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Open Access Publications from the University of California

Scholarly Works (37 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2018)

High altitude studies have been insightful in revealing putative hypoxia tolerant genes to improve pathological and physiological conditions that involve hypoxia or ischemia. Whole-genome sequencing of Ethiopian highlanders identified LIPE (Lipase E, Hormone Sensitive Type), as an important gene that allows them to live in chronic low oxygen conditions (Udpa et al., 2014). Using Drosophila melanogaster, ubiquitous knockdown of the ortholog of this gene, hormone-sensitive lipase (Hsl), results in increased tolerance to chronic hypoxia. To elucidate the tissue specificity of the adaptive phenotype, UAS-RNAi(Hsl) line was crossed with different cell- or tissue-specific GAL4 drivers to systematically knockdown this gene, without altering its expression in the rest of the organism. There was a remarkable difference in eclosion rates when Hsl was knocked down in eclosion hormone-expressing neurons, third instar larval fat body, oenocytes, and hemocytes, constituting a four-fold increase over controls (all knockdown crosses were >87%, P<0.005). Since Hsl plays a prominent role in lipid metabolism, its inhibition in the cell(s) or tissue(s) that produced the most profound response to hypoxia tolerance were quantified to provide evidence of any changes in triglyceride levels. An augmented triglyceride concentration was observed in Hsl knockdown crosses, specifically in, UAS-RNAi(Hsl) x da-GAL4 and UAS-RNAi(Hsl) x Eh-GAL4 with a 1.5- to 2-fold increase in hypoxia when compared with controls. This data suggests that lipid accumulation as a result of Hsl knockdown may be biochemically significant in promoting hypoxia tolerance.

  • 5 supplemental files
Cover page: Candidate Gene Identified in Humans Adapted to High-Altitude Depicts Hypoxia Tolerance in Drosophila melanogaster
  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2023)

Methadone is a synthetic opioid analgesic and a common pharmacological treatment for opioid use disorder (OUD) in pregnant women. Its clinical application blocks the effects of other opioids and attenuates withdrawal symptoms or behaviors leading to relapse or overdose. However, methadone’s ability to readily enter fetal circulation, accumulate in neural tissue, and cause long-term neurocognitive sequelae, has led to concerns regarding its effect on fetal brain development.

In the present research, human induced pluripotent stem cell-derived cortical organoid (hCO) technology was leveraged to probe the etiology of these deficits by understanding how methadone impacts the earliest mechanisms of cortical development.

Bulk mRNA sequencing of 2-month-old hCOs chronically treated with a clinically relevant dose of 1μM methadone revealed a robust transcriptional response to methadone associated with functional components of the synapse, including pre-synaptic vesicular trafficking/release and post-synaptic signal reception machinery, as well as the underlying extracellular matrix (ECM), and cilia. Co-expression network and predictive protein-protein interaction analyses demonstrated that these changes occurred in concert, centered around a regulatory axis of growth factors, developmental signaling pathways, and matricellular proteins (MCPs). Of the central MCPs, thrombospondin 1 (TSP1), which is necessary for synapse formation and maturation, was most prominently downregulated, with dose-dependent reductions in its protein levels. These results indicated that methadone exposure during cortical development alters transcriptional programs associated with synapse biology by modulating ECM and ciliary molecular mechanisms.

To further dissect methadone’s impact at the synapse, ultrastructural imaging was performed to visualize and quantify changes in synaptic features. Improving upon the limitations of prior ultrastructural studies of synapses in brain organoids, culture, imaging, and segmentation methodologies were modified to optimize the volumetric capture of synaptic features via electron microscopy. These techniques revealed that 10μM methadone reduces synaptic vesicle volumes as well as the range of vesicle morphologies in 3-month-old hCOs, which may have implications for the reductions in neurotransmission observed following methadone exposure.

Cumulatively, the findings presented in this dissertation provide novel insight into the molecular underpinnings of methadone’s effect on neurocognitive development, novel methods of studying these effects in brain organoids, and a basis for improving interventions for maternal OUD.

