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  Characterization of Hic-5 in Cancer Associated Fibroblasts: A Role in Extracellular Matrix Deposition and Remodeling

  Turner, Christopher; Goreczny, Gregory (2017)

  Hic-5 (TGFβ1i1) is a focal adhesion scaffold protein that has previously been implicated in many cancer-related processes. However, the contribution of Hic-5 during tumor progression has never been evaluated, in vivo. In Chapter 2 of this thesis, I crossed our Hic-5 knockout mouse with the MMTV-PyMT breast tumor mouse model to assess the role of Hic-5 in breast tumorigenesis. Tumors from the Hic-5 -/-;PyMT mouse exhibited an increased latency and reduced tumor growth. Immunohistochemical analysis of the Hic-5 -/-;PyMT tumors revealed that the tumor cells were less proliferative. However isolated tumor cells exhibit no difference in growth rate. Surprisingly, Hic-5 expression was restricted to the tumor stroma. Further analysis showed that Hic-5 regulates Cancer Associated Fibroblast (CAF) contractility and differentiation which resulted in a reduced ability to deposit and reorganize the extracellular matrix (ECM) in two-and three-dimensions. Furthermore, Hic-5 dependent ECM remodeling supported the ability of tumor cells to metastasize and colonize the lungs.The molecular mechanisms by which CAFs mediate ECM remodeling remains incompletely understood. In Chapter 3 of this thesis, I show that Hic-5 is required to generate fibrillar adhesions, which are specialized structures that are critical for the assembly of fibronectin fibers. Hic-5 was found to promote fibrillar adhesion formation through a newly characterized interaction with tensin1, a scaffold protein that binds to β1 integrin and actin. Furthermore, this interaction was mediated by Src-dependent phosphorylation of Hic-5 in two and three-dimensional matrix environments to prevent β1 integrin internalization and subsequent degradation in the lysosome. This work highlights the importance of the focal adhesion protein, Hic-5 during breast tumorigenesis and provides insight into the molecular machinery driving CAF-mediated ECM remodeling.
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  ANALYSIS OF THE VACUOLAR TYPE H+ ATPASE DURING FORMATION OF THE CILIATED ORGAN OF ASYMMETRY THAT ORIENTS THE LEFT-RIGHT BODY AXIS

  Amack, Jeffrey; Gokey, Jason (2015)
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  Bundling of cytoskeletal actin by the formin FMNL1 contributes to celladhesion and migration

  Blystone, Scott; Miller, Eric (2018)

  Metastasis is one of the leading causes of death in the world, affecting thousands every year. This is especially true of breast cancer, which can often result in the formation of secondary metastatic sites in the lung, liver, and bone marrow. There are many aspects to metastasis and an innumerable amount of molecular, biochemical, and cellular interactions contribute to its pathology. The ability of primary tumor cells to disseminate from the primary tumor, degrade the basement membrane, invade through the ECM, and eventually intravasate across the endothelial cell lining of the circulatory system or lymphatics requires a plethora of proteins, all working together in concert to achieve this. Nowhere in the cell is this more apparent than the actin cytoskeleton.Locomotion of cells requires several alterations in the actin cytoskeleton component of the cellular machinery. Generally speaking, cells must be able to polarize, form protrusions, adhere to the substratum, translocate, and then retract their tail, repeating this process as they continue to navigate to their destination. While there are many underlying aspects to this activity, spatiotemporal rearrangements of the actin cytoskeleton are key to the successful cellular motility. The mechanics behind dynamic actin cytoskeletal modifications are varied and complex, demonstrating the requirement for a variety of actin-associated, regulatory proteins.A crucial family of proteins involved in this process is the formin family of proteins. Formins are a relatively “new” group of actin modifiers which possess the unique ability to modify and generate linear actin filaments. While the members of this protein family all share some of the same actin modifying processes, many of these proteins also have functions exclusive to themselves. As a result, research into this field has blossomed and several novel features of different formins have been identified. Furthermore, alternative splice isoforms of several formins are often expressed in a variety of cell types, with specific functions attributed to each.The formin FMNL1 was originally identified in cells of a myeloid lineage and for many years was mostly thought to be involved in leukocyte adhesion and migration. Indeed, our lab has characterized many of the functions of this protein in both human and murine macrophages. However, as a result of the work in this dissertation, we have generated sufficient evidence suggesting that FMNL1 not only plays a role in breast cancer migration, but also exhibits functions unique to a specific alternative splice isoform of this protein.Our work on FMNL1 has pushed the field of study into this protein family in new directions. Herein, we have demonstrated that all three alternative splice isoforms of FMNL1 are expressed in a variety of cell types and the FMNL1ɣalternative splice isoform distinguishes itself from these isoforms via its ability to bundle linear actin filaments. Additionally, our data indicates that this is accomplished independently of the trademark FH2 domain, often thought to be the essential component of all formins. More specifically, we have identified a unique amino acid sequence in the C-terminal region of this isoform that most likely regulates this function. As a result, we have not only identified a potential therapeutic target for the treatment of metastasis via inhibition of cellular locomotion, but also pushed the field of formin research into a novel direction by providing insight which may foster new hypotheses and challenge classical theories regarding the relationship between formins and actin.
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  BRAIN SPECIFIC NEURAL EXTRACELLULAR MATRIX EXPRESSION AND MODIFICATIONS IN NEUROLOGICAL DISEASE AND DISORDERS

