INTRODUCTION:The ApoE receptor 2-Disabled homolog-1 (ApoER2-Dab1) pathway suppresses Tau phosphorylation as part of a multi-arm pathway that regulates cytoskeletal and synaptic integrity. We previously showed that multiple ApoER2-Dab1 pathway components accumulate in regions affected in early Alzheimer's disease (AD). Since the amygdala is a hub for emotional regulation and fear memory, we hypothesized that accumulation of ApoER2-Dab1 components in amygdala may correlate with cognitive or neuropsychiatric manifestations of AD. METHODS:We used single-marker and multiplex immunohistochemistry to label ApoER2-Dab1 components in amygdala from 32 cases spanning the clinicopathological spectrum of AD. RESULTS:Seven ApoER2-Dab1 pathway components accumulated in amygdala and correlated with histological progression and cognitive or neurobehavioral deficits in AD. ApoER2-Dab1 components accumulated within ApoER2-expressing neurons and dystrophic neurites surrounding ApoE-enriched extracellular plaques. DISCUSSION:Findings add to growing evidence implicating ApoER2-Dab1 disruption in neurodegeneration and suggest that ApoER2-Dab1 disruption in amygdala may contribute to neuropsychiatric manifestations of AD.

Evaporation of sweat on the skin surface is the major mechanism for dissipating heat in humans. The secretory capacity of sweat glands (SWGs) declines during aging, leading to heat intolerance in the elderly, but the mechanisms responsible for this decline are poorly understood. We investigated the molecular changes accompanying SWG aging in mice, where sweat tests confirmed a significant reduction of active SWGs in old mice relative to young mice. We first identified SWG-enriched mRNAs by comparing the skin transcriptome of Eda mutant Tabby male mice, which lack SWGs, with that of wild-type control mice by RNA-sequencing analysis. This comparison revealed 171 mRNAs enriched in SWGs, including 47 mRNAs encoding 'core secretory' proteins such as transcription factors, ion channels, ion transporters, and trans-synaptic signaling proteins. Among these, 28 SWG-enriched mRNAs showed significantly altered abundance in the aged male footpad skin, and 11 of them, including Foxa1, Best2, Chrm3, and Foxc1 mRNAs, were found in the 'core secretory' category. Consistent with the changes in mRNA expression levels, immunohistology revealed that higher numbers of secretory cells from old SWGs express the transcription factor FOXC1, the protein product of Foxc1 mRNA. In sum, our study identified mRNAs enriched in SWGs, including those that encode core secretory proteins, and altered abundance of these mRNAs and proteins with aging in mouse SWGs.

ABSTRACTBackgroundPhysical activity is essential for maintaining muscle mitochondrial function and aerobic capacity. The molecular mechanisms underlying such protective effects are incompletely understood, in part because it is difficult to separate the effects of disease status and physical activity. We explored the association of human skeletal muscle transcriptomic with four measures of energetics and mitochondria oxidative capacity in healthy individuals.MethodsUsing RNA sequencing of vastus lateralis muscle biopsies from 82 GESTALT participants (52 males, aged 22–89 years), we explored gene and splicing variant expression profiles associated with self‐reported physical activity, peak oxygen consumption (VO2 peak), muscle oxidative capacity (kPCr) and mitochondrial respiration (Mit‐O2 flux). The effect of aging on gene expression was examined in participants with low and high VO2 peak.ResultsThe four measures of energetics were negative correlated with age and generally intercorrelated. We identified protein‐coding genes associated with four energetic measures adjusting for age, muscle fiber‐ratio, sex and batch effect. Mitochondrial pathways were overrepresented across all energetic variables, albeit with little overlap at the gene level. Alternative spliced transcript isoforms associated with energetics were primarily enriched for cytoplasmic ribonucleoprotein granules. The splicing pathway was up‐regulated with aging in low but not in high fitness participants, and transcript isoforms detected in the low fitness group pertain to processes such as cell cycle regulation, RNA/protein localization, nuclear transport and catabolism.ConclusionsA consistent mitochondrial signature emerged across all energetic measures. Alternative splicing was enhanced in older, low fitness participants supporting the energy‐splicing axis hypothesis. The identified splicing variants were enriched in pathways involving the accumulation of ribonucleoproteins in cytoplasmic granules, whose function remains unclear. Further research is needed to understand the function of these proteoforms in promoting adaptation to low energy availability.

