👤 Brittany C Duncan

The accumulation of Aβ plaques and hyperphosphorylation of Tau neuropathologically characterize Alzheimer's disease (AD). Synaptic dysfunction and endoplasmic reticulum (ER) stress precede overt neuropathology. ER stress is characterized by the accumulation of unfolded/misfolded proteins, which leads to activation of the adaptive signaling pathway, the unfolded protein response (UPR). Chronic or unresolved ER stress, as in disease, is maladaptive and triggers the integrated stress response (ISR). We hypothesize that targeted attenuation of ISR activation would mitigate the early cognitive deficits and molecular pathology in the triple transgenic (3xTg) mouse model of AD. To test this hypothesis, we used an adeno-associated viral (AAV) vector to overexpress BiP, the key ER chaperone and UPR regulator, in the hippocampi of young 3xTg mice. BiP overexpression reduced phosphorylated PERK (pPERK), a marker of ISR activation, and increased synaptic proteins BDNF, PSD95, and choline acetyltransferase marker (ChAT). Hippocampal-dependent working memory, social memory, long-term spatial memory, and REM theta power were improved without changes in locomotion. BiP overexpression reduced neuroinflammation, as evidenced by a decrease in the astrocyte marker GFAP. Additionally, Aβ and Aβ42 levels were reduced in the hippocampus and cortex. Collectively, these findings indicate that modulation of ER stress via BiP overexpression ameliorates early cognitive and molecular alterations associated with AD. Show less

We previously reported that triazine thiols reduce apolipoprotein B (ApoB) secretion from human iPSC-derived hepatocytes (HLCs) and from humanized mice. To determine whether these compounds affected hepatocyte mRNA levels, we performed bulk RNA sequencing of HLCs treated with the triazine thiol DL-1 or with vehicle (DMSO) for 24 hours. Analyses revealed that in triazine thiol-treated cells, 145 mRNAs were reduced and 37 increased by ≥ 2-fold.  Several mRNAs encoding cysteine-rich metallothionines were upregulated, implying that HLCs respond to treatment by mounting a protective response through metal buffering. Show less

Bromodomain and extra-terminal domain (BET) proteins are therapeutic targets in several cancers including the most common malignant adult brain tumor glioblastoma (GBM). Multiple small molecule inhibitors of BET proteins have been utilized in preclinical and clinical studies. Unfortunately, BET inhibitors have not shown efficacy in clinical trials enrolling GBM patients. One possible reason for this may stem from resistance mechanisms that arise after prolonged treatment within a clinical setting. However, the mechanisms and timeframe of resistance to BET inhibitors in GBM is not known. To identify the temporal order of resistance mechanisms in GBM we performed quantitative proteomics using multiplex-inhibitor bead mass spectrometry and demonstrated that intrinsic resistance to BET inhibitors in GBM treatment occurs rapidly within hours and involves the fibroblast growth factor receptor 1 (FGFR1) protein. Additionally, small molecule inhibition of BET proteins and FGFR1 simultaneously induces synergy in reducing GBM tumor growth in vitro and in vivo. Further, FGFR1 knockdown synergizes with BET inhibitor mediated reduction of GBM cell proliferation. Collectively, our studies suggest that co-targeting BET and FGFR1 may dampen resistance mechanisms to yield a clinical response in GBM. Show less

Type 2 diabetes mellitus (T2DM) is a metabolic disease and comorbidity associated with several conditions, including cardiac dysfunction leading to heart failure with preserved ejection fraction (HFpEF), in turn resulting in T2DM-induced cardiomyopathy (T2DM-CM). However, the molecular mechanisms underlying the development of T2DM-CM are poorly understood. It is hypothesized that molecular alterations in myopathic genes induced by diabetes promote the development of HFpEF, whereas cardiac myosin inhibitors can rescue the resultant T2DM-mediated cardiomyopathy. To test this hypothesis, a Leptin receptor-deficient Show less

