The cast of clasts: catabolism and vascular invasion during bone growth, repair, and disease by osteoclasts, chondroclasts, and septoclasts
Three named cell types degrade and remove skeletal tissues during growth, repair, or disease: osteoclasts, chondroclasts, and septoclasts. A fourth type, unnamed and less understood, removes nonmineralized cartilage during development of secondary ossification centers. "Osteoclasts," best known and studied, are polykaryons formed by fusion of monocyte precursors under the influence of colony stimulating factor 1 (CSF)-1 (M-CSF) and RANKL. They resorb bone during growth, remodeling, repair, and disease. "Chondroclasts," originally described as highly similar in cytological detail to osteoclasts, reside on and degrade mineralized cartilage. They may be identical to osteoclasts since to date there are no distinguishing markers for them. Because osteoclasts also consume cartilage cores along with bone during growth, the term "chondroclast" might best be reserved for cells attached only to cartilage. "Septoclasts" are less studied and appreciated. They are mononuclear perivascular cells rich in cathepsin B. They extend a cytoplasmic projection with a ruffled membrane and degrade the last transverse septum of hypertrophic cartilage in the growth plate, permitting capillaries to bud into it. To do this, antiangiogenic signals in cartilage must give way to vascular trophic factors, mainly vascular endothelial growth factor (VEGF). The final cell type excavates cartilage canals for vascular invasion of articular cartilage during development of secondary ossification centers. The "clasts" are considered in the context of fracture repair and diseases such as arthritis and tumor metastasis. Many observations support an essential role for hypertrophic chondrocytes in recruiting septoclasts and osteoclasts/chondroclasts by supplying VEGF and RANKL. The intimate relationship between blood vessels and skeletal turnover and repair is also examined.
Identification of a Chemical Probe for Family VIII Bromodomains through Optimization of a Fragment Hit
The acetyl post-translational modification of chromatin at selected histone lysine residues is interpreted by an acetyl-lysine specific interaction with bromodomain reader modules. Here we report the discovery of the potent, acetyl-lysine-competitive, and cell active inhibitor PFI-3 that binds to certain family VIII bromodomains while displaying significant, broader bromodomain family selectivity. The high specificity of PFI-3 for family VIII was achieved through a novel bromodomain binding mode of a phenolic headgroup that led to the unusual displacement of water molecules that are generally retained by most other bromodomain inhibitors reported to date. The medicinal chemistry program that led to PFI-3 from an initial fragment screening hit is described in detail, and additional analogues with differing family VIII bromodomain selectivity profiles are also reported. We also describe the full pharmacological characterization of PFI-3 as a chemical probe, along with phenotypic data on adipocyte and myoblast cell differentiation assays.
Together, the IFT81 and IFT74 N-termini form the main module for intraflagellar transport of tubulin
The assembly and maintenance of most cilia and flagella rely on intraflagellar transport (IFT). Recent in vitro studies have suggested that, together, the calponin-homology domain within the IFT81 N-terminus and the highly basic N-terminus of IFT74 form a module for IFT of tubulin. By using Chlamydomonas mutants for IFT81 and IFT74, we tested this hypothesis in vivo Modification of the predicted tubulin-binding residues in IFT81 did not significantly affect basic anterograde IFT and length of steady-state flagella but slowed down flagellar regeneration, a phenotype similar to that seen in a strain that lacks the IFT74 N-terminus. In both mutants, the frequency of tubulin transport by IFT was greatly reduced. A double mutant that combined the modifications to IFT81 and IFT74 was able to form only very short flagella. These results indicate that, together, the IFT81 and IFT74 N-termini are crucial for flagellar assembly, and are likely to function as the main module for IFT of tubulin.
