Now showing items 1-20 of 216

    • A dual to the death: using novel host-directed antivirals to promote death of HCMV-infected myeloid cells through apoptosis and necroptosis

      Chan, Gary; Cheung, Jennifer (2022-06)
      Human cytomegalovirus (HCMV) is a ubiquitous member of the betaherpesvirus family, with seroprevalence rates ranging from 40-100% worldwide. Although primary infection is asymptomatic in most immunocompetent patients, HCMV is a significant cause of morbidity and mortality in the immunosuppressed and immunonaїve, including transplant recipients, patients with AIDS, and developing fetuses in utero. The diverse clinical presentations of HCMV are attributable to the pervasive systemic dissemination and extensive cellular tropism of the virus. Peripheral blood monocytes are believed to be the key cells responsible for HCMV dissemination from the initial site of infection to distant organ systems. Monocytes are normally short-lived, surviving for only 48 h in circulation before undergoing apoptosis. Previous work from our lab has shown that HCMV circumvents the short lifespan of monocytes by inducing a noncanonical activation of Akt to upregulate the expression of antiapoptotic proteins, thereby prolonging survival of infected monocytes. HCMV promotes survival in the absence of viral replication and lytic gene expression, rendering current direct-acting antivirals ineffective against quiescently infected monocytes. There are currently no antivirals that target quiescent or latent HCMV infection. We hypothesize that targeting host proteins that are essential for HCMV's induction of monocyte survival mechanisms will reduce viability of infected monocytes, ultimately reducing systemic viral dissemination. The studies in this thesis investigate novel host-directed antivirals targeted at two different cellular factors and their efficacy against quiescent HCMV infection in monocytes. The first host protein under scrutiny as an antiviral target is Sirtuin 2 (Sirt2), an NAD+-dependent deacetylase. Treatment with novel Sirt2 inhibitors promoted death of HCMV-infected monocytes as a cellular antiviral defense response through two concurrent regulated death pathways: apoptosis and necroptosis. HCMV has developed mechanisms to impede both death pathways, but inhibition of Sirt2 relieves the viral obstructions on both pathways by disrupting HCMV's unique phosphorylation on Akt. The second host protein targeted as a potential antiviral strategy is Mcl-1, an antiapoptotic member of the Bcl-2 family of proteins. HCMV-infected monocytes are dependent on Akt-dependent upregulation of Mcl-1 for survival during early infection. Treatment with Mcl-1 inhibitors blocked interaction between Mcl-1 and proapoptotic protein Bak, reducing viability of infected monocytes. Subsequent testing of Mcl-1 inhibitors in an ex vivo skin organ culture system resulted in a decrease in HCMV-infected cells that crawled out of the skin tissue, suggesting that Mcl-1 inhibition may reduce viral dissemination. Our studies lay down the groundwork for the investigation of novel host-directed antivirals, an approach that successfully targets quiescent HCMV infection in peripheral blood monocytes for the first time. Expanding the study of host-directed antivirals may bring the field one step closer to the possibility of a comprehensive antiviral regimen that is effective against all stages of viral infection.
    • Rational design of a genetically encoded fluorescent protein color switch using a modular, entropy-driven mechanism