PDF file 136K, Gene expression analysis of markers of proliferative and metastatic phenotypes

PDF file 255K, The Wnt5A mediated degradation of ROR1 is PKC-dependent

PDF file 103K, Decreasing ROR1 increases ROR2 and decreases proliferation in vivo

PDF file 110K, Figure legends for Supplementary Data

PDF file 165K, Wnt5A mediated degradation of ROR1 is not mediated through the lysosome

PDF file 234K, WNT5A induced degradation of ?-catenin requires SIAH2

PDF file 197K, Table of patient characteristics, and Wnt5A staining in paired samples pre and post-therapy

In metazoans, the largest sirtuin, SIRT1, is a nuclear protein implicated in epigenetic modifications, circadian signaling, DNA recombination, replication, and repair. Our previous studies have demonstrated that SIRT1 binds replication origins and inhibits replication initiation from a group of potential initiation sites (dormant origins). We studied the effects of aging and SIRT1 activity on replication origin usage and the incidence of transcription–replication collisions (creating R-loop structures) in adult human cells obtained at different time points during chronological aging and in cancer cells. In primary, untransformed cells, SIRT1 activity declined and the prevalence of R-loops rose with chronological aging. Both the reduction in SIRT1 activity and the increased abundance of R-loops were also observed during the passage of primary cells in culture. All cells, regardless of donor age or transformation status, reacted to the short-term, acute chemical inhibition of SIRT1 with the activation of excessive replication initiation events coincident with an increased prevalence of R-loops. However, cancer cells activated dormant replication origins, genome-wide, during long-term proliferation with mutated or depleted SIRT1, whereas, in primary cells, the aging-associated SIRT1-mediated activation of dormant origins was restricted to rDNA loci. These observations suggest that chronological aging and the associated decline in SIRT1 activity relax the regulatory networks that protect cells against excess replication and that the mechanisms protecting from replication–transcription collisions at the rDNA loci manifest as differentially enhanced sensitivities to SIRT1 decline and chronological aging.

PDF file 165K, Decreases in ROR2 expression increase tumor cell sensitivity to BRAF inhibitors

BACKGROUND:Von Willebrand factor (VWF) is elevated in sickle cell disease (SCD) and contributes to vaso-occlusion through its thrombogenic properties. VWF is regulated by ADAMTS13, a plasma protease that cleaves VWF into less bioactive multimers. Independent investigations have shown VWF to be elevated in SCD, whereas measurements of ADAMTS13 have been variable. OBJECTIVES:We assessed ADAMTS13 activity using multiple activity assays and measured levels of alternative VWF-cleaving proteases in SCD. METHODS/ PATIENTS:Plasma samples were collected from adult patients with SCD (n = 20) at a single institution when presenting for routine red cell exchange transfusion therapy. ADAMTS13 activity was measured by FRETS-VWF73, Technozym ADAMTS-13 Activity ELISA kit and a full-length VWF digestion reaction. Alternative VWF-cleaving proteases were identified by ELISA. A cell culture model was used to study the impact of SCD stimuli on endothelial ADAMTS13 and alternative VWF-cleaving proteases. RESULTS:ADAMTS13 activity was found to be moderately deficient across the SCD cohort as assessed by activity assays using a VWF A2 domain peptide substrate. However, SCD plasma showed preserved ability to digest full-length VWF, suggesting assay-discrepant results. Neutrophil and endothelial-derived proteases were found to be elevated in SCD plasma. Matrix metalloproteinase 9 specifically showed preferential cleavage of full-length VWF. Upregulation of alternative VWF-cleaving proteases occurred in endothelial cells exposed to SCD stimuli such as heme and hypoxia. CONCLUSIONS:This is the first demonstration of accessory plasma enzymes contributing to the regulation of VWF in a specific disease state and may have implications for assessing the VWF/ADAMTS13 axis in other settings.