  Matthews, Rick; Dwyer, Chrissa (2013)

  The central nervous system (CNS) is extraordinarily complex in both structure and function. The neural extracellular matrix (ECM) is one of the key classes ofmolecules that regulates thedevelopment of the CNS and maintains its structure and function in the adult.Thereby understanding the function of the neural ECMis key to understanding the CNS. The neural ECM is composed of several nervous-system specificproteins, which are hypothesized to uniquely contribute to the defining physiological functions of the CNS. However,work in this area has been hindered by the highly complex molecular properties of the neural ECM, which stem from alterations in expressionand modifications (resulting from glycosylation and proteolytic cleavage) of its constituents. Further defining mechanisms that alter the expression and modifications of neural ECM constituents are critical to fully understanding its complex array of functions. Often in neuropathologies, the neural ECM undergoes dynamic changes providing a valuable tool to further understand its function andthe opportunity to explore its contribution to disease pathology and utility as a therapeutic target. The work presented herein investigates the role of altered expression of the nervous-system specific ECM constituent, Brain Enriched Hyaluronan Binding (BEHAB)/ brevican(B/b), in glioma,and altered glycosylation of the nervous system enriched ECM constituent, RPTPζ/phosphacan, in O-mannosylrelated congenital muscular dystrophies (CMDs). Our work suggests that increased expression of B/b in the glioma tumor microenvironment (TEM) contributes to the pathological progression of these tumors, and reducing its expression is a valuable therapeutic strategy. Additionally, our work evaluates the transcriptional regulatory mechanisms leading to increases inB/b expression in glioma and highlights the potential value of these mechanisms as therapeutic targets. Our work also identifies the absence of O-mannosyl linked carbohydrates on RPTPζ/phosphacan in the brains of CMD models and suggests that altered glycosylation of RPTPζ/phosphacan may have a role in the neuropathologies underlying these disorders. Overall this work provides valuable insight intothe molecular complexities of the neural ECM stemming from changes in the expression and glycosylation of its constituents and furthers our understanding of its function in the normal CNS and in neuropathologies.
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  PHOSPHORYLATION AND UBIQUITINATION REGULATE PROTEIN PHOSPHATASE 5 ACTIVITY AND ITS PROSURVIVAL ROLE IN KIDNEY CANCER

  Mollapour, Mehdi; Dushukyan, Natela (2018)

  Protein Phosphatase 5 (PP5) is a serine/threonine phosphatase known to regulate many essential cellular functions including steroid hormone signaling, stress response, proliferation, apoptosis, and DNA repair. PP5 is a knownco-chaperone of the molecular chaperone heat shock protein 90 (Hsp90), and its regulation of Hsp90aidswiththe proper activation of Hsp90 clients and withsteroid hormone signaling.Hsp90 is also one of the strongest activators of PP5, as it releases the auto-inhibition of PP5 by interacting with the N-terminal tetratricopeptide repeat (TPR) domain of PP5. Our lab has recently shown that PP5 is phosphorylated at T362, and that this phosphorylation acts as an “on switch” resultingin the hyperactivation of PP5. Misregulation of this key phosphatase has been shown to aid in the tumor progression of ER-dependent and independent breast cancer. Elevated PP5 levels have also been linked to colorectalcancer, hepatocellular carcinoma (HCC), lymphoma, and prostate cancer. The work presented here reveals the pro-survival role that PP5 plays in kidney cancer. Clear cell renal cell carcinomas (ccRCC) are most often driven by mutations in the von Hippel-Lindau tumor suppressor (VHL). The data in this thesis shows that VHL binds and multi mono-ubiquitinates PP5 at two lysine residues K185 and K199. This post-translational modification negatively regulates PP5 likean “off switch” and ultimately leads to its degradation bythe proteasome. Mutations in the VHLgene that result in inactive mutants or a lack of VHL protein expression lead to ccRCC tumors. The data in this thesis shows that these VHL-nulltumors become dependent on elevated levels of PP5, and that both PP5 knockdown and inhibition lead to cancer cell death. The data further shows that the decrease in PP5 activity in VHL-null cells results in the induction of the extrinsic apoptotic pathway with a dramatic increase in the cleavage of PARP and caspases 3, 7, and 8.
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  POST-TRANSLATIONAL REGULATION OFCO-CHAPERONES AFFECTS HSP90 DRUG SENSITIVTY IN CANCER

