Medical Neuroscience Department Theses and Dissertations
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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.Item The Role of TREM2 and the Responses Mediated by Galectin-3 During Age-Related Myelin Degeneration(2025-03) McCray, Tyler Jacob; Oblak, Adrian L.; Bissel, Stephanie J.; Lahiri, Debomoy K.; Lamb, Bruce T.; McKinzie, David L.Aging is the greatest known risk factor for various neurodegenerative diseases. Myelin degeneration is an early pathological indicator of these diseases and normal part of aging; albeit, to a lesser extent. Despite this, little is known about how age-related degeneration could contribute to and impact development of neurodegenerative disease. Microglia participate in a variety of white matter events from demyelination to remyelination. The microglial innate immune receptor triggering receptor expressed on myeloid cells 2 (TREM2) has been implicated in regulating (de)myelination. We found in response to demyelination, TREM2 is required for large volumes of myelin debris and during extended periods of phagocytosis. In addition to lysosomal regulation, we showed TREM2 can modify the ER stress response prior to overt myelin debris preventing early microglial dysfunction. We found TREM2 is necessary for remyelination by recruiting reparative glia and mediating signaling that promotes OPC differentiation/maturation. One of the signaling factors involved, the β-galactosidase-binding protein galectin-3 (gal-3), was recently identified as a ligand for TREM2, however little is known about this interaction in the context of aging or neurodegenerative disease. Treating microglia with a pharmacological gal-3 inhibitor, we found overlapping functional deficits with Trem2-deficient microglia during myelin phagocytosis. These shared deficits included impaired myelin uptake, altered lysosomal function, ER stress, and lipid droplet accumulation that were rescued inTrem2-deficient microglia with the addition of recombinant gal-3. RNA-seq analyses revealed common genes and pathways affected that importantly included genes associated with the integrated stress response. Taken together, these data suggest Gal-3 mediates and throttles the TREM2-dependent stress response during age-related myelin degeneration. Further, it provides support for targeting TREM2 function early to augment reparative signaling preventing overt debris accumulation and/or promoting gal-3 to alleviate stress pathways that can lead to premature microglial dysfunction and onset of pathology.Item The Impact of INPP5D on Microglia Response to Tau Pathology in Alzheimer's Disease(2025-02) Soni, Dishaben Miteshkumar; Truitt, William A.; Oblak, Adrian L.; Lasagna-Reeves, Cristian; Bissel, Stephanie J.; Chu, ShaoyouAlzheimer’s Disease (AD), the most prevalent form of dementia, is neuropathologically defined by the extracellular buildup of amyloid-beta (Aβ) plaques, the formation of intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein, and progressive neuronal degeneration, ultimately leading to cognitive decline. Genetic studies have identified immune-related risk genes linked to AD, underscoring the regulatory role of microglia in AD pathogenesis. Among these genes, INPP5D, which is exclusively expressed by microglia in the brain, has been associated with an increased risk for AD. Elevated INPP5D expression in microglia correlates with amyloid-plaque burden in human AD brain tissue, and studies indicate that INPP5D deficiency modulates amyloid pathology, with effects differing by disease stage and model system. While INPP5D modulation has been shown to impact amyloid pathology, its influence on tau pathology remains largely unexplored. This dissertation seeks to illuminate the role of INPP5D in tau pathogenesis. Our initial studies demonstrated a positive correlation between INPP5D expression and tau-seeding activity in human AD brain samples. Likewise, we observed increased INPP5D expression associated with phospho-tau AT8 levels in PS19 mice, indicating a significant link between INPP5D and tau pathology. Building on these findings, we explored the effect of Inpp5d haplodeficiency on tau pathogenesis in PS19 mice, revealing that Inpp5d haplodeficiency recovered motor functions, mitigated tau pathology, lowered proinflammatory cytokine levels and altered microglial morphology without affecting the overall cellular composition. Transcriptomic analysis also showed the upregulation of genes involved in cell migration, immune response, angiogenesis, and wound healing. These results highlight a complex interplay between Inpp5d, tau pathology, and behavioral outcomes, supporting Inpp5d’s involvement in tau pathogenesis. To explore this further, we treated primary microglia isolated from Wildtype, Inpp5d+/-, and Inpp5d-/- mice with recombinant mutant tau-preformed fibrils and insolubletau extracted from PS19 mice brains. Our results revealed increased tau uptake in Inpp5d+/- and Inpp5d-/- microglia, suggesting that Inpp5d modulation enhances tau uptake, potentially influencing disease