      Loh, Stewart; John, Anna (2022-06)
      Engineered protein conformational switches have applications in cellular and in vitro biosensing, molecular diagnostics and artificial signaling systems in synthetic biology. They broadly consist of an input module and an output module that communicate via a conformational change. The overarching goal of this thesis is to tackle two major challenges in protein switch design - signal transduction, by coupling a target recognition domain to an output domain to produce a robust change in signal in addition to modularity, which allows the facile creation of sensors binding novel targets. Here, we attempted to test a rational design strategy that exploits two key protein engineering principles (1) loop entropy, by which long insertions into a loop of a host protein destabilizes the host due to an entropic cost associated with loop closure unless the inserted sequence adopts a folded structure; and (2) alternate frame folding (AFF), which allows a protein - green fluorescent protein variants(GFP), in this case - to switch between two mutually exclusive folds. Toward this goal, we first studied the effect of loop entropy at two different insertion sites in a GFP variant (chapter 2) using a well-characterized ribose binding protein as the input domain. We provide stability measurements using circular dichroism and fluorescence data to support our hypothesis of the application of the loop entropy principle in a GFP beta barrel scaffold. To provide a proof-of-concept of the combination of loop entropy and the AFF mechanism in a genetically encodable GFP scaffold, we chose an unstable, circularly permuted FK506-binding protein (cpFKBP) as the input recognition domain and inserted it in one of the two mutually exclusive folds of the GFP-AFF fusion protein (chapter 3). Upon addition of ligand, binding induced folding of the cpFKBP domain effects a conformational change in which the tenth beta strand of GFP exchanges, replacing Thr203 (green state) with Tyr203 (yellow state). We confirmed this mechanism in vitro by a ratiometric change in fluorescence output and observed that the process is slow and irreversible. We elucidate the biophysical principles underlying this mechanism by using denaturant and temperature to modulate the relative populations of the two folds in vitro. We also observed a faster and higher intensiometric response in mammalian cells which may be attributed to an alternate mechanism. We then harnessed this intensiometric response in a single fold of the fluorescent protein combined with a previously engineered monobody scaffold capable of binding a variety of targets (chapter 4). Altogether this work may have the potential to create a novel class of fluorescent protein biosensors comparable to existing single fluorescent protein-based biosensors currently available.
    • Targeting SHIP paralogs to promote microglial effector function in the CNS

      Thomas, Stephen J.; Pedicone, Chiara (2022-06)
      The two SH2-containing inositol 5'-phosphatases , SHIP1 (INPP5D) and SHIP2 (INPPL1), play an essential role in modulation of cellular signaling by transforming the PI3K product PI(3,4,5)P3 into PI(3,4)P2. PI3K signaling triggers activation of downstream signaling cascades that drive survival, effector functions, differentiation, and proliferation. SHIP1 can also mask the cytoplasmic tails of key receptors or their adaptor proteins such as DAP12, thus preventing PI3K recruitment to Trem2, a critical receptor for microglial function. Several GWAS studies correlated single nucleotide polymorphisms (SNPs) in INPP5D with Alzheimer's Disease (AD). However, it remains unclear whether these SNPs are deleterious or protective in AD and how they alter SHIP1 protein expression. SHIP2 overexpression has also been correlated with AD, suggesting that both SHIP1 and SHIP2 might be therapeutic targets. To study how SHIP1 and SHIP2 modulate microglial functions we used small molecule inhibitors and agonists of these enzymes. In our initial study we found that both SHIP paralogs are expressed in murine microglia and that Pan- SHIP1/2 inhibition increases lysosomal size and enhances microglial phagocytosis of Ab1-42 fibrils and dead neurons using both flow cytometry and confocal microscopy. Our lead Pan-SHIP1/2 inhibitor, K161, showed Blood Brain Barrier penetration as detected in the cerebral cortex of treated mice with mass spectrometry. K161 treatment of WT mice showed no difference in microglial frequency or lysosomal content in vivo; however, we observed a significant 2 increase in Ab1-42 and dead neurons phagocytosis ex vivo in microglia in K161- treated mice versus controls. Subsequently, we discovered a novel and highly potent SHIP1 selective agonist (K306) via artificial intelligence guided computational screening. We found that K306 can reduce the release of inflammatory cytokines in macrophages and microglial cells stimulated with LPS or Ab1-42. Interestingly, K306 didn't alter microglial phagocytic uptake of cargo, but did promote degradation of phagocytosed lipid-laden cargo - defining a novel role of SHIP1 in degradation of lipid cargo in microglia. These results highlight the importance of SHIP1 and SHIP2 in microglial biology and their modulation as therapeutics in different stages of neurodegenerative disease where microglia play a major role, such as AD.
    • Targeting wild-type and mutant p53 for cancer treatment