    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2018)
    Chronic Mountain Sickness (CMS) occurs in high-altitude residents with major neurological symptoms such as migraine headaches, dizziness and cognitive deficits. Recent work demonstrated that highlanders have increased intracellular pH (pHi) in their brain cells, perhaps for the sake of adaptation to hypoxemia and help to facilitate glycolysis, DNA synthesis, and cell cycle progression. Since there are well adapted (non-CMS) and maladapted (CMS) high-altitude dwellers, it is not clear whether pHi is differently regulated in these two high-altitude populations. In this work, we obtained induced pluripotent stem cell (iPSC)-derived astrocytes from both CMS and non-CMS highlanders who live in the Peruvian Andes (>14,000 ft) and studied pHi regulation in these astrocytes using pH-sensitive dye BCECF. Our results show that the steady-state pHi (ss pHi) is lower in CMS astrocytes compared with non-CMS astrocytes. In addition, the acid extrusion following an acid loading is faster and the pHi dependence of H+ flux rate becomes steeper in CMS astrocytes. Furthermore, the Na+ dependency of ss pHi is stronger in CMS astrocytes and the Na+/H+ exchanger (NHE) inhibitors blunted the acid extrusion in both CMS and non-CMS astrocytes. We conclude that (a) NHE contributes to the ss pHi stabilization and mediates active acid extrusion during the cytosolic acidosis in highlanders; (b) acid extrusion becomes less pHi sensitive in non-CMS (versus CMS) astrocytes which may prevent NHE from over-activated in the hypoxia-induced intracellular acidosis and render the non-CMS astrocytes more resistant to hypoxemia challenges.
      Cover page: Intracellular pH Regulation in iPSCs-derived Astrocytes from Subjects with Chronic Mountain Sickness
      • Thesis
      • Peer Reviewed
      UC San Diego Electronic Theses and Dissertations (2011)

      Damage caused by lack of oxygen is a key factor in many serious disease states ranging from stroke and heart attack to asthma and high altitude mountain sickness. Determining the pathways that can ameliorate injuries caused by hypoxia is key to developing treatments and prevention of hypoxic damage. This dissertation examines the role of Notch signaling in potentiating Drosophila survival under chronic low oxygen conditions.

      Pathway-level analysis of micro-array data of hypoxia-selected flies, the Notch signaling pathway was significantly up-regulated. Using the UAS-GAL4 system, the temporal and spatial specificity of Notch signaling was determined. The most robust results were found with glia-specific GAL4 drive, hence they were chosen for further characterization. Expression patterns of the GAL-4 glial drivers were confirmed by crossing them to a UAS-GFP transgenic fly line. Immunohistochemical analysis confirmed NICD (Notch Intracellular Domain) over-expression in glia cells. The developmental importance of Eaat1 glia was shown by crossing them to the UAS-reaper line; the results were embryonic lethal. Transcriptional up-regulation of Notch signaling was demonstrated by crossing flies stably over-expressing NICD with a reporter stock.

      To address the question of what mechanism Notch utilizes to potentiate survival during hypoxia, several hypotheses were considered. First, Notch signaling may allow Eaat1-expressing glia to form a stem cell niche. This niche, in turn, allows the replacement of damaged/dead neurons injured during hypoxia.

      Alternatively, other pathways may mediate the survival of these glial cells, which have been shown to be necessary to the survival of the whole organism. The metabolism of these glia could be altered via pyruvate dehydrogenase (PDH). Akt and NF-&kappaB are also known to interact with the Notch pathway. Perhaps canonical downstream target genes of Notch signaling, such as m-&alpha act as mechanism to allow survival of these crucial glia and thus the whole organism.

      To study Notch interacting with other pathways, flies stably over-expressing NICD under the Eaat1 driver were crossed to UAS-RNAi flies targeting genes involved in pathways known to potentiate survival during hypoxia. Both Relish and m-&alpha act downstream of Notch signaling to mediate survival, demonstrating novel means of Notch up-regulation leading to hypoxic survival.

      • 2 supplemental videos
      Cover page: The Role of Notch in Survival During Chronic Hypoxia
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2018)
      Oxygen plays a central role in cardiac energy metabolism. At high altitude where the ambient oxygen level is low, we found EDNRB is associated with human hypoxia adaptation. Our subsequent study in global heterozygous knockout mice (Ednrb-/+) revealed that cardiac function was conserved in these mice when exposed to extreme hypoxia. The major goal of this study was (i) to determine the functional role of cardiomyocyte EdnrB in maintaining cardiac function under hypoxic stress and (ii) to validate the phenotypes we detected in Ednrb-/+ mice using EDNRB blockers. Unlike the global knockouts, cardiac-specific heterozygote (EdnrBflox/+) and homozygote (EdnrBflox/flox) EdnrB knockout mice were phenotypically normal. When treated with graded low levels of oxygen (10% and 5% O2), both EdnrBflox/+ and EdnrBflox/flox were hypoxia tolerant. The cardiac indexes at 10% and 5% O2 for EdnrBflox/+ were significantly higher and lactate levels were significantly lower when compared to the cre-negative controls (P < 0.05). Simultaneously, mice treated with BQ-788 (EDNRB-specific blocker) had a significantly higher cardiac index (P < 0.005) and significantly lower lactate levels (P < 0.0001) than in control mice. A similar result was obtained with mice treated with Bosentan (non-specific). These data indicate that a lower level or complete lack of EdnrB in the cardiomyocytes significantly improves cardiac performance under extreme hypoxia, a novel role of cardiomyocyte EdnrB in the regulation of cardiac function. Furthermore, this rescue under extreme hypoxia can also be achieved using EDNRB-specific pharmacological agents, e.g., BQ-788. This systematically confirms, both genetically and pharmacologically, the protective role of a lower EDNRB under extreme hypoxia stress.