progression through altered microglial response. While further research is needed to clarify the mechanisms through which INPP5D influences tau pathogenesis, our findings highlight INPP5D as a promising therapeutic target for modulating tau pathology and improving microglial function in AD.Item The Role of Homeostatic Plasticity in Post-Traumatic Epilepsy(2024-12) Moore, Allison Chandler; Cummins, Theodore R.; Yoder, Kamen K.; Truitt, William A.Traumatic brain injury (TBI) can cause post-traumatic epilepsy (PTE), a condition characterized by chronic spontaneous seizures via aberrant hyperexcitability. Homeostatic plasticity is a phenomenon in which neurons adjust their neurotransmission in response to an imposed decrease or increase in activity to maintain a stable level of firing. It is possible that a compensatory response to TBI-induced loss of activity may lead to pathological hyperexcitability that results in PTE. We hypothesized that enhancing activity after TBI could treat PTE by suppressing the putative compensatory response. Using a neocortical (undercut) model of PTE, we tested the hypotheses that homeostatic plasticity contributes to PTE and that enhancing neuronal activity will mitigate PTE. Two sets of experiments were conducted. First, cortical pyramidal neuron activity was imaged in adult Thy1-GCaMP6 transgenic mice with undercut injury using in vivo two-photon microscopy multiple times for four weeks. Frequency of calcium transients of individual neurons was analyzed with a custom Matlab script (developed by A. Moore) and ImageJ. Graph theory metrics were applied to correlated time course data to quantify changes in network metrics over time and between the undercut and sham surgery mice. Second, to determine the effect of activity enhancement on PTE, undercut Thy1-Channelrhodopsin 2 transgenic mice were assigned to one of three conditions: no treatment, daily intraperitoneal injection of D-cycloserine (DCS), or optogenetic stimulation for 10 days. Post-treatment, continuous wireless electroencephalography (EEG) recordings monitored spontaneous seizure activity for two weeks. Seizure susceptibility was measured using the pentylenetetrazol (PTZ) test. Seizure frequency was determined manually after applying standard signal processing methods. The feasibility of machine learning algorithms to identify seizures was explored. The project underscores the need for modern standardized and objective analyses of PTE in rodent models. Our data suggest that local connectivity in layers II/III pyramidal neurons exhibits small world network architecture during the latent period (weeks 1-4 after undercut injury) and that enhancing cortical activity with DCS or LED 4-6 weeks after undercut injury can reduce spontaneous seizure behaviors and paroxysmal EEG features associated with different seizure types. This work provides the foundation for future therapies for PTE that activate excitatory cortical neurons.Item Translational Responses of Motor Neurons to Neurodegeneration in ALS Identifies FGF21 as a Critical Myogenic Regulatory Factor That Slows Disease Progression(2021-12) Stansberry, Wesley Michael; Oblak, Adrian; Landreth, Gary; Lasagna-Reeves, Cristian; Pierchala, Brian; Walker, ChandlerThe neuromuscular junction (NMJ) is a chemical synapse that is the site of skeletal muscle innervation by spinal motor neurons and the maintenance of the NMJ is critical for preserving musculoskeletal homeostasis. Under normal physiological conditions, spinal motor neurons have significant regenerative potential and can regrow axons in response to peripheral nerve injury. In diseases such as amyotrophic lateral sclerosis (ALS), the NMJ is dismantled and motor neurons selectively degenerate resulting in progressive muscle wasting and eventual fatal paralysis. Interestingly, some motor neurons are more resistant to degeneration, with slow motor neurons persisting for longer and, in some cases, reinnervating fast, vacant NMJ endplates, underscoring the vital role of motor neurons in supporting skeletal muscle in disease states. In this dissertation we explore the role of motor neurons in skeletal muscle maintenance in ALS. We adapted the RiboTag methodology developed by Sanz et al. to perform ribosomal profiling of motor neurons in two mouse models of ALS. In chapter two we evaluated the translatome of spinal motor neurons in the Ubqln2P497S proteostasis model. The most significant finding from this study was the dramatic downregulation of muscle-related transcripts in motor neuron cell bodies in ALS, raising the possibility of motor neurons translating mRNAs previously thought to be muscle cell-type specific in direct support of the skeletal muscles they innervate. In chapter three, another RiboTag screen comparing a sciatic nerve crush model of acute injury and the Sod1G93A ALS model identified Fgf21, a metabolic and stress-inducible hormone, as one of the most upregulated ALS-specific transcripts. Transgenic mouse models where Fgf21 is conditionally knocked out in Sod1G93A motor neurons showed reduced motor neuron survival and NMJ innervation. Behavioral and survival trials with Sod1G93A mice showed a dramatic reduction in locomotion and lifespan when Fgf21 was conditionally knocked out of motor neurons. Taken together, these data suggest Fgf21 functions in a neuroprotective capacity in ALS pathology. Here we evaluate the functions of Fgf21 and the mechanisms by which it promotes motor neuron survival and skeletal muscle innervation and metabolism.