Ovulation is a cyclical biological rupture event fundamental to fertilisation and endocrine function. During this process, the somatic support cells that surround the germ cell undergo a remodelling process that culminates in breakdown of the follicle wall and release of a mature egg. Ovulation is driven by known proteolytic and inflammatory pathways as well as structural alterations to the follicle vasculature and the fluid-filled antral cavity. Ovulation is one of several types of systematic remodelling that occur in the human body that can be described as rupture. Although ovulation is a physiological form of rupture, other types of rupture occur in the human body which can be pathological, physiological, or both. In this review, we use intracranial aneurysms and chorioamniotic membrane rupture as examples of rupture events that are pathological or both pathological and physiological, respectively, and compare these to the rupture process central to ovulation. Specifically, we compared existing transcriptomic profiles, immune cell functions, vascular modifications, and biomechanical forces to identify common processes that are conserved between rupture events. In our transcriptomic analysis, we found 12 differentially expressed genes in common among two different ovulation data sets and one intracranial aneurysm data set. We also found three genes that were differentially expressed in common for both ovulation data sets and one chorioamniotic membrane rupture data set. Combining analysis of all three data sets identified two genes (Angptl4 and Pfkfb4) that were upregulated across rupture systems. Some of the identified genes, such as Rgs2, Adam8, and Lox, have been characterised in multiple rupture contexts, including ovulation. Others, such as Glul, Baz1a, and Ddx3x, have not yet been characterised in the context of ovulation and warrant further investigation as potential novel regulators. We also identified overlapping functions of mast cells, macrophages, and T cells in the process of rupture. Each of these rupture systems share local vasoconstriction around the rupture site, smooth muscle contractions away from the site of rupture, and fluid shear forces that initially increase and then decrease to predispose one specific region to rupture. Experimental techniques developed to study these structural and biomechanical changes that underlie rupture, such as patient-derived microfluidic models and spatiotemporal transcriptomic analyses, have not yet been comprehensively translated to the study of ovulation. Review of the existing knowledge, transcriptomic data, and experimental techniques from studies of rupture in other biological systems yields a better understanding of the physiology of ovulation and identifies avenues for novel studies of ovulation with techniques and targets from the study of vascular biology and parturition. Show less

A series of 10-alkoxy-Anthryl-isoxazole-pyrrole-doubletails (RO-AIMs) were synthesized using a crown ether assisted nucleophilic aromatic substitution followed by a modified Schotten-Baumann reaction. The novel RO-AIMs described here exhibit robust growth inhibition for the human SNB19 CNS glioblastoma cell line, and biphenyl analog 8c had activity in the nanomolar regime, which represents the most efficacious compound in the AIM series to date. Computational modeling for RO-AIMs binding in a ternary complex with c-myc quadruplex DNA and its helicase DHX36 is presented which represents our current working hypothesis. Show less

In cats, mutations in myosin binding protein C (encoded by the MYBPC3 gene) have been associated with hypertrophic cardiomyopathy (HCM). However, the molecular mechanisms linking these mutations to HCM remain unknown. Here, we establish Drosophila melanogaster as a model to understand this connection by generating flies harboring MYBPC3 missense mutations (A31P and R820W) associated with feline HCM. The A31P and R820W flies displayed cardiovascular defects in their heart rates and exercise endurance. We used RNA-seq to determine which processes are misregulated in the presence of mutant MYBPC3 alleles. Transcriptome analysis revealed significant downregulation of genes encoding small nucleolar RNA (snoRNAs) in exercised female flies harboring the mutant alleles compared to flies that harbor the wild-type allele. Other processes that were affected included the unfolded protein response and immune/defense responses. These data show that mutant MYBPC3 proteins have widespread effects on the transcriptome of co-regulated genes. Transcriptionally differentially expressed genes are also candidate genes for future evaluation as genetic modifiers of HCM as well as candidate genes for genotype by exercise environment interaction effects on the manifestation of HCM; in cats as well as humans. Show less

The purpose of this review is to contextualize findings from the first 25 years of PTSD genetics research, focusing on the most robust findings and interpreting results in light of principles that have emerged from modern genetics studies. Genome-wide association studies (GWAS) encompassing tens of thousands of participants enabled the first molecular genetic heritability and genetic correlation estimates for PTSD in 2017. In 2018, highly promising loci for PTSD were reported, including variants in and near the CAMKV, KANSL1, and TCF4 genes. Twin studies from 25 years ago established that PTSD is genetically influenced and foreshadowed the molecular genetic findings of today. Discoveries that were impossible with smaller studies have been achieved via collaborative/team-science efforts. Most promisingly, individual genomic loci offer entirely novel clues about PTSD etiology, providing the raw material for transformative discoveries, and the future of PTSD research is bright. Show less

