Browsing by Author "Baucum, AJ"

Now showing 1 - 8 of 8
  • Thumbnail Image
    Item
    20S proteasome assembly: alternative pathways and complexes
    (2017) Hammack, Lindsay J.; Kusmierczyk, Andrew R.; Mosley, Amber L.; Randall, Stephen; Baucum, AJ
    The 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.
  • Thumbnail Image
    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, AJ
    Retinal 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.
  • Thumbnail Image
    Item
    Converging Effects of Chronic Pain and Binge Alcohol Consumption on Corticostriatal Neurons and the Effects of Acute Alcohol Exposure on the Medial Prefrontal Cortex
    (2024-07) Yin, Yuexi; Atwood, Brady K.; Baucum, AJ; Hopf, Woody; McKinzie, David L.; Sheets, Patrick L.
    Chronic pain and alcohol use disorder (AUD) are highly comorbid, but whether the two conditions share common brain pathways is unclear. Prior work shows that the anterior insular cortex (AIC) is involved in both chronic pain and alcohol use disorder. However, circuit-specific changes elicited by the combination of pain and alcohol use remain understudied. The goal of this work was to elucidate the converging effects of binge alcohol consumption and chronic pain on AIC neurons that send projections to the dorsolateral striatum (DLS). Here, we used the Drinking-in-the-Dark paradigm to model binge-like alcohol drinking in mice that underwent spared nerve injury (SNI). We found that SNI male mice with no prior alcohol exposure consumed less alcohol compared to sham mice. Electrophysiological analyses showed that AIC-DLS neurons from SNI-alcohol male mice displayed increased neuronal excitability and increased frequency of miniature excitatory postsynaptic currents. However, mice exposed to alcohol prior to SNI consumed similar amounts of alcohol compared to sham mice following SNI. Together, our data suggest that the pain and alcohol interaction can sensitize the AIC-DLS circuit in mice, which may be critical in understanding how chronic pain alters motivated behaviors associated with alcohol. My second goal was to assess the acute pharmacological effects of alcohol on prodynorphin-expressing neurons in the prelimbic cortex (PLPdyn+), a subregion of the medial prefrontal cortex (mPFC). Kappa opioid receptor (KOR) system dysregulation contributes to alcohol addiction. Prodynorphin (Pdyn) is the precursor peptide to the endogenous opioid ligand for KORs. Early studies demonstrated that acute alcohol exposure elevates Pdyn mRNA expression in the mPFC. However, its functional effects on Pdyn-expressing neurons are not known. Here, we used whole-cell patch-clamp electrophysiology in acute brain slices and glutamate-uncaging via laser scanning photo to map local excitatory and inhibitory inputs onto PL neurons. We found that acute alcohol increases local inhibitory inputs to both layer 2/3 PLPdyn+ and PLPdyn- neurons but has no effect on excitatory inputs. Under untreated conditions, PLPdyn+ neurons show stronger local excitatory inputs compared to PLPdyn- neurons. Overall, these data suggest that acute alcohol intoxication inhibits intracortical circuit of PL neurons regardless of neuronal subtypes.
  • Thumbnail Image
    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, Matthew
    Accurate 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.
  • Thumbnail Image
    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.
  • Thumbnail Image
    Item
    Investigating TRPV4 Signaling in Choroid Plexus Culture Models
    (2022-05) Hulme, Louise; Blazer-Yost, Bonnie; Baucum, AJ; Mastracci, Teresa; Belecky-Adams, Teri
    Hydrocephalus 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.
  • Thumbnail Image
    Item
    The APOE Pathway as a Modulator of Amyloid Pathology in Alzheimer's Disease Models
    (2025-03) Tate, Mason Douglas; Baucum, AJ; Kim, Jungsu; Bissel, Stephanie J.; Lasagna Reeves, Cristian A.; Oblak, Adrian L.
    Alzheimer’s disease (AD) is characterized by the accumulation of beta-amyloid (Aβ) peptides and amyloid plaque deposition. The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for sporadic AD, with apoE protein crucial for brain lipid transport. ATP-binding cassette subfamily A member 1 (ABCA1), another risk gene, loads lipids onto apoE, highlighting the importance of lipid homeostasis in AD. MicroRNA-33 regulates the expression of ABCA1 and apoE lipidation, although its effect on amyloid pathology is unknown. Additionally, apoE variants can modulate AD risk. The apoEε4R251G variant eliminates the increased risk associated with the APOEε4 allele. This variant is located within the lipid binding domain of apoE, however its roles in lipid homeostasis and amyloid pathology remain unexplored. This dissertation investigates the role of apoE in amyloid pathology. We first used microRNA-33 knockout mice within an amyloidosis mouse model to determine if increased ABCA1 and apoE lipidation affect amyloid pathology. We demonstrate that deleting microRNA-33 reduced Aβ levels and plaque deposition. Through our multi-omics approach, we identified that microRNA-33 regulates microglial function, and mechanistically confirmed in vitro that inhibition of microRNA-33 increased microglial migration and Aβ phagocytosis. We next explored if the astrocyte-specific deletion of microRNA-33 could similarly reduce amyloid pathology. While the loss of microRNA-33 in astrocytes increased ABCA1 levels, we did not observe an increase in apoE lipidation. Furthermore, the astrocyte-specific deletion of microRNA-33 did not reduce amyloid pathology to the extent seen in the whole-body knockouts, suggesting a critical role for microglial microRNA-33 or a synergistic effect across cell types. Finally, we investigated if the astrocytic expression of the novel R251G apoE variant modulated apoE lipid pathways and amyloid pathology in an amyloidosis mouse model. We show that apoEε4R251G exhibits increased lipid binding compared to apoEε4. Additionally, the R251G variant reduced levels of Aβ and plaque deposition. Furthermore, astrocytes expressing apoEε4R251G colocalized more around plaques compared to apoEε4 mice, suggesting that astrocytes might be influencing the changes observed in amyloid pathology. Collectively, our results highlight the role of apoE lipid homeostasis in AD and potential therapeutic targets that can modulate apoE function and mitigate amyloid pathology.
  • Thumbnail Image
    Item
    The Impact of Abstinence from Chronic Alcohol Consumption on the Mouse Striatal Proteome: Sex and Subregion-Specific Differences
    (2024-10) Duffus, Brittnie-lee Marie; Atwood, Brady; Oblak, Adrian; Mao, Yao-Ying; Baucum, AJ; Fischer, Kathryn
    Alcohol 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.