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.Item Modulation of Intralocular Pressure by Tuning Transcriptional Control of Lipid Synthesis(2024-06) Wang, Ting; Morral, Nuria; Pattabiraman, Padmanabhan; Corson, Timothy W.; Landreth, Gary E.; Perrin, Benjamin J.Glaucoma is an age-related optic neuropathy and is one of the leading causes of irreversible blindness. Primary open-angle glaucoma (POAG) is the predominant subtype of glaucoma. Elevated intraocular pressure (IOP) is a major risk factor for POAG and lowering IOP is the most effective therapeutic strategy. IOP is maintained by the balance of aqueous humor (AH) generation by the ciliary body and drainage by conventional outflow pathway including trabecular meshwork (TM). TM is a highly contractile and mechanosensitive tissue, and its contractility regulated by the actin cytoskeleton and extracellular matrix (ECM) is directly related to IOP regulation. Using multiomics analysis in human TM (HTM) cells, I identified that mechanical stretch caused the activation of sterol regulatory element binding proteins (SREBPs) related-lipid biogenesis pathways. Further, using immunofluorescence, and constitutive activation of each SREBP isoform, I discovered the mechanosensing role of SREBPs in HTM cells and mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo resulted in significant IOP lowering. Conversely, significantly elevated IOP was observed after using the pharmacological activator of SREBPs by clozapine and constitutive activation of SREBPs ex vivo and in vivo, respectively. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways and phospholipids, cholesterol, and triglyceride levels. The increased lipid biogenesis was found after constitutive activation of SREBP isoforms in HTM cells but with slightly different effects between each isoform. Further, I showed that fatostatin mitigated actin polymerization machinery and stabilization, and identified that SREBPs activation is a critical regulator of ECM engagement to the matrix sites. Lastly, I identified that cholesterol levels play an important role in regulating actin polymerization, focal adhesion formation, cell-ECM interactions, and membrane tension in HTM cells. Therefore, we have established the direct connection between cholesterol and TM contractility. Overall, I postulate that lowering de novo lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics.Item The Role of the Lung-Brain Axis in the Ozone-Impaired Amyloid Associated Astrocytic and Vascular Phenotype(2024-06) Ahmed, Chandrama; Oblak, Adrian; Block, Michelle; Baucum, A. J.; Bissel, Stephanie J.; Nass, Richard M.Air pollution has been associated with an increased risk of Alzheimer’s Disease (AD). Studies show ozone (O3), a major component of urban air pollution, can exacerbate amyloid pathology. However, O3 reacts in its entirety with lung epithelial lining after inhalation, hence does not translocate to brain. Studies have implicated the lung−brain axis in O3 induced central nervous system (CNS) pathology. However, the mechanistic underpinnings of its role in amyloid pathology is obscure. Here, we explored the impact of O3 on the astrocytic and vascular response to amyloid plaque in 5xFAD mice and its link to the O3 lung response. O3 exposure increased GFAP positive astrocyte density correlating with increased plaque burden in the cortex. Focusing on the plaque microenvironment, we found O3 qualitatively altered plaque associated astrocytes, evidenced by both proteomic and transcriptomic changes. Along with loss of protein expression, proteomic changes reflected increased cell-cell interaction in plaque microenvironment. Specifically, we found increased astrocyte-microglia contact selectively in periplaque space from O3 exposure. Transcriptional analysis of periplaque astrocytes revealed an accelerated shift towards disease associated astrocyte (DAA) phenotype. Elevated circulating HMGB1 was previously found from O3 exposure. In this study we demonstrate deleting HMGB1 selectively in peripheral myeloid cells and not in CNS microglia ameliorates the lung immune response to O3 as well as downregulates DAA marker in the CNS, providing a potential link between peripheral HMGB1 and O3 induced astrocytic dysregulation. On examining vascular response to O3 we found increased vascular amyloid accumulation associated with an altered vascular proteomic profile. Our analysis indicates O3 potentially disrupts vascular function such as amyloid clearance. Taken together, our study demonstrates that astrocyte and neurovasculature are contributors to O3 lung-brain axis with important implications towards amyloid pathology