Analogous to the c-Myc (Myc)/Max family of bHLH-ZIP transcription factors, there exists a parallel regulatory network of structurally and functionally related proteins with Myc-like functions. Two related Myc-like paralogs, termed MondoA and MondoB/carbohydrate response element-binding protein (ChREBP), up-regulate gene expression in heterodimeric association with the bHLH-ZIP Max-like factor Mlx. Myc is necessary to support liver cancer growth, but not for normal hepatocyte proliferation. Here, we investigated ChREBP's role in these processes and its relationship to Myc. Unlike Myc loss, ChREBP loss conferred a proliferative disadvantage to normal murine hepatocytes, as did the combined loss of ChREBP and Myc. Moreover, hepatoblastomas (HBs) originating in Show less

Genome-wide association studies have struggled to identify functional genes and variants underlying complex phenotypes. We recruited a multi-ethnic cohort of healthy volunteers (n = 91) and used their tissue to generate induced pluripotent stem cells (iPSCs) and hepatocyte-like cells (HLCs) for genome-wide mapping of expression quantitative trait loci (eQTLs) and allele-specific expression (ASE). We identified many eQTL genes (eGenes) not observed in the comparably sized Genotype-Tissue Expression project's human liver cohort (n = 96). Focusing on blood lipid-associated loci, we performed massively parallel reporter assays to screen candidate functional variants and used genome-edited stem cells, CRISPR interference, and mouse modeling to establish rs2277862-CPNE1, rs10889356-DOCK7, rs10889356-ANGPTL3, and rs10872142-FRK as functional SNP-gene sets. We demonstrated HLC eGenes CPNE1, VKORC1, UBE2L3, and ANGPTL3 and HLC ASE gene ACAA2 to be lipid-functional genes in mouse models. These findings endorse an iPSC-based experimental framework to discover functional variants and genes contributing to complex human traits. Show less

Neuronal ceroid lipofuscinoses (NCLs), a group of genetically distinct fatal neurodegenerative disorders with no treatment or cure, are clinically characterised by progressive motor and visual decline leading to premature death. While the underlying pathological mechanisms are yet to be precisely determined, the diseases share several common features including inflammation, lysosomal lipofuscin deposits and lipid abnormalities. An important hallmark of most common neurodegenerative disorders including Alzheimer's, Parkinson's and motor neuron diseases is deregulation of biologically active metal homeostasis. Metals such as zinc, copper and iron are critical enzyme cofactors and are important for synaptic transmission in the brain, but can mediate oxidative neurotoxicity when homeostatic regulatory mechanisms fail. We previously demonstrated biometal accumulation and altered biometal transporter expression in 3 animal models of CLN6 NCL disease. In this study we investigated the hypothesis that altered biometal homeostasis may be a feature of NCLs in general using 3 additional animal models of CLN1, CLN3 and CLN5 disease. We demonstrated significant accumulation of the biometals zinc, copper, manganese, iron and cobalt in these mice. Patterns of biometal accumulation in each model preceded significant neurodegeneration, and paralleled the relative severity of disease previously described for each model. Additionally, we observed deregulation of transcripts encoding the anti-oxidant protein, metallothionein (Mt), indicative of disruptions to biometal homeostasis. These results demonstrate that altered biometal homeostasis is a key feature of at least 4 genetically distinct forms of NCL disease. Show less

To determine the sensitivity of portal venous phase contrast-enhanced CT for the detection of renal stones. This retrospective study included 97 CT examinations of the abdomen without and with intravenous contrast, including 85 (87.6%) examinations with at least one renal stone on the "gold standard" noncontrast images, as scored by a single radiologist. Three other radiologists each independently reviewed only the contrast-enhanced images from all 97 examinations and recorded all renal stones. Reviewer sensitivity for stones was categorized by stone diameter. Reviewer sensitivity and specificity for stone disease were also calculated on a per-kidney basis. The 97 cases included a total of 238 stones ≥1 mm, with a mean (±SD) of 1.2 ± 1.9 stones per kidney and a stone diameter of 3.5 ± 3.0 mm. Pooling data for the three reviewers, sensitivity for all stones was 81%; sensitivity for stones ≥2, ≥3, ≥4, and ≥5 mm was 88%, 95%, 99%, and 98%, respectively. Sensitivity for stone disease on a per-kidney basis was 94% when considering all stones; when considering only stones ≥2, ≥3, and ≥4 mm, sensitivity was 96%, 99%, and 100%, respectively. Specificity for stone disease on a per-kidney basis was 98% overall, 99% when considering only stones ≥2 mm, and 100% when considering only stones ≥3 mm. Contrast-enhanced CT is highly sensitive for the detection of renal stones ≥3 mm in diameter and less sensitive for smaller stones. In cases where the clinical diagnosis is uncertain and performance of a CT examination is being contemplated, intravenous contrast utilization would allow assessment for stone disease while also optimizing evaluation for other conditions. Show less