Despite being densely packed with chromatin, nuclear bodies and a nucleoskeletal network, the nucleus is a remarkably dynamic organelle. Chromatin loops form and relax, RNA transcripts and transcription factors move diffusively, and nuclear bodies move. We show here that RNA splicing speckled domains (splicing speckles) fluctuate in constrained nuclear volumes and remodel their shapes. Small speckles move in a directed way toward larger speckles with which they fuse. This directed movement is reduced upon decreasing cellular ATP levels or inhibiting RNA polymerase II activity. The random movement of speckles is reduced upon decreasing cellular ATP levels, moderately reduced after inhibition of SWI/SNF chromatin remodeling and modestly increased upon inhibiting RNA polymerase II activity. To define the paths through which speckles can translocate in the nucleus, we generated a pressure gradient to create flows in the nucleus. In response to the pressure gradient, speckles moved along curvilinear paths in the nucleus. Collectively, our results demonstrate a new type of ATP-dependent motion in the nucleus. We present a model where recycling splicing factors return as part of small sub-speckles from distal sites of RNA processing to larger splicing speckles by a directed ATP-driven mechanism through interchromatin spaces.
The flaviviruses dengue virus (DENV) and Zika virus (ZIKV) are severe health threats with rapidly expanding ranges. To identify the host cell dependencies of DENV and ZIKV, we completed orthologous functional genomic screens using RNAi and CRISPR/Cas9 approaches. The screens recovered the ZIKV entry factor AXL as well as multiple host factors involved in endocytosis (RAB5C and RABGEF), heparin sulfation (NDST1 and EXT1), and transmembrane protein processing and maturation, including the endoplasmic reticulum membrane complex (EMC). We find that both flaviviruses require the EMC for their early stages of infection. Together, these studies generate a high-confidence, systems-wide view of human-flavivirus interactions and provide insights into the role of the EMC in flavivirus replication.
Researchers at the University of Massachusetts Medical School’s Systems and Psychosocial Advances Research Center (SPARC) assembled a team of Deaf and hearing researchers, clinicians, filmmakers, actors, artists, and Deaf people in recovery to develop Signs of Safety – a population-specific client toolkit and therapist companion guide that supplements Seeking Safety.
See the embedded video to view the contents of this document in American Sign Language (ASL).
Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder resulting from motor neuron death. Approximately 10% of cases are familial (FALS), typically with a dominant inheritance mode. Despite numerous advances in recent years, nearly 50% of FALS cases have unknown genetic aetiology. Here we show that mutations within the profilin 1 (PFN1) gene can cause FALS. PFN1 is crucial for the conversion of monomeric (G)-actin to filamentous (F)-actin. Exome sequencing of two large ALS families showed different mutations within the PFN1 gene. Further sequence analysis identified 4 mutations in 7 out of 274 FALS cases. Cells expressing PFN1 mutants contain ubiquitinated, insoluble aggregates that in many cases contain the ALS-associated protein TDP-43. PFN1 mutants also display decreased bound actin levels and can inhibit axon outgrowth. Furthermore, primary motor neurons expressing mutant PFN1 display smaller growth cones with a reduced F/G-actin ratio. These observations further document that cytoskeletal pathway alterations contribute to ALS pathogenesis.
Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3' untranslated regions (UTRs) or in 'strongly distal' 3' UTRs. Stau1 also interacts with actively translating ribosomes and with mRNA coding sequences (CDSs) and 3' UTRs in proportion to their GC content and propensity to form internal secondary structure. On mRNAs with high CDS GC content, higher Stau1 levels lead to greater ribosome densities, thus suggesting a general role for Stau1 in modulating translation elongation through structured CDS regions. Our results also indicate that Stau1 regulates translation of transcription-regulatory proteins.
The type III RNAse, Dicer, is responsible for the processing of microRNA (miRNA) precursors into functional miRNA molecules, non-coding RNAs that bind to and target messenger RNAs for repression. Dicer expression is essential for mouse midbrain development and dopaminergic (DAergic) neuron maintenance and survival during the early post-natal period. However, the role of Dicer in adult mouse DAergic neuron maintenance and survival is unknown. To bridge this gap in knowledge, we selectively knocked-down Dicer expression in individual DAergic midbrain areas, including the ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) via viral-mediated expression of Cre in adult floxed Dicer knock-in mice (Dicer(flox/flox)). Bilateral Dicer loss in the VTA resulted in progressive hyperactivity that was significantly reduced by the dopamine agonist, amphetamine. In contrast, decreased Dicer expression in the SNpc did not affect locomotor activity but did induce motor-learning impairment on an accelerating rotarod. Knock-down of Dicer in both midbrain regions of adult Dicer(flox/flox) mice resulted in preferential, progressive loss of DAergic neurons likely explaining motor behavior phenotypes. In addition, knock-down of Dicer in midbrain areas triggered neuronal death via apoptosis. Together, these data indicate that Dicer expression and, as a consequence, miRNA function, are essential for DAergic neuronal maintenance and survival in adult midbrain DAergic neuron brain areas.
Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remain controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than approximately 590 nt, and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs.
Functional Characterization of Novel PFN1 Mutations Causative for Familial Amyotrophic Lateral Sclerosis: A Dissertation
Amyotrophic lateral sclerosis (ALS) is a progressive adult neurodegenerative disease that causes death of both upper and lower motor neurons. Approximately 90 percent of ALS cases are sporadic (SALS), and 10 percent are inherited (FALS). Mutations in the PFN1 gene have been identified as causative for one percent of FALS. PFN1 is a small actin-binding protein that promotes actin polymerization, but how ALS-linked PFN1 mutations affect its cognate functions or acquire gain-of-function toxicity remains largely unknown.
To elucidate the contribution of ALS-linked PFN1 mutations to neurodegeneration, we have characterized these mutants in both mammalian cultured cells and Drosophila models. In mammalian neuronal cells, we demonstrate that ALS-linked PFN1 mutants form ubiquitinated aggregates and alter neuronal morphology. We also show that ALS-linked PFN1 mutants have partial loss-of-function effects on actin polymerization in growth cones of mouse primary motor neurons and larval neuromuscular junctions (NMJ) in Drosophila. In Drosophila, we also observe that PFN1 level influences integrity of adult motor neurons, as demonstrated by locomotion, lifespan, and leg NMJ morphology.
In sum, the work presented in this dissertation has shed light on PFN1- linked ALS pathogenesis by demonstrating a loss-of-function mechanism. We have also developed a Drosophila PFN1 model that will serve as a valuable tool to further uncover PFN1-associated cellular pathways that mediate motor neuron functions.
Function of the β4 Integrin in Cancer Stem Cells and Tumor Formation in Breast Cancer: A Masters Thesis
The integrin α6β4 (referred to as β4) is expressed in epithelial cells where it functions as a laminin receptor. Integrin β4 is important for the organization and maintenance of epithelial architecture in normal cells. Particularly, β4 is shown to be essential for mammary gland development during embryogenesis. Integrin β4 also plays important roles in tumor formation, invasion and metastasis in breast cancer. However, the mechanism of how integrin β4 mediates breast tumor formation has not been settled. A few studies suggest that integrin β4 is involved in cancer stem cells (CSCs), but the mechanism is not clear. To address this problem, I examined the expression of β4 in breast tumors and its potential role involved in regulating CSCs. My data shows that β4 is expressed heterogeneously in breast cancer, and it is not directly expressed in CSCs but associated with a basal epithelial population. This work suggests that β4 can regulate CSCs in a non-cell-autonomous manner through the interactions between β4+ non-CSC population and β4- CSC population. My data also shows that β4 expression is associated with CD24+CD44+ population in breast tumor. To further study the role of β4 in breast cancer progression, I generated a β4 reporter mouse by inserting a p2A-mCherry cassette before ITGB4 stop codon. This reporter mouse can be crossed with breast tumor models to track β4+ population during tumor progression.
Gene Therapy for Amyotrophic Lateral Sclerosis: An AAV Delivered Artifical MicroRNA Against Human SOD1 Increases Survival and Delays Disease Progression of the SOD1<sup>G93A</sup> Mouse Model: A Dissertation
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, atrophy, paralysis and death within five years of diagnosis. About ten percent of cases are inherited, of which twenty percent are due to mutations in the superoxide dismutase 1 (SOD1) gene. Since the only FDA approved ALS drug prolongs survival by just a few months, new therapies for this disease are needed. Experiments in transgenic ALS mouse models have shown that decreasing levels of mutant SOD1 protein alters and in some cases entirely prevents disease progression. We explored this potential therapeutic approach by using a single stranded AAV9 vector encoding an artificial microRNA against human SOD1 injected bilaterally into the cerebral lateral ventricles of neonatal SOD1G93A mice. This therapy extended median survival from 135 to 206 days (a 50% increase) and delayed hind limb paralysis. Animals remained ambulatory until endpoint, as defined by a sharp drop in body weight. Treated animals had a reduction of mutant human SOD1 mRNA levels in upper and lower motor neurons. As compared to untreated SOD1G93A mice, the AAV9 treated mice also had significant improvements in multiple parameters including the number of motor neurons, diameter of ventral root axons, and degree of neuroinflammation in the spinal cord. These studies clearly show that an AAV9-delivered artificial microRNA is a translatable therapeutic approach for ALS.