      Stewart N Loh; Blayney, Alan John (2021)
    • Charting Neurotypical Change in Complement and Cytokine Levels Across Postnatal Human Cortical Development

      Sager, Rachel (2021-12)
      A burgeoning body of evidence supports a role for immune signals in neurotypical human brain development. Furthermore, associations between neuroinflammation in development and the subsequent increased risk for psychiatric disorders indicate that an excess of immune signaling early in life damages brain function later in life. In this dissertation, I examined the postnatal expression of two major immune signaling families: complement and cytokines; and the relative contributions of neural cell types to the cortical transcriptome. I used high-throughput microarray, quantitative reverse transcription PCR, immunohistochemistry and multiplex immunoassays. I found coordinated increases in glial cell marker, complement, and cytokine transcripts from birth until the typical age of entry into school (age 5). There were two main patterns of change in gene expression encoding immune signals and their receptors: an early postnatal peak in toddlers followed by a decline in expression levels (C1Q, C3, IL-1β, CD11B, IL-1R1, IL-18) and an early postnatal increase in toddlers, followed by additional increases in adolescents and young adults (IL-6, TNF-α). Complement inhibitor mRNAs were also differentially expressed across postnatal human life, increasing before reaching a plateau around school age (CD46, CD55, CR1,) or peaking in young adulthood (SERPING1, CD59). This suggests sustained complement inhibition during adolescence. The multiple cytokine and complement family members that peaked in toddlers suggest a period of dominant immune signaling from age two to five in humans. This may be related to the proliferation or maturation of glia during early postnatal development, whereas the cytokines seen increasing in adolescents and young adults are contemporaneous with periods of proposed increases in synaptic elimination. These findings open up additional avenues of investigation into the role of immune signaling in normal mammalian brain development and support that time periods of normative increases in developmental immune factor signaling overlap with known 'windows of vulnerability' to manifesting autism and schizophrenia.
    • The Role of Laminins in the Retinal Vascular Basement Membrane

      Brunken, William J.; Watters, Jared (2021-11)
      The vascular basement membrane (vBM) of the central nervous system (CNS) is a highly specialized structure that is composed of various extracellular matrix (ECM) proteins and has many functions, including providing a point of adhesion for the cells of the vasculature, serving as a physical barrier, and providing an interface for communication with endothelial cells. One family of ECM molecules, laminins, are responsible for many of these specialized functions. There are 16 known isoforms of laminin, each consisting of a single α, β, and γ-chain. The distribution of these isoforms in the CNS vBM, however, remains unknown. Here, we used the retina to examine the distribution of laminin chains in the CNS vBM throughout development, as well as the roles of β2-containing laminins in vBM organization and γ3-containing laminins in arterial morphogenesis. The results presented in Chapter 2 demonstrate that there are dramatic changes in the temporal and spatial patterning of many of the laminin chains in the retinal vasculature throughout development, particularly the α2, α5, and γ3-chains. We deleted a key component of the CNS vBM, the laminin β2-chain, to gain a deeper understanding of how laminins affect vBM structure. Deletion of the β2-chain leads to decreased expression of several partner chains, including α2, α5, and γ3. Interestingly, the deletion of laminin β2 also leads to increased deposition of two other ECM molecules, agrin and perlecan, in the BMs of retinal veins and arteries, respectively. We also provide strong evidence that astrocytes contribute laminin 221 to the retinal vBM and that this laminin may directly regulate AQP4 expression in vascular associated astrocytic endfeet. The results presented in Chapter 3 demonstrate that laminins are involved in regulating arterial morphogenesis. Specifically, we found that γ3-containing laminins signal through dystroglycan to induce Dll4-Notch signaling, leading to decreased vascular branching and increased smooth muscle coverage: hallmarks of the arterial phenotype. Taken together, the work presented here further elucidates the structural and functional roles for laminins in the CNS vBM.