Here we report that the transcription factor cyclic AMP-responsive element-binding protein H (CREB-H, encoded by CREB3L3) is required for the maintenance of normal plasma triglyceride concentrations. CREB-H-deficient mice showed hypertriglyceridemia secondary to inefficient triglyceride clearance catalyzed by lipoprotein lipase (Lpl), partly due to defective expression of the Lpl coactivators Apoc2, Apoa4 and Apoa5 (encoding apolipoproteins C2, A4 and A5, respectively) and concurrent augmentation of the Lpl inhibitor Apoc3. We identified multiple nonsynonymous mutations in CREB3L3 that produced hypomorphic or nonfunctional CREB-H protein in humans with extreme hypertriglyceridemia, implying a crucial role for CREB-H in human triglyceride metabolism. Show less

Bardet-Biedl syndrome is a pleiotropic disorder with 14 BBS genes identified. BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, and BBS9 form a complex called the BBSome, which is believed to recruit Rab8(GTP) to the primary cilium and promote ciliogenesis. The second group, the chaperonin-like proteins BBS6, BBS10, and BBS12, have been defined as a vertebrate-specific branch of the type II chaperonin superfamily. These may play a role in the regulation of BBSome assembly. Using sequence analysis, the role of BBS6, 10 and 12 was assessed in the patient population comprising 93 cases from 74 families. Systemic and ocular phenotypes were defined. In the study, chaperonin-like BBS gene mutations accounted for the disease in approximately 36.5% of BBS families. A total of 38 different non-polymorphic exonic sequence variants were identified in 40.5% of BBS families (41.9% cases), of which 26 were novel (68%). Six cases had mutations present in more than one chaperonin-like BBS gene. One case with four mutations in BBS10 had a phenotype of overall greater severity. The phenotypes observed were beyond the classic BBS phenotype as they overlapped with characteristics of MKKS (congenital heart defect, vaginal atresia, hydrometrocolpos, cryptorchidism), as well as Alström syndrome (diabetes, hearing loss, liver abnormalities, endocrine anomalies, cardiomyopathy). While overlap between the MKKS and BBS phenotypes has previously been reported for cases with BBS6 mutations, we also observed MKKS phenotypes involving BBS10 and BBS12 and Alström-like phenotypes associated with mutations in BBS1, BBS2, BBS6, BBS7, BBS9, BBS10 and BBS12 for the first time. Show less

Wiring of the nervous system is a multi-step process involving complex interactions of the growing fibre with its tissue environment and with neighbouring fibres. Nogo-A is a membrane protein enriched in the adult central nervous system (CNS) myelin, where it restricts the capacity of axons to grow and regenerate after injury. During development, Nogo-A is also expressed by neurons but its function in this cell type is poorly known. Here, we show that neutralization of neuronal Nogo-A or Nogo-A gene ablation (KO) leads to longer neurites, increased fasciculation, and decreased branching of cultured dorsal root ganglion neurons. The same effects are seen with antibodies against the Nogo receptor complex components NgR and Lingo1, or by blocking the downstream effector Rho kinase (ROCK). In the chicken embryo, in ovo injection of anti-Nogo-A antibodies leads to aberrant innervation of the hindlimb. Genetic ablation of Nogo-A causes increased fasciculation and reduced branching of peripheral nerves in Nogo-A KO mouse embryos. Thus, Nogo-A is a developmental neurite growth regulatory factor with a role as a negative regulator of axon-axon adhesion and growth, and as a facilitator of neurite branching. Show less

Studies in Drosophila and vertebrate systems have demonstrated that heparan sulfate proteoglycans (HSPGs) play crucial roles in modulating growth factor signaling. We have isolated mutations in sister of tout velu (sotv), a gene that encodes a co-polymerase that synthesizes HSPG glycosaminoglycan (GAG) chains. Our phenotypic and biochemical analyses reveal that HS levels are dramatically reduced in the absence of Sotv or its partner co-polymerase Tout velu (Ttv), suggesting that both copolymerases are essential for GAG synthesis. Furthermore, we find that mutations in sotv and ttv impair Hh, Wg and Decapentaplegic (Dpp) signaling. This contrasts with previous studies that suggested loss of ttv compromises only Hh signaling. Our results may contribute to understanding the biological basis of hereditary multiple exostoses (HME), a disease associated with bone overgrowth that results from mutations in EXT1 and EXT2, the human orthologs of ttv and sotv. Show less

