human choroid plexus papilloma (HIBCPP) cell line as a model of the blood-cerebrospinal fluid barrier (BCSFB) via morphology, tightness, and polarization of transporters in choroid plexus epithelia (CPe), and 2) to utilize Ussing-style electrophysiology to elucidate signaling pathways associated with the activation of the transient receptor potential vanilloid 4 (TRPV4) channel involved in cerebrospinal fluid (CSF) secretion. RT-PCR was implemented to determine gene expression of cell fate markers, junctional complex proteins, and transporters of interest. Scanning electron microscopy and confocal three-dimensional renderings of cultures grown on permeable supports were utilized to delineate the morphology of the brush border, junctional complexes, and polarization of key transporters. Electrophysiology was used to understand and explore TRPV4-mediated signaling in the HIBCPP cell line, considering both short-circuit current (Isc) and conductance responses. HIBCPP cells grown under optimized culture conditions exhibited minimal multilayering, developed an intermediate resistance monolayer, retained differentiation properties, and expressed, and correctly localized, junctional proteins and native transporters. We found that activation of TRPV4 resulted in a robust, multiphasic change in electrogenic ion flux and increase in conductance accompanied by substantial fluid secretion. This response appears to be modulated by a number of different effectors, implicating phospholipase C (PLC), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K) in TRPV4-mediated ion flux. The HIBCPP cell line is a representative model of the human BCSFB, which can be utilized for studies of transporter function, intracellular signaling, and regulation of CSF production.
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    Developmental signaling pathways in adult energy homeostasis
    (2021-08) Antonellis, Patrick; Berbari, Nicolas; Baucum, A.J.; Adams, Andrew; Perrin, Benjamin
    Many signaling pathways which are classically understood for their roles in early development are also known to be involved in tissue maintenance and adult energy homeostasis. Furthermore, dysfunction of these signaling pathways results in human diseases such as cancer. An in depth understanding of how developmentally important signaling pathways function in the adult will provide mechanistic insights into disease and potential new therapeutic targets. Here in Chapter 1, the Wnt, fibroblast growth factor (FGF), and Hedgehog (Hh) signaling pathways are discussed and examples of their relevance in development, adult homeostasis, and disease are provided. Wnt signaling provides an example of this concept as it has well described roles during both development and adult metabolism. Work included in Chapter 2, investigates the regulation of adult energy homeostasis by a member of the endocrine FGF family, FGF19. The three endocrine FGFs, FGF19 (FGF15 in mice), FGF21, and FGF23 have well described roles in the regulation of metabolic processes in adults. While FGF23 is primarily involved in the regulation of phosphate and vitamin D homeostasis, FGF19 and FGF21 have shown similar pharmacological effects on whole body metabolism. Here, the importance of adaptive thermogenesis for the pharmacological action of FGF19 is explored. Using UCP1KO animals we show that whole-body thermogenesis is dispensable for body weight loss following FGF19 treatment. Finally, the potential involvement of Hh signaling in mediating the hyperphagia driven obesity observed in certain ciliopathies is explored in Chapter 3. Emerging evidence suggests cilia play an important role in the regulation of feeding behavior. In mammals, the hedgehog pathway is dependent on the primary cilium as an organizing center and defects in hedgehog signaling share some clinical symptoms of ciliopathies. Here, we characterized the expression of core pathway components in the adult hypothalamus. We show that neurons within specific nuclei important for regulation of feeding behavior express Hh ligand and members of its signaling pathway. We also demonstrate that the Hh pathway is transcriptionally upregulated in response to an overnight fast. This work provides an important foundation for understanding the functional role of Hh signaling in regulation of energy homeostasis. In its entirety, this work highlights the emerging clinical relevance of developmentally critical pathways in diseases associated with dysfunction of adult tissue homeostasis, such as obesity.
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    Hedgehog Signaling Regulates Apical Actin Morphology
    (Office of the Vice Chancellor for Research, 2016-04-08) Anderson, Matthew; Hege, Melissa; Berbari, Nicolas; Perrin, Benjamin
    Stereocilia are highly patterned actin based cell protrusions found on the apical surface of auditory hair cells. They are formed mainly from bundled filamentous actin and its associated actin cross-linking proteins. Interestingly, stereocilia develop around another cell appendage, the microtubule based kinocilium, which is the primary cilium for a hair cell. Primary cilia are found on most somatic cells and play a significant role in the regulation and proper transduction of the Hedgehog (Hh) pathway. In the current study, we are testing the hypothesis that Hh pathway activity can alter actin bundling and elongation. In support of this idea, ectopic activation or repression of Hh signaling changed the morphology of stereocilia in vivo. To further test our hypothesis, we used a CL4 porcine kidney epithelial cell culture system stably expressing the actin crosslinking protein ESPN fused to green fluorescent protein. These cells serve as an in vitro model of apical actin protrusions similar to mature stereocilia in vivo. We manipulated Hh signaling in these cells using both a genetic and a pharmacological approach. In the pharmacological approach, CL4 cells were treated with the hedgehog agonist (Purmophamine) and antagonist (Cyclopamine), at varying concentrations for 48 hours. Genetically, the Hh pathway was ectopically activated by overexpressing the transcription factor Gli1, Gli2, Gli3, and SmoA1 repressed by expressing Gli3R. Immunofluorescent (IF) and scanning electron microscopy (SEM) revealed that CL4 cells dramatically altered the apical actin structures under these conditions. In particular, activating Gli transcription decreased apical actin-based structures while antagonizing activity resulted in more actinbased protrusions. This data strongly supports the hypothesis that the Hh signaling pathway can regulate the actin cytoskeleton.