During routine genome duplication, many potential replication origins remain inactive or 'dormant'. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that are distinguished from baseline (i.e. ordinarily active) origins by their preferential association with two phospho-isoforms of the helicase component MCM2. During normal unperturbed cell growth, baseline origins, but not dormant origins, associate with a form of MCM2 that is phosphorylated by DBF4-dependent kinase (DDK) on serine 139 (pS139-MCM2). This association facilitates the initiation of DNA replication from baseline origins. Concomitantly, SIRT1 inhibits Ataxia Telangiectasia and Rad3-related (ATR)-kinase-mediated phosphorylation of MCM2 on serine 108 (pS108-MCM2) by deacetylating the ATR-interacting protein DNA topoisomerase II binding protein 1 (TOPBP1), thereby preventing ATR recruitment to chromatin. In cells devoid of SIRT1 activity, or challenged by replication stress, this inhibition is circumvented, enabling ATR-mediated S108-MCM2 phosphorylation. In turn, pS108-MCM2 enables DDK-mediated phosphorylation on S139-MCM2 and facilitates replication initiation at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational MCM modifications that reflect a balance between SIRT1 activity and ATR signaling.

Background Compared to normal cells, cancer cells exhibit a higher level of oxidative stress, which primes key cellular and metabolic pathways and thereby increases their resilience under oxidative stress. This higher level of oxidative stress also can be exploited to kill tumor cells while leaving normal cells intact. In this study we have found that isovalerylspiramycin I (ISP I), a novel macrolide antibiotic, suppresses cancer cell growth and tumor metastases by targeting the nucleolar protein selenoprotein H (SELH), which plays critical roles in keeping redox homeostasis and genome stability in cancer cells. Methods We developed ISP I through genetic recombination and tested the antitumor effects using primary and metastatic cancer models. The drug target was identified using the drug affinity responsive target stability (DARTS) and mass spectrum assays. The effects of ISP I were assessed for reactive oxygen species (ROS) generation, DNA damage, R-loop formation and its impact on the JNK2/TIF-IA/RNA polymerase I (POLI) transcription pathway. Results ISP I suppresses cancer cell growth and tumor metastases by targeting SELH. Suppression of SELH induces accumulation of ROS and cancer cell-specific genomic instability. The accumulation of ROS in the nucleolus triggers nucleolar stress and blocks ribosomal RNA transcription via the JNK2/TIF-IA/POLI pathway, causing cell cycle arrest and apoptosis in cancer cells. Conclusions We demonstrated that ISP I links cancer cell vulnerability to oxidative stress and RNA biogenesis by targeting SELH. This suggests a potential new cancer treatment paradigm, in which the primary therapeutic agent has minimal side-effects and hence may be useful for long-term cancer chemoprevention.