Hereditary multiple exostoses (HME), a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The corresponding gene products, exostosin-1 (EXT1) and exostosin-2 (EXT2), are type II transmembrane glycoproteins which form a Golgi-localized heterooligomeric complex that catalyzes the polymerization of heparan sulfate (HS). Although the majority of the etiological mutations in EXT are splice-site, frameshift, or nonsense mutations that result in premature termination, 12 missense mutations have also been identified. Furthermore, two of the reported etiological missense mutations (G339D and R340C) have been previously shown to abrogate HS biosynthesis (McCormick et al. 1998). Here, a functional assay that detects HS expression on the cell surface of an EXT1-deficient cell line was used to test the remaining missense mutant exostosin proteins for their ability to rescue HS biosynthesis in vivo. Our results show that EXT1 mutants bearing six of these missense mutations (D164H, R280G/S, and R340S/H/L) are also defective in HS expression, but surprisingly, four (Q27K, N316S, A486V, and P496L) are phenotypically indistinguishable from wild-type EXT1. Three of these four "active" mutations affect amino acids that are not conserved among vertebrates and invertebrates, whereas all of the HS-biosynthesis null mutations affect only conserved amino acids. Further, substitution or deletion of each of these four residues does not abrogate HS biosynthesis. Taken together, these results indicate that several of the reported etiological mutant EXT forms retain the ability to synthesize and express HS on the cell surface. The corresponding missense mutations may therefore represent rare genetic polymorphisms in the EXT1 gene or may interfere with as yet undefined functions of EXT1 that are involved in HME pathogenesis. Show less

To gain entry into the host, viruses use host cell surface molecules that normally serve as receptors for other ligands. Herpes simplex virus type 1 (HSV-1) uses heparan sulphate (HS) glycosaminoglycans (GAGs) as receptors for initial attachment to the host cell surface. HS GAGs are both ubiquitous and structurally diverse, and normally serve as critical mediators of interactions between the cell and the extracellular environment. We have used the HS binding ability of HSV-1 to identify the function of a cellular gene, EXT1, which is involved in HS polymerisation. Cellular factors that affect virus growth and replication are often key regulators of the cell cycle and EXT1 is no different-humans with inherited mutations in EXT1 have developmental defects that lead to bone tumours (hereditary multiple exostoses, HME) and sometimes chondrosarcomas. Thus, as a result of using HSV-1 as a molecular probe, a functionally orphaned disease gene now has a defined function. These findings highlight the utility of viruses for investigating important cellular processes. Show less

Hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The proteins encoded by these genes, EXT1 and EXT2, are endoplasmic reticulum-localized type II transmembrane glycoproteins that possess or are tightly associated with glycosyltransferase activities involved in the polymerization of heparan sulfate. Here, by testing a cell line with a specific defect in EXT1 in in vivo and in vitro assays, we show that EXT2 does not harbor significant glycosyltransferase activity in the absence of EXT1. Instead, it appears that EXT1 and EXT2 form a hetero-oligomeric complex in vivo that leads to the accumulation of both proteins in the Golgi apparatus. Remarkably, the Golgi-localized EXT1/EXT2 complex possesses substantially higher glycosyltransferase activity than EXT1 or EXT2 alone, which suggests that the complex represents the biologically relevant form of the enzyme(s). These findings provide a rationale to explain how inherited mutations in either of the two EXT genes can cause loss of activity, resulting in hereditary multiple exostoses. Show less

Bone development is a highly regulated process sensitive to a wide variety of hormones, inflammatory mediators and growth factors. One of the most common hereditary skeletal dysplasias, hereditary multiple exostoses (HME), is an autosomal dominant disorder characterized by skeletal malformations that manifest as bony, benign tumours near the end of long bones. HME is usually caused by defects in either one of two genes, EXT1 and EXT2, which encode enzymes that catalyse the biosynthesis of heparan sulphate, an important component of the extracellular matrix. Thus, HME-linked bone tumours, like many other skeletal dysplasias, probably result from disruptions in cell surface architecture. However, despite the recent success in unravelling functions for several members of the EXT gene family, significant challenges remain before this knowledge can be used to develop new approaches for the diagnosis and treatment of disease. Show less