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Browsing by Author "Baucum, AJ"
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Item 20S proteasome assembly: alternative pathways and complexes(2017) Hammack, Lindsay J.; Kusmierczyk, Andrew R.; Mosley, Amber L.; Randall, Stephen; Baucum, AJThe ubiquitin-proteasome system is responsible for the targeted degradation of proteins within the cell. The 26S proteasome, which is the protease of this system, is a high molecular weight complex consisting of 33 subunits that arrange to form two smaller complexes the 19S regulatory particle (RP) and the 20S core particle (CP). The 19S RP can bind one or both ends of the 20S CP and is responsible for recognizing the ubiquitinated substrates. After recognition, the 19S RP will subsequently deubiquitinate, unfold, and translocate the substrates into the proteolytic 20S CP. The 20S CP consists of seven unique alpha and seven unique beta subunits that arrange into four stacked rings, with two alpha rings capping two beta rings. Assembly of the alpha(1-7)beta(1-7)beta(1-7)alpha(1-7) structure begins with the formation of an alpha ring and proceeds through specific assembly intermediates. This process is assisted by assembly chaperone proteins that promote on pathway interactions to efficiently construct the 20S CP. In this dissertation, three new findings are described which further characterize the proteasome assembly pathway. First, novel non-canonical complexes comprised of proteasome subunit alpha4 were identified in vivo, revealing proteasome subunits can assemble into complexes outside of the proteasome. Second, Hsp70 proteins, Ssa1/2, were shown to assist in the assembly of 20S CPs, adding to the growing list of proteins guiding proteasome assembly. Third, a novel complex was identified which is believed to represent a new proteasome assembly intermediate.Item Axonal Outgrowth and Pathfinding of Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells(2020-08) Fligor, Clarisse; Meyer, Jason; Marrs, James; Belecky-Adams, Teri; Jones, Kathryn; Baucum, AJRetinal ganglion cells (RGCs) serve as a vital connection between the eye and the brain with damage to their axons resulting in loss of vision and/or blindness. Reti- nal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, providing a valuable model of RGC development in vitro. The working hypothesis of these studies is that hPSC-derived RGCs are capable of extensive outgrowth and display target specificity and pathfinding abilities. Initial efforts focused on charac- terizing RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner express- ing a compliment of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful to investigate and model the extensive axonal outgrowth necessary to reach post-synaptic targets. As such, additional efforts aimed to elucidate factors promoting axonal outgrowth. Results demonstrated significant enhancement of axonal outgrowth through modulation of both substrate composi- tion and growth factor signaling. Furthermore, RGCs possessed guidance receptors that are essential in influencing outgrowth and pathfinding. Subsequently, to de- termine target specificity, aggregates of hPSC-derived RGCs were co-cultured with explants of mouse lateral geniculate nucleus (LGN), the primary post-synaptic target of RGCs. Axonal outgrowth was enhanced in the presence of LGN, and RGCs dis- played recognition of appropriate targets, with the longest neurites projecting towards LGN explants compared to control explants or RGCs grown alone. Generated from xvii the fusion of regionally-patterned organoids, assembloids model projections between distinct regions of the nervous system. Therefore, final efforts of these studies focused upon the generation of retinocortical assembloids in order to model the long-distance outgrowth characteristic of RGCs. RGCs displayed extensive axonal outgrowth into cortical organoids, with the ability to respond to environmental cues. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC develop- ment, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing outgrowth as well as modeling long-distance projections and pathfinding abilities.Item Defining the Role of Lysine Acetylation in Regulating the Fidelity of DNA Synthesis(2020-12) Ononye, Onyekachi Ebelechukwu; Balakrishnan, Lata; Watson, John; Baucum, AJ; Turchi, John; Bochman, MatthewAccurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol α), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase α tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol α tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) Saccharomyces cerevisiae helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.Item Increased Excitability of Pyramidal Neurons in the Secondary Motor Cortex Enhances Cocaine-Seeking(2023-09) Huang, Donald; Atwood, Brady K.; Baucum, AJ; Hopf, Woody; Ma, Yao-Ying; Sheets, Patrick L.Cocaine addiction is a brain disorder characterized by chronic relapse. Although drug-seeking behaviors have been recognized to be associated with relapse, the role of the motor cortex, including the primary (M1) and secondary (M2) motor cortex, which are functionally important mediators of complex behaviors remains unclear in addiction. Here we use a rat cocaine intravenous self-administration (IVSA) model to investigate the intrinsic excitability of pyramidal neurons in the medial prefrontal cortices and motor cortices during withdrawal. Cocaine IVSA-trained rats performed a cocaine-seeking test on withdrawal day (WD) 1 or WD 45. Relative to WD 1 an increase in cocaineseeking was detected on WD 45. Whole-cell patch clamp recordings revealed an increase in intrinsic excitability in pyramidal neurons in Layer 2 of the secondary motor cortex (M2-L2) in cocaine trained rats on WD 45. Using a pharmacological approach, bath application of GABAA receptor agonist, muscimol, dosedependently (0.1 mM, 0.3 mM, and 1.0 mM) decreased the excitability of M2-L2 pyramidal neurons in cocaine IVSA-trained rats on WD 45. Pharmacological inactivation of M2-L2 by bilateral intra-M2 injection of muscimol (324 ng/1.0 μl) attenuated cocaine-seeking on WD 45. A chemogenetic approach was used to validate that M2-L2 pyramidal neurons play a contributing role in the increase in cocaine-seeking, a microinjection of rAAV5-CaMKIIa-hM4di-mCherry was performed to express Gi-DREADD receptors on M2-L2 pyramidal neurons. Activating Gi-DREADD with an intraperitoneal injection of compound 21 on WD 45 attenuated cocaine-seeking. To elucidate the mechanism that contributes to the increased excitability of M2-L2 pyramidal neurons, an analysis of the action potential kinetics revealed that calcium-activated small conductance potassium (SK) channel-mediated medium afterhyperpolarization amplitude decreased in cocaine vs. saline IVSA-trained rats on WD 45. SK channel activation by 1-EBIO (300 μm) increased the medium afterhyperpolarization amplitude and decreased the excitability of M2-L2 pyramidal neurons in cocaine IVSA-trained rats. Furthermore, intra-M2 injection of 1-EBIO on WD 45 attenuated cocaine-seeking. These experiments suggest that cocaine IVSA-training-induced persistent changes in M2-L2 pyramidal neurons' intrinsic excitability contributes to enhanced cocaine-seeking. Our results provide evidence targeting the SK channels in the superficial layer for M2 could be an important therapeutic approach for preventing cocaine relapse.Item Investigating TRPV4 Signaling in Choroid Plexus Culture Models(2022-05) Hulme, Louise; Blazer-Yost, Bonnie; Baucum, AJ; Mastracci, Teresa; Belecky-Adams, TeriHydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear. Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative in vitro model is critical to elucidate the signaling pathways orchestrating CSF production in the CP. This research aims to characterize an in vitro culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.