  Mollapour, Mehdi; Dunn, Diana (2017)

  Heat Shock Protein-90 (Hsp90) is a molecular chaperone critical to thestability and activity of over 200 proteins known as “clients” including many oncogenes. Hsp90 chaperone function is linked to its ability to hydrolyze ATP and Hsp90 drugs inhibit its activity leading to the degradation of clients, thus making Hsp90 an attractive target for cancer therapy. The Hsp90 chaperone cycle is fine-tuned by another group of proteins called co-chaperones. They modifythe cycle, allowing Hsp90 to chaperone different pools of clients. Post-translational modifications (PTM) of Hsp90 and its co-chaperones can also regulate the chaperone cycle, and affect Hsp90 drug sensitivity. Here it is shownthat c-Abl kinase phosphorylates Y223in the co-chaperoneAha1, promotingits interaction with and stimulation of Hsp90 ATPase activity. Pharmacologic inhibition of c-Abl prevents the Aha1-Hsp90 interaction thereby, hypersensitizing cancer cells to Hsp90 inhibitors.Another co-chaperone of Hsp90, protein phosphatase-5 (PP5), mediates thede-phosphorylation of the co-chaperone Cdc37which is an essentialprocessfor the activation of kinase clients. The crystal structure of phospho-Cdc37 bound to the catalytic domain of PP5revealed elements of substrate specificity within the phosphatase cleft. Hyperactivityandhypoactivity of PP5 increasedHsp90 binding to its inhibitor, providing insight into increasingthe efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.PP5 is autoinhibited by intramolecular interactions that can be activated by anumber of cellular factors, includingHsp90. Casein kinase-1δ (CK1δ)-mediated phosphorylation of T362-PP5, was identified as an integral step for PP5 activation, independent of binding to Hsp90. Additionally, the tumor suppressor von Hippel-Lindau (VHL), the substrate recognition component of the VCB-E3-ubiquitin ligase, was found to interact with and multi-monoubiquitinate K185/K199-PP5 for proteasomal degradationin an oxygen-independent manner. Furthermore, VHL-deficient clear cell renal cell carcinoma (ccRCC) cell lines or patient tumors exhibit elevated PP5 levels. Down-regulation of PP5 caused apoptosis inccRCC cells, suggesting a prosurvival role for PP5 in ccRCC.Thisevidence suggests that inhibition of the enzymes that target and catalyze the PTM of Hsp90 and co-chaperones can act synergistically with Hsp90 inhibitors, providingnovel therapeutic strategiesto enhance the efficacy of Hsp90 inhibitors in cancer cells.
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  MITOCHONDRIAL ELECTRON TRANSPORT CHAIN ACTIVITY IN SYSTEMIC LUPUS ERYTHEMATOSUS

  Perl, Andras; Doherty, Edward (2014)

  Systemic lupus erythematosus (SLE) is an autoimmune disorder, characterized by T cell and B cell dysfunction. SLE mitochondria have been shown to be dysfunctional with increased mass, mitochondrial potential, decreased ATP, elevated reactive oxygen species (ROS) and reactive nitrogen species (RNS) concentrations, and altered Ca2+ stores. Drug treatments that target the mitochondria have shown efficacy in treating SLE. Here we have investigated electron transport chain (ETC) activity in SLE, to better understand the causes of mitochondrial dysfunction in SLE. We have found that mitochondrial complexes I and IV of the ETC have elevated respiration in SLE compared to healthy controls after both overnight resting and anti-CD3/CD28 stimulation. We have also shown that SLE complex I is resistant to NO inhibition of respiration. SLE peripheral blood lymphocytes (PBL) have increased S-nitrosylation (SNO) while immunoprecipitated complex I had decreased SNO of proteins compared to healthy controls. The drug Nacetylcysteine (NAC) was able to inhibit complex I activity in SLE, and was found to reduce the amount of complex I protein NDUFS3 after 15 minutes as measured by western blotting. These results have led us to the conclusion that SLE mitochondrial complex I is in an active form which is resistant to SNO and is driving the production of ROS and RNS that are associated with SLE. The drug NAC is able to inhibit complex I respiration which may have therapeutic efficacy by reducing the ROS and RNS stress in SLE.
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  Preclinical Development of Anti-Cancer Drugs from Natural Products.