Eukaryotic genomes can produce two types of transcripts: protein-coding and non-coding RNAs (ncRNAs). Cryptic ncRNA transcripts are bona fide RNA Pol II products that originate from bidirectional promoters, yet they are degraded by the RNA exosome. Such pervasive transcription is prevalent across eukaryotes, yet its regulation and function is poorly understood.
We hypothesized that chromatin architecture at cryptic promoters may regulate ncRNA transcription. Nucleosomes that flank promoters are highly enriched in two histone marks: H3-K56Ac and the variant H2A.Z, which make nucleosomes highly dynamic. These histone modifications are present at a majority of promoters and their stereotypic pattern is conserved from yeast to mammals, suggesting their evolutionary importance. Although required for inducing a handful of genes, their contribution to steady-state transcription has remained elusive. In this work, we set out to understand if dynamic nucleosomes regulate cryptic transcription and how this is coordinated with the RNA exosome.
Remarkably, we find that H3-K56Ac promotes RNA polymerase II occupancy at a large number of protein coding and noncoding loci, yet neither histone mark has a significant impact on steady state mRNA levels in budding yeast. Instead, broad effects of H3-K56Ac or H2A.Z on levels of both coding and ncRNAs are only revealed in the absence of the nuclear RNA exosome. We show that H2A.Z functions with H3-K56Ac in chromosome folding, facilitating formation of Chromosomal Interaction Domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA levels, perhaps in part by regulating higher order chromatin structures. Together, these chromatin factors achieve a balance of RNA exosome activity (yin; negative) and Pol II (yang; positive) to maintain transcriptional homeostasis.
Recently, the medial habenula-interpeduncular (MHb-IPN) axis has been hypothesized to modulate anxiety although neuronal populations and molecular mechanisms regulating affective behaviors in this circuit are unknown. Here we show that MHb cholinergic neuron activity directly regulates anxiety-like behavior. Optogenetic silencing of MHb cholinergic IPN inputs reduced anxiety-like behavior in mice. MHb cholinergic neurons are unique in that they robustly express neuronal nicotinic acetylcholine receptors (nAChRs), although their role as autoreceptors in these neurons has not been described. nAChRs are ligand-gated cation channels that are activated by the excitatory neurotransmitter, acetylcholine (ACh), as well as nicotine, the addictive component of tobacco smoke. We expressed novel nAChR subunits that render nAChRs hypersensitive to ACh, ACh detectors, selectively in MHb cholinergic neurons of adult mice. Mice expressing these ACh detectors exhibited increased baseline anxiety-like behavior that was alleviated by blocking the mutant receptors. Under stressful conditions, such as during nicotine withdrawal, nAChRs were functionally upregulated in MHb cholinergic neurons mediating an increase in anxiety-like behavior. Together, these data indicate that MHb cholinergic neurons regulate anxiety via signaling through nicotinic autoreceptors and point toward nAChRs in MHb as molecular targets for novel anxiolytic therapeutics.
Optimizing RNA Library Preparation to Redefine the Translational Status of 80S Monosomes: A Dissertation
Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved. This optimized method was used in an adapted ribosome-profiling approach to sequence mRNA footprints protected either by 80S monosomes or polysomes in S. cerevisiae. Contrary to popular belief, we show that 80S monosomes are translationally active as demonstrated by strong three-nucleotide phasing of monosome footprints across open reading frames. Most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on upstream ORFs, canonical ORFs shorter than ~590 nucleotides and any ORF for which the total time required to complete elongation is substantially shorter than the time required for initiation. Additionally, endogenous NMD targets tend to be monosome-enriched. Thus, rather than being inactive, 80S monosomes are significant contributors to overall cellular translation.
