Faculty Bibliography

Like many animals and humans, reproduction in the nematode C. elegans declines with age. This decline is the cumulative result of age-related changes in several steps of germline function, many of which are highly accessible for experimental investigation in this short-lived model organism. Here we review recent work showing that a very early and major contributing step to reproductive decline is the depletion of the germline stem and progenitor cell pool. Since many cellular and molecular aspects of stem cell biology and aging are conserved across animals, understanding mechanisms of age-related decline of germline stem and progenitor cells in C. elegans has broad implications for aging stem cells, germline stem cells, and reproductive aging.

Single molecule analysis of replicating DNA (SMARD) is a powerful methodology that allows in vivo analysis of replicating DNA; identification of origins of replication, assessment of fork directionality, and measurement of replication fork speed. SMARD, which has been extensively used to study replication of nuclear DNA, involves incorporation of thymidine analogs to nascent DNA chains and their subsequent visualization through immune detection. Here, we adapt and fine-tune the SMARD technique to the specifics of human and mouse mitochondrial DNA. The mito-SMARD protocol allows researchers to gain in vivo insight into mitochondrial DNA (mtDNA) replication at the single molecule level and with high resolution.

Biomarkers of neurodegeneration and neuronal injury have the potential to improve diagnostic accuracy, disease monitoring, prognosis, and measure treatment efficacy. Neurofilament proteins (NfPs) are well suited as biomarkers in these contexts because they are major neuron-specific components that maintain structural integrity and are sensitive to neurodegeneration and neuronal injury across a wide range of neurologic diseases. Low levels of NfPs are constantly released from neurons into the extracellular space and ultimately reach the cerebrospinal fluid (CSF) and blood under physiological conditions throughout normal brain development, maturation, and aging. NfP levels in CSF and blood rise above normal in response to neuronal injury and neurodegeneration independently of cause. NfPs in CSF measured by lumbar puncture are about 40-fold more concentrated than in blood in healthy individuals. New ultra-sensitive methods now allow minimally invasive measurement of these low levels of NfPs in serum or plasma to track disease onset and progression in neurological disorders or nervous system injury and assess responses to therapeutic interventions. Any of the five Nf subunits - neurofilament light chain (NfL), neurofilament medium chain (NfM), neurofilament heavy chain (NfH), alpha-internexin (INA) and peripherin (PRPH) may be altered in a given neuropathological condition. In familial and sporadic Alzheimer's disease (AD), plasma NfL levels may rise as early as 22 years before clinical onset in familial AD and 10 years before sporadic AD. The major determinants of elevated levels of NfPs and degradation fragments in CSF and blood are the magnitude of damaged or degenerating axons of fiber tracks, the affected axon caliber sizes and the rate of release of NfP and fragments at different stages of a given neurological disease or condition directly or indirectly affecting central nervous system (CNS) and/or peripheral nervous system (PNS). NfPs are rapidly emerging as transformative blood biomarkers in neurology providing novel insights into a wide range of neurological diseases and advancing clinical trials. Here we summarize the current understanding of intracellular NfP physiology, pathophysiology and extracellular kinetics of NfPs in biofluids and review the value and limitations of NfPs and degradation fragments as biomarkers of neurodegeneration and neuronal injury.

Investigations into the regulatory mechanisms controlling cholesterol homeostasis have proven fruitful in identifying low-density lipoprotein (LDL)-lowering therapies to reduce the risk of atherosclerotic cardiovascular disease. A major advance was the discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9), a secreted protein that binds the LDL receptor (LDLR) on the cell surface and internalizes it for degradation, thereby blunting its ability to take up circulating LDL. The discovery that loss-of-function mutations in PCSK9 lead to lower plasma levels of LDL cholesterol and protection from cardiovascular disease led to the therapeutic development of PCSK9 inhibitors at an unprecedented pace. However, there remain many gaps in our understanding of PCSK9 regulation and biology, including its posttranscriptional control by microRNAs. Using a high-throughput region(3'-UTR) of human microRNA library screen, we identified microRNAs targeting the 3' untranslated region of human PCSK9. The top 35 hits were confirmed by large-format PCSK9 3'-UTR luciferase assays, and 10 microRNAs were then selected for further validation in hepatic cells, including effects on PCSK9 secretion and LDLR cell surface expression. These studies identified seven novel microRNAs that reduce PCSK9 expression, including miR-221-5p, miR-342-5p, miR-363-5p, miR-609, miR-765, and miR-3165. Interestingly, several of these microRNAs were also found to target other genes involved in LDLR regulation and potently upregulate LDLR cell surface expression in hepatic cells. Together, these data enhance our understanding of post-transcriptional regulators of PCSK9 and their potential for therapeutic manipulation of hepatic LDLR expression.

TNFα/TNFR signaling plays a critical role in the pathogenesis of various inflammatory and autoimmune diseases, and anti-TNFα therapies have been accepted as the effective approaches for treating several autoimmune diseases. Progranulin (PGRN), a multi-faced growth factor-like molecule, directly binds to TNFR1 and TNFR2, particularly to the latter with higher affinity than TNFα. PGRN derivative Atsttrin is composed of three TNFR-binding domain of PGRN and exhibits even better therapeutic effects than PGRN in several inflammatory disease models, including collagen-induced arthritis. Herein we describe the detailed methodology of using (1) ELISA-based solid phase protein-protein interaction assay to demonstrate the direct binding of Atsttrin to TNFR2 and its inhibition of TNFα/TNFR2 interaction; and (2) tartrate-resistant acid phosphatase (TRAP) staining of in vitro osteoclastogenesis to reveal the cell-based anti-TNFα activity of Atsttrin. Using the protocol described here, the investigators should be able to reproducibly detect the physical inhibition of TNFα binding to TNFR and the functional inhibition of TNFα activity by Atsttrin and various kinds of TNF inhibitors.

Gap junctions are molecular structures that allow communication between neighboring cells. It has been shown that gap junctional intercellular communication (GJIC) is notoriously reduced in cancer cells compared to their normal counterparts. Ouabain, a plant derived substance, widely known for its therapeutic properties on the heart, has been shown to play a role in several types of cancer, although its mechanism of action is not yet fully understood. Since we have previously shown that ouabain enhances GJIC in epithelial cells (MDCK), here we probed whether ouabain affects GJIC in a variety of cancer cell lines, including cervico-uterine (CasKi, SiHa and Hela), breast (MDA-MB-321 and MCF7), lung (A549), colon (SW480) and pancreas (HPAF-II). For this purpose, we conducted dye transfer assays to measure and compare GJIC in monolayers of cells with and without treatment with ouabain (0.1, 1, 10, 50 and 500 nM). We found that ouabain induces a statistically significant enhancement of GJIC in all of these cancer cell lines, albeit with distinct sensitivity. Additionally, we show that synthesis of new nucleotides or protein subunits is not required, and that Csrc, ErK1/2 and ROCK-Rho mediate the signaling mechanisms. These results may contribute to explaining how ouabain influences cancer.

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9-mediated internalization of ALK-1, BMP-9-dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9-induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9-mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

Type I and III interferons induce expression of the "myxovirus resistance proteins" MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles ("biomolecular condensates"). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40-50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20-30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.