  Huang, Ying; Sun, Qing (2014)

  Cancer has been and will continue to be the common concern in the United States and worldwide. As a conventional treatment to fight cancer, new anti-cancer drugs with more efficiency and less toxicity are extremely required. In this study, we have identified two novel compounds with anti-cancer properties from two traditional Chinese medicinal plants. One is Lappaol F that was extracted from the seeds of the plant Actium Lapp L., which has been used in China for centuries as anti-viral and anti-bacterial medicine. Another is M-9 that was extracted from the stem of Marsdenia tenacissima,a plant that has been applied to treat inflammation and cancer in China. Our results showed that Lappaol F inhibited cancer cell growth by regulating a series of cell cycle related proteins and inducing cell cycle arrest at G1 and/or G2 phase. p21 played a critical role in Lappaol F-induced cyclin B1 and cyclin-dependent kinase 1 (CDK1) suppression as well as G2arrest. Lappaol F also induced cell death in a number of cancer cells through caspases activation. Lappaol F-mediated cell growth inhibition was p53-independent. Notably, results from animal studies showed that Lappaol F effectively inhibited tumor growth in vivo, while being well tolerated by the mice. Thus, Lappaol F has a strong potential to be developed as a novel anti-cancer chemotherapeutic. Our studies showed that M-9 successfully sensitized several tumor cells but not non-tumorigenic cells to paclitaxel (Taxol) treatment. Additionally, M-9 reversed chemotherapeutic resistance in a number of multidrug resistant cells. Further results suggested that M-9 functioned, at least to a certain extent, via inhibiting drug efflux by competitively binding to P-glycoprotein (P-gp), a protein that accounts for multidrug resistance. Importantly, results from the in vivostudies demonstrated that M-9 strongly enhanced Taxol-induced growth suppression against xenografts derived from HeLa cells. Moreover, mice tolerated the treatment of Taxol and M-9 well. Therefore, M-9 is a novel chemosensitizer candidate to overcome P-gp-mediated multidrug resistance. Taken together, our studies provide a solid basis for further development of these two compounds as anti-cancer remedies.
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  RELN AS A CANDIDATE GENE FOR AUTISM SPECTRUM DISORDER (ASD)

  Howell, Brian; Lammert, Dawn (2017)

  Autism spectrum disorder (ASD) affects approximately 1 in 45 people, and is characterized by deficits in social communication and repetitive behaviors. Sequencing advancements have enabled the identification of numerous candidate genes, but precisely how these genes contribute to ASD remains largely unknown. RELNis consistently implicated as a candidate gene for autism. The encoded secreted glycoprotein, Reelin is important for proper brain developmental and postnatal synapse function. Here we examine the molecular and cellular consequences of the de novo RELNmutation R2290C. This mutation falls in a conserved arginine-amino acid-arginine (RXR) motif that is found within the Reelin subrepeat structure. Several other ASD patient mutations fall with in this consensus and all examined reduce Reelin secretion. Based on this we tested two hypothesis: (1) that the mutations reduce Reelin signaling and (2) that they have a gain-of-function consequence, such as ER stress. Using an engineered cell line with a heterozygous RELNR2290C mutation and the RELN Orleans (Orl) mouse line that produces nearly full length Reelin that is defective for secretion, we found evidence for both increased Dab1 and increased PDIA1 expression. Since, like most genes implicated in ASD RELNlikely acts in a multifactorial manner, we investigated whether second site mutations might contribute to ASD-related behaviors. Towards this end we crossed the heterozygous Orl and Shank3b mice to model two hits that are present in at least one ASD proband. We found that the resulting double heterozygousmice had impaired socialization and altered ultrasonic vocalizations. Furthermore, forebrain and cerebellar lysates showed increased PSD-95, identifying a potentially common mechanism and therapeutic target for ASD. These studies are the first to investigate the biological relevance of RELNcoding mutations in ASD.
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  A matter of life and death: human cytomegalovirus induction of monocyte survival and differentiation into macrophages through manipulation of the PI3K/Akt pathway

  Chan, Gary; Cojohari, Olesea (2017)

  Human cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus infecting up to 80% of the US population and reaching 100% seroprevalence in many parts of the world. In mostindividuals HCMV infection is usually asymptomatic. In contrast, in immunodeficient or immunonaive people, such as transplant recipients and the developing fetus, the virus is a major cause of morbidity and mortality. During a primary infection, HCMVcan spread very effectively in the body infecting many organ types and monocytes are believed to be the principal cell type responsible for HCMV dissemination throughout the body. Monocytes, however, are naturally programmed to undergo apoptosis after 48h in the circulation and are not permissive for viral replication. Our lab has shown that in order to combat these biological hurdles, HCMV promotes survival of these short-lived cells past their 48h “viability gate”. Besides inducing survival, the virus also mediates the differentiation of monocytes into macrophages skewed towards an M1 pro-inflammatory phenotype with select M2 anti-inflammatory features, which are long-lived cells, permissive for viral replication. However, the mechanisms used by HCMV to concomitantly induce survival and macrophage differentiation -two linked but separate processes, are not fully understood. The studies in this thesis reveal that upon binding and entry, HCMV initiates a survival program in monocytes by inducing a rapid and sustained activation of the PI3K/Akt pathway, which isdifferent from that induced by myeloid growth factors. Moreover, after inducing cellular survival across the 48-h viability gate, the virus also employsthe PI3K/Akt pathway to regulate caspase 3 activation which mediatesthe atypical M1/M2 polarization. Our work suggests that virus not only makes use of the PI3K/Akt pathway, but manipulates it at multiple levels toallow for viral-specific downstream functional changes.Deciphering how the virus uniquely maneuvers signaling pathways in monocytes to drive their survival and differentiation might allow us to develop new treatments targeting HCMV-infected monocytes and preventing viral spread and disease.
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  The Role of TET Methylcytosine Dioxygenase 2 in Myeloid Malignancies