BACKGROUND:Sporadic Alzheimer's disease (sAD) lacks a unifying hypothesis that can account for the lipid peroxidation observed early in the disease, enrichment of ApoE in the core of neuritic plaques, hallmark plaques and tangles, and selective vulnerability of entorhinal-hippocampal structures. OBJECTIVE:We hypothesized that 1) high expression of ApoER2 (receptor for ApoE and Reelin) helps explain this anatomical vulnerability; 2) lipid peroxidation of ApoE and ApoER2 contributes to sAD pathogenesis, by disrupting neuronal ApoE delivery and Reelin-ApoER2-Dab1 signaling cascades. METHODS:In vitro biochemical experiments; Single-marker and multiplex fluorescence-immunohistochemistry (IHC) in postmortem specimens from 26 individuals who died cognitively normal, with mild cognitive impairment or with sAD. RESULTS:ApoE and ApoER2 peptides and proteins were susceptible to attack by reactive lipid aldehydes, generating lipid-protein adducts and crosslinked ApoE-ApoER2 complexes. Using in situ hybridization alongside IHC, we observed that: 1) ApoER2 is strongly expressed in terminal zones of the entorhinal-hippocampal 'perforant path' projections that underlie memory; 2) ApoE, lipid aldehyde-modified ApoE, Reelin, ApoER2, and the downstream Reelin-ApoER2 cascade components Dab1 and Thr19-phosphorylated PSD95 accumulated in the vicinity of neuritic plaques in perforant path terminal zones in sAD cases; 3) several ApoE/Reelin-ApoER2-Dab1 pathway markers were higher in sAD cases and positively correlated with histological progression and cognitive deficits. CONCLUSION:Results demonstrate derangements in multiple ApoE/Reelin-ApoER2-Dab1 axis components in perforant path terminal zones in sAD and provide proof-of-concept that ApoE and ApoER2 are vulnerable to aldehyde-induced adduction and crosslinking. Findings provide the foundation for a unifying hypothesis implicating lipid peroxidation of ApoE and ApoE receptors in sAD.

Background The p97/valosin-containing protein (VCP) complex is a crucial factor for the segregation of ubiquitinated proteins in the DNA damage response and repair pathway. Objective We investigated whether blocking the p97/VCP function can inhibit the proliferation of RepID-deficient cancer cells using immunofluorescence, clonogenic survival assay, fluorescence-activated cell sorting, and immunoblotting. Result p97/VCP was recruited to chromatin and colocalized with DNA double-strand breaks in RepID-deficient cancer cells that undergo spontaneous DNA damage. Inhibition of p97/VCP induced death of RepID-depleted cancer cells. This study highlights the potential of targeting p97/VCP complex as an anticancer therapeutic approach. Conclusion Our results show that RepID is required to prevent excessive DNA damage at the endogenous levels. Localization of p97/VCP to DSB sites was induced based on spontaneous DNA damage in RepID-depleted cancer cells. Anticancer drugs targeting p97/VCP may be highly potent in RepID-deficient cells. Therefore, we suggest that p97/VCP inhibitors synergize with RepID depletion to kill cancer cells.

Atherosclerosis is an aging-related disease characterized by the buildup of plaque and hardening of the arteries over time. Previous studies have identified senescent cells in mouse and human atherosclerotic plaques. Cellular senescence, a hallmark of cells in aging tissues, is marked by irreversible growth arrest, elevated lysosomal activity, and increased production of p16, p21, p53 and SASP factors such as cytokines (e.g. IL-6, IL-8), chemokines (e.g. CCL2), adhesion molecules (e.g. ICAM-1), and angiogenic factors (e.g. VEGF). VSMCs cultured from atherosclerotic plaques express numerous markers of senescence, suggesting an important role for senescent cells in the development of vascular disease; however, the mechanisms that regulate senescence in vascular cells remain mostly unknown. In order to deepen our understanding of VSMC senescence, we assessed the changes in gene expression following exposure to senescence-inducing hypoxia. We identified a number of transcripts that were differentially expressed in senescent VSMCs using a microarray, and one transcript, GDF15 mRNA, encoding the growth factor GDF15, involved in the regulation of the cell cycle, was strongly and continuously induced with the progression of senescence. GDF15 has previously been found to be a novel SASP factor secreted in response to cellular stress and evidence suggests it can have pro- and anti-apoptotic and pro- and anti-inflammatory roles depending on the context and stage of vascular disease progression. Paracrine signaling to the surrounding cells (endothelial, macrophages, VSMCs, etc.) via SASP is crucial to the development of atherosclerosis in senescent cells, but little is known about the role of GDF15 in this process. We tested VSMCs with the traditional senescence model (Doxorubicin) and confirmed the significant increase in GDF15 mRNA conserved across different senescence systems in VSMCs, including hypoxia-induced senescence. Further, knockdown of GDF15 in pre-senescent VSMCs resulted in increased cell proliferation and decreased levels of senescence markers p21 and IL-6 mRNAs. Overall, our preliminary data suggests that VSMCs induce GDF15 steadily during the progression to senescence and that reducing GDF15 can help slow the progression of senescence in pre-senescent cells. We hypothesize that enhanced GDF15 expression in senescent VSMCs promotes inflammation and atherosclerosis development, and that therapeutically targeting GDF15 could offer new avenues for attenuating vascular disease. To confirm this, we will further investigate the role of GDF15 in senescent VSMCs using immunofluorescence and murine models of atherosclerosis to determine the mechanism by which GDF15 uniquely contributes to vascular cell senescence and increased disease progression.