      Key messages

      Under normal condition, cardiomyocytes-specific EdnrB knockout mice, both heterozygote and homozygote, are phenotypically normal. Under hypoxic condition, a lower level or complete deletion of cardiomyocyte EdnrB conserves cardiac function by maintaining high cardiac index. Similarly, mice treated with both specific (BQ-788) and non-specific (Bosentan) EDNRB blockers are tolerant to hypoxia by maintaining better cardiac function. The oxygen perfusion under extreme hypoxia is better in the mice with lower EDNRB, as depicted by lower lactate level at 5% oxygen. Our current study systematically confirms, both genetically and pharmacologically, the protective role of a lower EDNRB under extreme hypoxia stress. Overall, it supports our hypothesis that studies on human hypoxia adaptation provide new insight to common disease pathogenesis and treatments.
        Cover page: Cardiac-specific knockout and pharmacological inhibition of Endothelin receptor type B lead to cardiac resistance to extreme hypoxia
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2021)
        The microbiota plays a critical role in regulating organismal health and response to environmental stresses. Intermittent hypoxia and hypercapnia, a condition that represents the main hallmark of obstructive sleep apnea in humans, is known to induce significant alterations in the gut microbiome and metabolism, and promotes the progression of atherosclerosis in mouse models. To further understand the role of the microbiome in the cardiovascular response to intermittent hypoxia and hypercapnia, we developed a new rodent cage system that allows exposure of mice to controlled levels of O2 and CO2 under gnotobiotic conditions. Using this experimental setup, we determined the impact of the microbiome on the transcriptional response to intermittent hypoxia and hypercapnia in the left ventricle of the mouse heart. We identified significant changes in gene expression in both conventionally reared and germ-free mice. Following intermittent hypoxia and hypercapnia exposure, we detected 192 significant changes in conventionally reared mice (96 upregulated and 96 downregulated) and 161 significant changes (70 upregulated and 91 downregulated) in germ-free mice. Only 19 of these differentially expressed transcripts (∼10%) were common to conventionally reared and germ-free mice. Such distinct transcriptional responses imply that the host microbiota plays an important role in regulating the host transcriptional response to intermittent hypoxia and hypercapnia in the mouse heart.
          Cover page: Microbiota Modulates Cardiac Transcriptional Responses to Intermittent Hypoxia and Hypercapnia
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2021)
          Hypoxia is a critical pathological element in many human diseases, including ischemic stroke, myocardial infarction, and solid tumors. Of particular significance and interest of ours are the cellular and molecular mechanisms that underlie susceptibility or tolerance to low O2. Previous studies have demonstrated that Notch signaling pathway regulates hypoxia tolerance in both Drosophila melanogaster and humans. However, the mechanisms mediating Notch-conferred hypoxia tolerance are largely unknown. In this study, we delineate the evolutionarily conserved mechanisms underlying this hypoxia tolerant phenotype. We determined the role of a group of conserved genes that were obtained from a comparative genomic analysis of hypoxia-tolerant D.melanogaster populations and human highlanders living at the high-altitude regions of the world (Tibetans, Ethiopians, and Andeans). We developed a novel dual-UAS/Gal4 system that allows us to activate Notch signaling in the Eaat1-positive glial cells, which remarkably enhances hypoxia tolerance in D.melanogaster, and, simultaneously, knock down a candidate gene in the same set of glial cells. Using this system, we discovered that the interactions between Notch signaling and bnl (fibroblast growth factor), croc (forkhead transcription factor C), or Mkk4 (mitogen-activated protein kinase kinase 4) are important for hypoxia tolerance, at least in part, through regulating neuronal development and survival under hypoxic conditions. Becausethese genetic mechanisms are evolutionarily conserved, this group of genes may serve as novel targets for developing therapeutic strategies and have a strong potential to be translated to humans to treat/prevent hypoxia-related diseases.
            Cover page: Genetic interactions regulate hypoxia tolerance conferred by activating Notch in excitatory amino acid transporter 1-positive glial cells in Drosophila melanogaster