  Mohi, Golam; Nath, Dipmoy (2017)

  TET methylcytosine dioxygenase 2 (TET2) catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an intermediate stage in the DNA demethylation processthat controls the transcription of genes. TET2 is highly expressed in the hematopoietic system and is suggestedto regulate the maintenance and differentiation of hematopoietic stem/progenitors. Mutations in TET2 has been found in different hematological malignancies including Acute Myeloid Leukemia (AML), Chronic Myelomonocytic Leukemia (CMML), Myeloproliferative Neoplasms (MPN), Myelodysplastic Syndrome (MDS), etc. However, the mechanisms by which TET2 controls hematopoiesis and contributes to myeloidmalignancies remainunknown. The direct targets of TET2 have not been elucidated yet. In order to identify the direct targets of TET2, we have performed chromatin immunoprecipitation using TET2 specific antibody followed by genome-wide sequencing. We have found enrichment in binding of TET2 in the promoters of SHP1, SOCS3 and PLZF among other targets. Interestingly, the expression of these genes is also significantly downregulated in the hematopoietic progenitors of TET2 deficient mice. Furthermore, we have found that CMML patients with TET2 mutations also have decreased expression of these genes. Knockdown of TET2 resulted in downregulation of these genes in leukemic Molm14 and murine Ba/F3 cells. Conversely, overexpression of TET2 in monocytic U937 cells increased the expression of these genes. Using methylation specific PCR, we also have observed increased methylation in the promoters of SHP1, SOCS3 and PLZF in TET2 deficient Molm14 and Ba/F3 cells. Also, using methyl/hydroxymethyl-DNA immunoprecipitation, we observed an increase in the 5mC level and decrease in the 5hmC level in the promoter region of these genes suggesting that TET2 directly regulates the expression of these genes by regulating the methyl and hydroxymethyl level of the promoter of these genes. Although loss of function of TET2 has been associated with multiple hematopoietic malignancies, TET2 is most commonly mutated in CMML with almost 50% patients bearing TET2 mutations. Interestingly, TET2 mutations are frequently associated with CBL mutations in CMML. In order to assess the concurrent effects of TET2 and CBL deficiencies, we generated TET2 CBL double knockout mice. We observed that simultaneous deletion of TET2 and CBL resulted in increased leukocytes and neutrophil and enhanced splenomegaly compared to control mice. The double knockout mice showed increase in the granulocyte macrophage progenitors and a significant expansion of the stem progenitor cell population. The overall survival of these mice also reduced substantially. It suggests that concurrent deletion of TET2 and CBL increased the severity of the CMML like disease in mice and thus TET2 and CBL deletion may cooperate in the pathogenesis of CMML. Although most studies suggest a tumor suppressor function of TET2, we also have found a tumor promoter function, especially in MLL rearranged leukemia. We have found that knockdown of TET2 resulted in decreased proliferation in MLL-AF9 positive Molm14 leukemia cells and murine Ba/F3 MLL-AF9 expressing cells. Conversely overexpression of TET2 significantly increased the proliferation level of Molm14 cells. In orderto understand the in vivo role of TET2 in MLL-AF9 mediated leukemia, we performed a retroviral BMT experiment. Whereas the expression of MLL-AF9 in wild type BM resulted in marked increase in WBC and NE and splenomegaly, the deletion of TET2 reduced the white blood cell and neutrophil count and also caused reduction in the spleen size. MLL-AF9 overexpression resulted in the increase in hematopoietic stem/progenitor cells and granulocyte macrophage progenitors and granulocyte/monocyte precursors which was significantly reduced in TET2 KO MLL-AF9 mice. There was a significant reduction of the hematopoietic colony formation ability mediated by MLL-AF9 in TET2 KO mice. The overall survival of the knockout mice was markedly improved compared to the control MLL-AF9 mice. Together these results suggest a tumor promoter role of TET2. Taken together, all the results indicate a dual role of TET2 in myeloid leukemia.
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  ANTIVIRAL ACTIVITY AND MECHANISM OF ACTION OF A NOVEL URACIL ANALOG FOR VARICELLA-ZOSTER VIRUS

  Moffat, Jennifer; DE, CHANDRAV (2015)