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    The mechanism of triglyceride partitioning – how the ANGPTL3-4-8 system of proteins orchestrates tissue energy distribution
    (2020-12) Pottanat, Thomas G.; Berbari, Nicolas; Konrad, Robert; Perrin, Benjamin; Skalnik, David
    The incidence of Metabolic Syndrome (MetS) is increasing worldwide and accompanied by elevated risks for cardiovascular disease (CVD) and other subsequent comorbidities. MetS is associated with increased circulating triglycerides. A key enzyme involved in triglyceride (TG) clearance is lipoprotein lipase (LPL) whose activity is modulated by a variety of factors. Recent literature has identified the importance of angiopoietin-like proteins (ANGPTL) as regulators of LPL activity and has hypothesized a model in which three of these proteins interact with LPL to regulate the partitioning of TG metabolism from adipose to skeletal muscle. The work detailed in this dissertation adds to the model of ANGPTL regulation of LPL by establishing how ANGPTL8 modulates the ability of ANGPTL3 and ANGPTL4 to inhibit LPL activity in the bloodstream and localized environments, respectively. In the updated model, elevated insulin concentrations result in increased hepatic ANGPTL3/8 secretion and increased ANGPTL4/8 in adipose tissue. ANGPTL3/8 works as an endocrine molecule to inhibit skeletal muscle LPL from hydrolyzing circulating TG. Simultaneously, ANGPTL4/8 works in a paracrine mechanism to bind LPL on the endothelial vasculature adjacent to adipose tissue to alleviate ANGPTL4-mediated LPL inhibition and also prevent ANGPTL3/8 inhibition of localized LPL. Thus, in the postprandial state free fatty acids (FFA) from the hydrolysis of TG are directed into adipocytes for storage. Under fasting conditions, ANGPTL8 production is decreased in adipocytes and hepatocytes. This decreased production results in diminished ANGPTL4/8 and ANGPTL3/8 secretion from their respective tissues. As a result, ANGPTL4 inhibits adipocyte localized LPL activity while ANGPTL3 at physiological concentrations has minimal effect on LPL activity. Furthermore, any ANGPTL3/8 which is produced has its LPL-inhibitory ability diminished by the circulating apolipoprotein ApoA5. LPL is more active in skeletal muscle compared to adipose tissue where energy is shunted towards utilization in the muscle and away from storage in adipose tissue. A complete understanding of LPL regulation by ANGPTL proteins can potentially provide therapeutics targets for MetS.
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    The role of ADF and cofilin in auditory sensory cell development
    (2020-12) McGrath, Jamis; Perrin, Benjamin; Cummins, Theodore; Belecky-Adams, Teri; Balakrishnan, Lata; Zhao, Bo
    Our ability to hear relies on sensory cells found in the inner ear that transduce sound into biological signals. Microvilli-like protrusions called stereocilia are bundled on the apical surfaces of these cells and allow them to respond to sound-evoked vibrations. The architecture of the stereocilia bundle is highly patterned to ensure normal hearing. Filaments of polymerized actin proteins are bundled in parallel into large cylindrical structures that define the dimensions of stereocilia. This network is then anchored to the cell by inserting into another actin-based structure called the cuticular plate, which forms a gel-like structure and facilitates the mechanical properties of the bundle. The shape of the bundle is determined through tissue-level and intrinsic polarization signaling pathways. Auditory brainstem-evoked response testing, immunofluorescence imaging, scanning electron microscopy, and biochemical labeling techniques were used to study how the ADF/cofilin family of actin filament severing and depolymerizing proteins contributes to the development of the stereocilia bundle. Loss of these proteins disrupts the normal bundle patterning process, changes the lengths and widths of stereocilia, and alters the regulation of filament ends near the ion channel at stereocilia tips that is responsible for mechanotransduction. The activity of this channel regulates ADF/cofilins and the actin at stereocilia tips. Aberrant actin growth in actin networks beneath the stereocilia bundle influences the bundle patterning process, causes dysmorphic bundles to form. This work identifies that ADF/cofilins are necessary during auditory sensory cell development to facilitate normal bundle patterning and establishes this protein family as a molecular link between mechanotransduction and stereocilia bundle maturation.