      Amack, Jeffrey, D.; Santra, Peu (2021-11)
      Vacuolar type H+-ATPase (V-ATPase) is a ubiquitously expressed enzyme complex that pumps protons across membranes. The proton-motive force generated by V-ATPase is used by cells to acidify intracellular compartments. Additionally, certain specialized tissue types have V-ATPase on plasma membranes where it secretes H+into the extracellular space. While V-ATPase activity is essential for several cellular functions, our understanding of cell-type specific functions for V-ATPase remains limited. Here, I focused on investigating V-ATPase functions in mechanosensory hair cells. Hair cells are functional units of mammalian auditory and vestibular systems. Consequently, hair cell loss leads to permanent deafness. Mutation in specific V-ATPase subunits causes sensorineural deafness in human, however, the mechanism is not well understood. I used zebrafish as model vertebrate to investigate how loss of V-ATPase function impacts hair cells. Using a combination of genetic mutations, pharmacological manipulations and live imaging of hair cells in vivo, I found that V-ATPase activity is critical for hair cell survival. Analysis of molecular markers and cellular morphologies indicates hair cells in V-ATPase mutants undergo a caspase-independent, necrosis-like death. V-ATPase mutant hair cells show a significant decrease in mitochondrial membrane potential (mPTP). On modulating mPTP pharmacologically, V-ATPase mutants show a modest but consistent improvement of hair cell survival. These results indicate mitochondrial dysfunction contributes to hair cell death in V-ATPase mutants. Next, I generated a novel cilia pH biosensor and found that hair cell kinocilia have a more basic pH than other primary cilia in zebrafish embryos. Interestingly, my collaborators and I discovered that V-ATPase subunits localize to hair cell kinocilia in zebrafish and mice, which suggests cell-type specific functions for V-ATPase in kinocilia. pH maintenance in kinocilia may be an essential function that contributes to proper kinocilia length and/or function. In conclusion, this work has uncovered a function for V-ATPase activity that is critical for hair cell survival, in part by maintaining mitochondrial health, and a function that mediates hair cell kinocilia form and function. The work presented in this thesis advances our understanding of V-ATPase functioning in hearing loss, more broadly elucidates new in vivo cell-type specific V-ATPase functions.
    • Meta-Analysis of Alzheimer's Disease Risk with Obesity, Diabetes, and Related Disorders

      Profenno, Louis A.; Porsteinsson, Anton P.; Faraone, Stephen V. (Elsevier BV, 2010-03)
      Background: Late-onset Alzheimer’s disease (AD) is a multifactorial and heterogeneous disorder with major risk factors including advanced age, presence of an apolipoprotein E 4 (APOE4) allele, and family history of AD. Other risk factors may be obesity and diabetes and related disorders, which are highly prevalent. Methods: We reviewed longitudinal epidemiological studies of body mass, diabetes, metabolic syndrome, and glucose and insulin levels on risk for AD. We conducted meta-analyses of the results from these studies. Results: For obesity assessed by body mass index, the pooled effect size for AD was 1.59 (95% confidence interval [CI] 1.02–2.5; z 2.0; p .042), and for diabetes, the pooled effect size for AD was 1.54 (95% CI 1.33–1.79; z 5.7; p .001). Egger’s test did not find significant evidence for publication bias in the meta-analysis for obesity (t 1.4, p .21) or for diabetes (t .86, p .42). Since these disorders are highly comorbid, we conducted a meta-analysis combining all studies of obesity, diabetes, and abnormal glucose or insulin levels, which yielded a highly significant pooled effect size for AD of 1.63 (95% CI 1.39 –1.92; z 5.9; p .001). Conclusions: Obesity and diabetes significantly and independently increase risk for AD. Though the level of risk is less than that with the APOE4 allele, the high prevalence of these disorders may result in substantial increases in future incidence of AD. Physiological changes common to obesity and diabetes plausibly promote AD.