  The alphaherpesvirus varicella-zoster virus (VZV) is widespread in humans. VZV causes primary and recurrent diseases that are preventable with live vaccines and are treatable with antiviral drugs. New antiviral drugs for varicella-zoster virus (VZV) with increased potency are needed, especially to prevent post-herpetic neuralgia. The purpose of this project was to evaluate β-L-1-[5-(E-2-Bromovinyl)-2-(hydroxymethyl)-1,3-dioxolan-4-yl)] uracil (L-BHDU) and 5′-O-valyl-L-BHDU for efficacy, safety, resistance, and mechanism of action in three models of VZV replication: primary human foreskin fibroblasts (HFFs), skin organ culture (SOC) and in SCID-Hu mice with skin xenografts. We found that L-BHDU and valyl-L-BHDU were safe and effective against VZV in culture and in a mouse model. Herpes simplex virus Type 1 was also sensitive to LBHDU in cultured cells. The mechanism of action of L-BHDU and its effect on drugdrug interactions were not known. Given its similar structure to brivudine (BVdU), we addressed whether L-BHDU, like BVdU, inhibits 5-fluorouracil (5-FU) metabolism. LBHDU did not interfere with 5FU metabolism, indicating that L-BHDU is a safer drug than BVdU. However, L-BHDU antagonized the activity of acyclovir (ACV), BVdU and foscarnet (PFA) in cultured cells, which was due to competition for phosphorylation by VZV thymidine kinase (TK). The mechanism of action of L-BHDU was studied by evaluating its activity against related α-herpesviruses and by analyzing resistant VZV strains. VZV strains resistant to L-BHDU (L-BHDUR) were cross-resistant to ACV and BVdU but not to PFA and cidofovir (CDV). Whole genome sequencing of L-BHDUR strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV TK. The purified L-BHDUR TKs were enzymatically inactive and failed to phosphorylate the drug. In wild type VZV- infected cells, L-BHDU was converted to L-BHDU mono- and diphosphate forms; cells infected with L-BHDUR virus did not phosphorylate the drug. We also investigated whether addition of nucleosides reversed LBHDU inhibition of VZV in dividing and quiescent HFFs. Excess thymidine and uridine, but not purines, in proportion to L-BHDU restored VZV replication only in dividing cells, suggesting that the active form of L-BHDU interfered with pyrimidine biosynthesis. Like other herpesviruses, VZV infection induced thymidine triphosphate (dTTP) in confluent cells while L-BHDU treatment decreased the dTTP pool. Some herpesviruses raise dNTP pools by inducing cellular enzymes. However, VZV infection did not increase cellular thymidylate synthase (TS) expression to facilitate viral replication. Furthermore, the active form of L-BHDU did not interfere with cellular metabolism, suggesting a viral target. Further studies are required to identify the target(s) of L-BHDU active form(s).
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  The p53-Zn2+ Energy Landscape and Metallochaperone Hypothesis

  Loh, Stewart; Blanden, Adam (2017)

  p53 is a tumor suppressor protein found mutated in essentially half of human cancers, and dysfunctional in nearly all human cancers. Each DNA-binding domain of the protein contains a critical tetrahedrally coordinated Zn2+. In this work, we present a quantitative thermodynamic model describing the energetics of the p53-Zn2+ interaction, as well as the mechanism of action of a new class of therapeutic compounds we call synthetic zinc metallochaperones (ZMC) that restore proper structure and function to many mutant p53s by delivering Zn2+ to the protein in the cell. We combine recombinant protein expression and in vitrobiophysical characterization with cell biology, molecular biology, medicinal chemistry, and live cell imaging to address these issues. Our model for both the mechanism of action of ZMCs and the p53-Zn2+interaction are broadly based on the Metallochaperone Hypothesis originally proposed by our group in 2010. We find that the core tenants of the Metallochaperone Hypothesis are accurate, and have expanded that model to quantitatively describe the link between p53-Zn2+ binding and protein stability noted for decades in the field. We find that at physiological temperature and Zn2+ concentrations, wild-type p53 has a folding energy of ~0 kcal mol-1, and as such is exquisitely sensitive to inactivation by mutation, and rapidly changes the fraction folded in response to changes in Zn2+ concentration. We demonstrate that ZMCs are ionophores, transport Zn2+ from the extracellular space into cells, and rescue mutant p53 by increasing the intracellular free Zn2+ concentration. This increase in Zn2+ stabilizes the mutant proteins via the same mechanism previously described for substrate stabilization of enzymes, and is only seen in a "Goldilocks Zone" of Zn2+ concentrations and ZMC Kds. This presents a fundamentally new way to interact with and reactivate mutant p53s, and raises questions about the potential for biological exploitation of this interaction for signaling or other functions.
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  Agonal factors distort gene-expression patterns in human postmortem brains

  Dai, Jiacheng; Chen, Yu; Chen, Chao; Liu, Chunyu (Cold Spring Harbor Laboratory, 2020-07-12)