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2021)
            To detect the genomic mechanisms underlying evolutionary dynamics of adaptation in sexually reproducing organisms, we analyze multigenerational whole genome sequences of Drosophila melanogaster adapting to extreme O2 conditions over an experiment conducted for nearly two decades. We develop methods to analyze time-series genomics data and predict adaptive mechanisms. Here, we report a remarkable level of synchronicity in both hard and soft selective sweeps in replicate populations as well as the arrival of favorable de novo mutations that constitute a few asynchronized sweeps. We additionally make direct experimental observations of rare recombination events that combine multiple alleles on to a single, better-adapted haplotype. Based on the analyses of the genes in genomic intervals, we provide a deeper insight into the mechanisms of genome adaptation that allow complex organisms to survive harsh environments.
              Cover page: Multiple mechanisms drive genomic adaptation to extreme O2 levels in Drosophila melanogaster
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2020)
              The SLC22 family of transporters is widely expressed, evolutionarily conserved, and plays a major role in regulating homeostasis by transporting small organic molecules such as metabolites, signaling molecules, and antioxidants. Analysis of transporters in fruit flies provides a simple yet orthologous platform to study the endogenous function of drug transporters in vivo. Evolutionary analysis of Drosophila melanogaster putative SLC22 orthologs reveals that, while many of the 25 SLC22 fruit fly orthologs do not fall within previously established SLC22 subclades, at least four members appear orthologous to mammalian SLC22 members (SLC22A16:CG6356, SLC22A15:CG7458, CG7442 and SLC22A18:CG3168). We functionally evaluated the role of SLC22 transporters in Drosophila melanogaster by knocking down 14 of these genes. Three putative SLC22 ortholog knockdowns-CG3168, CG6356, and CG7442/SLC22A-did not undergo eclosion and were lethal at the pupa stage, indicating the developmental importance of these genes. Additionally, knocking down four SLC22 members increased resistance to oxidative stress via paraquat testing (CG4630: p < 0.05, CG6006: p < 0.05, CG6126: p < 0.01 and CG16727: p < 0.05). Consistent with recent evidence that SLC22 is central to a Remote Sensing and Signaling Network (RSSN) involved in signaling and metabolism, these phenotypes support a key role for SLC22 in handling reactive oxygen species.
                Cover page: Drosophila SLC22 Orthologs Related to OATs, OCTs, and OCTNs Regulate Development and Responsiveness to Oxidative Stress
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2022)
                The molecular mechanisms leading to high-altitude pulmonary hypertension (HAPH) remains poorly understood. We previously analyzed the whole genome sequence of Kyrgyz highland population and identified eight genomic intervals having a potential role in HAPH. Tropomodulin 3 gene (TMOD3), which encodes a protein that binds and caps the pointed ends of actin filaments and inhibits cell migration, was one of the top candidates. Here we systematically sought additional evidence to validate the functional role of TMOD3. In-silico analysis reveals that some of the SNPs in HAPH associated genomic intervals were positioned in a regulatory region that could result in alternative splicing of TMOD3. In order to functionally validate the role of TMOD3 in HAPH, we exposed Tmod3-/+ mice to 4 weeks of constant hypoxia, i.e. 10% O2 and analyzed both functional (hemodynamic measurements) and structural (angiography) parameters related to HAPH. The hemodynamic measurements, such as right ventricular systolic pressure, a surrogate measure for pulmonary arterial systolic pressure, and right ventricular contractility (RV- ± dP/dt), increases with hypoxia did not separate between Tmod3-/+ and control mice. Remarkably, there was a significant increase in the number of lung vascular branches and total length of pulmonary vascular branches (P < 0.001) in Tmod3-/+ after 4 weeks of constant hypoxia as compared with controls. Notably, the Tmod3-/+ endothelial cells migration was also significantly higher than that from the wild-type littermates. Our results indicate that, under chronic hypoxia, lower levels of Tmod3 play an important role in the maintenance or neo-vascularization of pulmonary arteries.
                  Cover page: Heterozygous Tropomodulin 3 mice have improved lung vascularization after chronic hypoxia.
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