Phase III Preclinical Trials in Translational Stroke Research: Community Response on Framework and Guidelines
The multicenter phase III preclinical trial concept is currently discussed to enhance the predictive value of preclinical stroke research. After public announcement, we collected a community feedback on the concept with emphasis on potential design features and guidelines by an anonymous survey. Response analysis was conducted after plausibility checks by applying qualitative and quantitative measures. Most respondents supported the concept, including the implementation of a centralized steering committee. Based on received feedback, we suggest careful, stepwise implementation and to leave selected competencies and endpoint analysis at the discretion of participating centers. Strict application of quality assurance methods is accepted, but should be harmonized. However, received responses also indicate that the application of particular quality assurance models may require more attention throughout the community. Interestingly, clear and pragmatic preferences were given regarding publication and financing, suggesting the establishing of writing committees similar to large-scale clinical trials and global funding resources for financial support. The broad acceptance among research community encourages phase III preclinical trial implementation.
Spatial learning is known to depend on protein synthesis in the hippocampus. Whereas the role of the hippocampus in spatial memory is established, the biochemical and molecular mechanisms underlying this process are poorly understood. To comprehend the complex pattern of protein expression induced by spatial learning, we analyzed alterations in the rat hippocampus proteome after 7 days of spatial learning in the Morris water maze. Forty Wistar rats were randomized into two groups. Animals of group A learned to localize a hidden platform in the water maze. Animals of group B served as controls and spent exactly the same time in the water maze as animals of group A. However, no platform was used in this test and the rats could not learn to localize the target. After the last trial, hydrophilic proteins from the hippocampus were isolated. A proteome-wide study was performed, based on two-dimensional gel electrophoresis and mass spectrometry. Compared with non-learning animals, 53 (70%) proteins were downregulated and 23 (30%) proteins were upregulated after 7 days in rats with spatial learning. The overall changes in protein expression, as quantified by the induction factor, ranged from -1.62 (downregulation to 62%) to 2.10 (upregulation by 110%) compared with controls (100%). Most identified proteins exhibit known functions in vesicle transport, cytoskeletal architecture, and metabolism as well as neurogenesis. These findings indicate that learning in the Morris water maze has a morphological correlate on the proteome level in the hippocampus.
Electrophysiology and neuronal integrity following systemic arterial hypotension in a rat model of unilateral carotid artery occlusion
Patients with carotid artery stenosis may be particularly susceptible to hypotension-associated cerebral ischemia and subsequent neurological sequelae. Measuring somatosensory evoked potentials (SEP), electroencephalogram (EEG), direct current (DC) potential, and histology, we compared the temporal evolution of cortical functional perturbations as well as neuronal integrity in a model of unilateral carotid artery occlusion and systemic hypobaric hypotension (HH) at the lower limit of cerebral blood flow autoregulation (50 mm Hg). Serial measurements of EEG power spectra as well as SEP-amplitudes and latencies of N10.3 were performed before, during, and up to 60 min after 30 min-HH (n=7) or the control condition (n=7) in male Wistar rats. In two additional groups (with [n=7] or without [n=7] HH), cortical spreading depressions (CSD) were elicited to ascertain their contribution to brain injury. Hematoxilin-Eosin (HandE) staining was used to assess neuronal cell death at 5 days after surgery. Relative to baseline, HH attenuated ipsilateral EEG power spectrum (by maximally 62%), increased SEP-latencies (by approximately 6-10%) and amplitudes (by approximately 57-70%), and induced selective neuronal cell death in the cerebral cortex and hippocampus (P < 0.05 vs. contralateral). Spontaneous CSD occurred in approximately 30% of HH-animals. Repolarization of the DC-potential during HH was significantly prolonged relative to normotensive conditions (10.3+/-11.5 min, P < 0.001). Our model may help to understand underlying pathophysiology and improve outcome in a clinical subset of patients with carotid artery stenosis and transient systemic hypotension.