  Agonal factors, the conditions that occur just prior to death, can impact the molecular quality of postmortem brains, influencing gene expression results. Nevertheless, study designs using postmortem brain tissue rarely, if ever, account for these factors, and previous studies had not documented nor adjusted for agonal factors. Our study used gene expression data of 262 samples from ROSMAP with the following terminal states recorded for each donor: surgery, fever, infection, unconsciousness, difficulty breathing, and mechanical ventilation. Performed differential gene expression and weighted gene co-expression network analyses (WGCNA), fever and infection were the primary contributors to brain gene expression changes. Fever and infection also contributed to brain cell-type specific gene expression and cell proportion changes. Furthermore, the gene expression patterns implicated in fever and infection were unique to other agonal factors. We also found that previous studies of gene expression in postmortem brains were confounded by variables of hypoxia or oxygen level pathways. Therefore, correction for agonal factors through probabilistic estimation of expression residuals (PEER) or surrogate variable analysis (SVA) is recommended to control for unknown agonal factors. Our analyses revealed fever and infection contributing to gene expression changes in postmortem brains and emphasized the necessity of study designs that document and account for agonal factors.
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  Risk variants and polygenic architecture of disruptive behavior disorders in the context of attention-deficit/hyperactivity disorder

  Demontis, Ditte; Walters, Raymond K.; Rajagopal, Veera M.; Waldman, Irwin D.; Grove, Jakob; Als, Thomas D.; Dalsgaard, Søren; Ribasés, Marta; Bybjerg-Grauholm, Jonas; Bækvad-Hansen, Maria; et al. (Springer Science and Business Media LLC, 2021-01-25)

  Attention-Deficit/Hyperactivity Disorder (ADHD) is a childhood psychiatric disorder often comorbid with disruptive behavior disorders (DBDs). Here, we report a GWAS meta-analysis of ADHD comorbid with DBDs (ADHD + DBDs) including 3802 cases and 31,305 controls. We identify three genome-wide significant loci on chromosomes 1, 7, and 11. A meta-analysis including a Chinese cohort supports that the locus on chromosome 11 is a strong risk locus for ADHD + DBDs across European and Chinese ancestries (rs7118422, P = 3.15×10−10, OR= 1.17). We find a higher SNP heritability for ADHD + DBDs (h2 SNP = 0.34) when compared to ADHD without DBDs (h2 SNP = 0.20), high genetic correlations between ADHD + DBDs and aggressive (rg = 0.81) and anti-social behaviors (rg = 0.82), and an increased burden (polygenic score) of variants associated with ADHD and aggression in ADHD + DBDs compared to ADHD without DBDs. Our results suggest an increased load of common risk variants in ADHD + DBDs compared to ADHD without DBDs, which in part can be explained by variants associated with aggressive behavior.
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  Consortium neuroscience of attention deficit/hyperactivity disorder and autism spectrum disorder: The ENIGMA adventure

  Hoogman, Martine; Rooij, Daan; Klein, Marieke; Boedhoe, Premika; Ilioska, Iva; Li, Ting; Patel, Yash; Postema, Merel C.; Zhang‐James, Yanli; Anagnostou, Evdokia; et al. (Wiley, 2020-05-18)

  Neuroimaging has been extensively used to study brain structure and function in individuals with attention deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) over the past decades. Two of the main shortcomings of the neuroimaging literature of these disorders are the small sample sizes employed and the heterogeneity of methods used. In 2013 and 2014, the ENIGMA-ADHD and ENIGMA-ASD working groups were respectively, founded with a common goal to address these limitations. Here, we provide a narrative review of the thus far completed and still ongoing projects of these working groups. Due to an implicitly hierarchical psychiatric diagnostic classification system, the fields of ADHD and ASD have developed largely in isolation, despite the considerable overlap in the occurrence of the disorders. The collaboration between the ENIGMA-ADHD and -ASD working groups seeks to bring the neuroimaging efforts of the two disorders closer together. The outcomes of case–control studies of subcortical and cortical structures showed that subcortical volumes are similarly affected in ASD and ADHD, albeit with small effect sizes. Cortical analyses identified unique differences in each disorder, but also considerable overlap between the two, specifically in cortical thickness. Ongoing work is examining alternative research questions, such as brain laterality, prediction of case–control status, and anatomical heterogeneity. In brief, great strides have been made toward fulfilling the aims of the ENIGMA collaborations, while new ideas and follow-up analyses continue that include more imaging modalities (diffusion MRI and resting-state functional MRI), collaborations with other large databases, and samples with dual diagnoses.
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  Evidence for the multigenic inheritance of schizophrenia

  Freedman, Robert; Leonard, Sherry; Olincy, Ann; Kaufmann, Charles A.; Malaspina, Dolores; Cloninger, C. Robert; Svrakic, Dragan; Faraone, Stephen V.; Tsuang, Ming T. (Wiley, 2002-08-21)