progression and identifies peripheral myeloid HMGB1 as its potential modulator. Further studies are required to fully understand the consequences of this impact and its role in amyloid pathology.Item Contributions of the Presynaptic Protein Bassoon to Tau Pathogenesis and Neurodegeneration(2024-05) Patel, Henika Sanjaybhai; Oblak, Adrian; Lasagna-Reeves, Cristian; McKinzie, David; Kim, Jungsu; Webb, Ian; Murray, MelissaNeurodegenerative tauopathies, characterized by the aggregation of misfolded tau protein, pose a significant clinical and scientific challenge. A high-molecular-weight (HMW) tau species is known to be involved in spreading tau pathology. However, the nature and composition of this species remain elusive, hindering targeted interventions. There are four main chapters in this dissertation. The first chapter highlights the existing knowledge about tau and its role in neurodegenerative tauopathies and discusses the possible contribution of protein interactors in the pathogenesis of tau pathology. The second chapter investigates the association between pathological hallmarks and functional deficits in the aged PS19 tauopathy model. The findings indicate that a diverse spectrum of pathological tau species may underly different symptoms and that neuroinflammation might contribute to functional deficits independent of tau pathology. In the third chapter, we isolated and characterized the HMW tau species with seeding capabilities from the PS19 brains. Using unbiased quantitative mass spectrometry analysis, we identified Bassoon (BSN), a presynaptic protein, as a crucial interactor of the HMW tau seed. BSN overexpression exacerbated tau-seeding and toxicity both in vitro and in the Drosophila model of tauopathy. Conversely, the downregulation of BSN reduced tau spreading and overall disease pathology in the PS19 mice, indicating the important role of BSN in taumediated pathogenesis. In chapter four, we studied the disease-associated p.Pro3866Ala missense mutation in BSN and further evaluated the mechanisms through which BSN could induce toxicity and neurodegeneration. Using CRISPR-Cas9 technology, we developed a knock-in mouse model harboring the BSN P3866A missense mutation in the endogenous murine Bsn. We observed somatic BSN accumulation suggesting that the P3866A mutation might be enhancing the aggregation propensity of BSN and provide a conducive environment to promote tau aggregation. Furthermore, we observed dysregulation in protein degradation pathways, neuroinflammation, and enhanced synapse elimination by microglia. These findings underscore the pivotal role of BSN in providing a favorable environment for tau aggregation and influencing the properties of the tau seed, thereby contributing to neurodegenerative processes. Overall, our results indicate that targeting BSN could be a potential therapeutic intervention for neurodegenerative diseases.Item Brain Responses to Sugar: Implications for Alcohol Use Disorder and Obesity(2024-05) Alessi, Jonathan P.; Yoder, Karmen K.; Kareken, David A.; Džemidžić, Mario; Considine, Robert V.; Harezlak, JaroslawObesity and alcohol use may together account for 640,000 adult deaths each year in the United States. In both cases, overconsumption drives untoward effects. Alcohol use and obesity also both relate to sweet liking, as sugar consumption is consistently linked to weight gain and intense sweet liking has been linked to an inherited risk for alcohol use disorder (AUD). However, the neural underpinnings of these associations are largely unknown. Thus, we used sugar-sweetened water administration during functional magnetic resonance imaging (fMRI) to probe these relationships in two studies. In the first, we tested the relationship between a known AUD risk factor, subjective response to alcohol, and the brain response to both sucrose and monetary reward in 140 young adults. We found a significant positive correlation between the enjoyable component of subjective responses to a standardized intravenous alcohol exposure and activation to high-concentration sucrose (but not monetary reward) in the right dorsal anterior insula and the supplementary motor area, supporting a role for these regions in AUD risk. In the second study, we investigated the neural mechanisms of sweet liking decreases following bariatric surgery, the most effective obesity treatment. Here, we evaluated the change in brain activation to sucrose in 24 women before (BMI 47.0 + 6.9 kg/m2) and 21 women after (BMI 37.6 + 6.5 kg/m2) bariatric surgery and compared the pre- and post-surgical activation patterns to those of 21 normal to overweight (BMI 23.5 + 2.5 kg/m2) control participants. Brain activation did not differ between controls and surgery participants at either time point. However, activation to sucrose in reward, but not sensory, regions decreased significantly after surgery, consistent with reduced drive to consume sweet foods. Together, these studies highlight the utility of quantifying brain responses to sweet taste as a method to understand the mechanisms underlying overconsumptive behavior.