The spindle assembly checkpoint (SAC) prevents premature chromosome segregation by inactivating the anaphase promoting complex/cyclosome (APC/C) until all chromosomes are properly attached to mitotic spindles. Here we identify a role for Cullin–RING ubiquitin ligase complex 4 (CRL4), known for modulating DNA replication, as a crucial mitotic regulator that triggers the termination of the SAC and enables chromosome segregation. CRL4 is recruited to chromatin by the replication origin binding protein RepID/DCAF14/PHIP. During mitosis, CRL4 dissociates from RepID and replaces it with RB Binding Protein 7 (RBBP7), which ubiquitinates the SAC mediator BUB3 to enable mitotic exit. During interphase, BUB3 is protected from CRL4-mediated degradation by associating with promyelocytic leukemia (PML) nuclear bodies, ensuring its availability upon mitotic onset. Deficiencies in RepID, CRL4 or RBBP7 delay mitotic exit, increase genomic instability and enhance sensitivity to paclitaxel, a microtubule stabilizer and anti-tumor drug.

In eukaryotic cells, a transcribed pre‐mRNA is spliced by the spliceosome, a catalytic machinery comprising proteins and noncoding RNAs. Splicing typically removes introns, but it can also generate circular RNAs (circRNAs). The body of the circRNA is fully present in the parent pre‐mRNA; the only unique sequence is found where the ends of the linear RNA are ligated—the junction. Initially, circRNAs were thought to be inert byproducts of this splicing process. However, in recent years, it has been discovered that circRNAs may have important functions within the cell, including acting as a microRNA “sponge” that limits the functions of the miRNA. Through this and other functions, circRNAs have been implicated in many diseases including cancer.The goal of this study is to visualize a particular circRNA in the cell. We have adopted the BaseScope technology (Advanced Cell Diagnostics, Newark, CA) to locate and visualize a circRNA via in situ hybridization. This technology utilizes a “ZZ”‐shaped probe to target specific sequences in RNA and greatly amplify a chromogenic signal. This method allows for probe hybridization to small RNA sequences, which is essential for visualization of circRNA, as the probe is specific to the circRNA junction sequence and will not hybridize to the pre‐mRNA. We have performed this method in HeLa cells to identify the circRNA circPCNX, a circRNA that associates with the cancer‐related RNA‐binding protein AUF1. We have also visualized PCNX mRNA, the linear counterpart of circPCNX. Ongoing BaseScope analysis reveals punctate dots, each representing a single instance of circPCNX, which allows for quantification of RNA copy numbers as well as visualization of their subcellular localization. Preliminary findings suggest that circPCNX exists in low abundancy in HeLa cells, while PCNX mRNA is found to be more abundant, and exists in both the cytoplasm and nucleus of HeLa cells. This method provides both descriptive and quantitative analyses of circPCNX that complement computational strategies to investigate circPCNX levels and localization in HeLa cells. Efforts to visualize circPCNX and other circRNAs shed light on the dynamics and functions of circRNAs and allow us to harness their therapeutic potential.Support or Funding InformationThis work was supported by the National Institute on Aging Intramural Research Program of the NIH.