  Schizophrenia is assumed to have complex inheritance because of its high prevalence and sporadic familial transmission. Findings of linkage on different chromosomes in various studies corroborate this assumption. It is not known whether these ®endings represent heterogeneous inheritance, in which various ethnic groups inherit illness through different major gene effects, or multigenic inheritance, in which affected individuals inherit several common genetic abnormalities. This study therefore examined inheritance of schizophrenia at different genetic loci in a nationally collected European American and African American sample. Seventy-seven families were previously genotyped at 458 markers for the NIMH Schizophrenia Genetics Initiative. Initial genetic analysis tested a dominant model, with schizophrenia and schizoaffective disorder, depressed type, as the affected phenotype. The families showed one genome-wide significant linkage (Z ¼ 3.97) at chromosome 15q14, which maps within 1 cM of a previous linkage at the a7-nicotinic receptor gene. Chromosome 10p13 showed suggestive linkage (Z ¼ 2.40). Six others (6q21, 9q32, 13q32, 15q24, 17p12, 20q13) were positive, with few differences between the two ethnic groups. The probability of each family transmitting schizophrenia through two genes is greater than expected from the combination of the independent segregation of each gene. Two trait-locus linkage analysis supports a model in which genetic alleles associated with schizophrenia are relatively common in the general population and affected individuals inherit risk for illness through at least two different loci.
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  Treatment of nonpsychotic relatives of patients with schizophrenia: Six case studies

  Tsuang, Ming T.; Stone, William S.; Tarbox, Sarah I.; Faraone, Stephen V. (Wiley, 2002-11-27)

  There is growing support for the notion that the genetic liability for schizophrenia could be manifested in brain dysfunction, even without the full manifestations of schizophrenia [Meehl, 1962, 1989; Seidman, 1997; Faraone et al., 2001]. This liability is characterized clinically by neurologic, neurobiological, psychiatric, neuropsychological, and psychosocial impairments in nonpsychotic, first-degree relatives of people with schizophrenia and includes eye tracking dysfunction [Levy et al., 1994], allusive thinking [Catts et al., 1993], neurologic signs [Erlenmeyer-Kimling et al., 1982], biochemical abnormalities [Callicott et al., 1998], char acteristic auditory evoked potentials [Friedman and Squires-Wheeler, 1994], neuroimaging assessed brain abnormalities [Seidman et al., 1997], and neuropsycho logical impairment [Kremen et al., 1994]. Paul Meehl introduced the term ‘‘schizotaxia’’ in 1962 to describe the genetic predisposition to schizophrenia [Meehl, 1962], and we have modified the concept to take account of subsequent research [Faraone et al., 2000]. The concept of schizotaxia raises at least three fundamental issues: 1) What is the conceptual basis of schizotaxia? 2) Is it a valid syndrome? and 3) perhaps most importantly from the point of view of the eventual prevention of schizo phrenia, is it treatable? In this paper, we review the model of schizotaxia by focusing first on its nature and extent. We then describe preliminary research criteria for its diagnosis in nonpsychotic relatives of schizo phrenic patients, followed by a presentation of our initial attempts to treat schizotaxia. Finally, prospects for the future focus on the need to validate the proposed syndrome further and on the clinical implications of treating schizotaxia.
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  Report from the third international meeting of the Attention-Deficit Hyperactivity Disorder Molecular Genetics Network

  Faraone, Stephen V. (Wiley, 2002-03-18)
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  Linkage of chromosome 13q32 to schizophrenia in a large veterans affairs cooperative study sample

  Faraone, Stephen V.; Skol, Andrew D.; Tsuang, Debby W.; Bingham, Stephen; Young, Keith A.; Prabhudesai, Sarita; Haverstock, Susan L.; Mena, Felicitas; Menon, Aerath Sri Kumar; Bisset, Darren; et al. (Wiley, 2002-07-29)

  Several prior reports have suggested that chromosomal region 13q32 may harbor a schizophrenia susceptibility gene. In an attempt to replicate this finding, we assessed linkage between chromosome 13 markers and schizophrenia in 166 families, each with two or more affected members. The families, assembled from multiple centers by the Department of Veterans Affairs Cooperative Studies Program, included 392 sampled affected subjects and 216 affected sib pairs. By DSM-III-R criteria, 360 subjects (91.8%) had a diagnosis of schizophrenia and 32 (8.2%) were classified as schizoaffective disorder, depressed. The families had mixed ethnic backgrounds. The majority were northern European-American families (n¼62, 37%), but a substantial proportion were African- American kindreds (n¼60, 36%). Chromosome 13 markers, spaced at intervals of approximately 10cMover the entire chromosome and 2–5 cM for the 13q32 region were genotyped and the data analyzed using semiparametric affected only linkage analysis. For the combined sample (with race broadly defined and schizophrenia narrowly defined) the maximum LOD score was 1.43 (Z-score of 2.57; P¼0.01) at 79.0 cM between markers D13S1241 (76.3 cM) and D13S159 (79.5 cM). Both ethnic groups showed a peak in this region. The peak is within 3 cMof the peak reported by Brzustowicz et al. [1999: Am J Hum Genet 65:1096–1103].

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