The role of inhibition of osteocyte apoptosis in mediating orthodontic tooth movement and periodontal remodeling: a pilot study.

BACKGROUND: Orthodontic tooth movement (OTM) has been shown to induce osteocyte apoptosis in alveolar bone shortly after force application. However, how osteocyte apoptosis affects orthodontic tooth movement is unknown. The goal of this study was to assess the effect of inhibition of osteocyte apoptosis on osteoclastogenesis, changes in the alveolar bone density, and the magnitude of OTM using a bisphosphonate analog (IG9402), a drug that affects osteocyte and osteoblast apoptosis but does not affect osteoclasts. MATERIAL AND METHODS: Two sets of experiments were performed. Experiment 1 was used to specifically evaluate the effect of IG9402 on osteocyte apoptosis in the alveolar bone during 24 h of OTM. For this experiment, twelve mice were divided into two groups: group 1, saline administration + OTM24-h (n=6), and group 2, IG9402 administration + OTM24-h (n=6). The contralateral unloaded sides served as the control. The goal of experiment 2 was to evaluate the role of osteocyte apoptosis on OTM magnitude and osteoclastogenesis 10 days after OTM. Twenty mice were divided into 4 groups: group 1, saline administration without OTM (n=5); group 2, IG9402 administration without OTM (n=5); group 3, saline + OTM10-day (n=6); and group 4, IG9402 + OTM10-day (n=4). For both experiments, tooth movement was achieved using Ultra Light (25g) Sentalloy Closed Coil Springs attached between the first maxillary molar and the central incisor. Linear measurements of tooth movement and alveolar bone density (BVF) were assessed by MicroCT analysis. Cell death (or apoptosis) was assessed by terminal dUTP nick-end labeling (TUNEL) assay, while osteoclast and macrophage formation were assessed by tartrate-resistant acid phosphatase (TRAP) staining and F4/80+ immunostaining. RESULTS: We found that IG9402 significantly blocked osteocyte apoptosis in alveolar bone (AB) at 24 h of OTM. At 10 days, IG9402 prevented OTM-induced loss of alveolar bone density and changed the morphology and quality of osteoclasts and macrophages, but did not significantly affect the amount of tooth movement. CONCLUSION: Our study demonstrates that osteocyte apoptosis may play a significant role in osteoclast and macrophage formation during OTM, but does not seem to play a role in the magnitude of orthodontic tooth movement.

mTOR deletion in neural crest cells disrupts cardiac outflow tract remodeling and causes a spectrum of cardiac defects through the mTORC1 pathway.

A critical cell type participating in cardiac outflow tract development is a subpopulation of the neural crest cells, the cardiac neural crest cells (NCCs), whose defect causes a spectrum of cardiovascular abnormalities. Accumulating evidence indicates that mTOR, which belongs to the PI3K-related kinase family and impacts multiple signaling pathways in a variety of contexts, plays a pivotal role for NCC development. Here, we investigated functional roles of mTOR for cardiac neural crest development using several lines of mouse genetic models. We found that disruption of mTOR caused NCC defects and failure of cardiac outflow tract separation, which resulted in a spectrum of cardiac defects including persistent truncus arteriosus, ventricular septal defect and ventricular wall defect. Specifically, mutant neural crest cells showed reduced migration into the cardiac OFT and prematurely exited the cell cycle. A number of critical factors and fundamental signaling pathways, which are important for neural crest and cardiomyocyte development, were impaired. Moreover, actin dynamics was disrupted by mTOR deletion. Finally, by phenotyping the neural crest Rptor and Rictor knockout mice respectively, we demonstrate that mTOR acts principally through the mTORC1 pathway for cardiac neural crest cells. Altogether, these data established essential roles of mTOR for cardiac NCC development and imply that dysregulation of mTOR in NCCs may underline a spectrum of cardiac defects.

Oral and Gut Microbial Diversity and Immune Regulation in Patients with HIV on Antiretroviral Therapy.

Despite evidence of a chronic inflammatory phenotype in people living with HIV (PLWH) on antiretroviral therapy (ART), the role of oral microbiota in chronic immune activation has not been fully explored. We aimed to determine the relationship between oral and gut microbiome diversity and chronic systemic inflammation in ART-treated PLWH with prevalent severe periodontitis, an inflammatory condition commonly associated with HIV infection. We assessed bacterial and fungal communities at oral and gastrointestinal sites in a cohort (n = 52) of primarily postmenopausal women on ART using 16S rRNA and internal transcribed spacer (ITS) sequencing and measured cellular and soluble markers of inflammation and immune dysfunction. Linear mixed-effect regression and differential abundance analyses were used to associate clinical characteristics and immunological markers with bacterial and fungal diversity and community composition. Bacterial α-diversity in plaque, saliva, and gut was associated with different immunological markers, while mycobial diversity was not associated with soluble or cellular biomarkers of immune stimulation or T cell dysfunction. Furthermore, lipopolysaccharide-positive (LPS+) bacteria previously linked to inflammatory outcomes were enriched at oral sites in patients with severe periodontitis. Fungal α-diversity was reduced in plaque from teeth with higher clinical attachment loss, a marker of periodontitis, and in saliva and plaque from patients with a history of AIDS. Our results show that both bacterial and fungal oral microbiome communities likely play a role in chronic systemic immune activation in PLWH. Thus, interventions targeting both inflammation and the microbiome, particularly in the oral cavity, may be necessary to reduce chronic immune dysregulation in patients with HIV.IMPORTANCE A feedback loop between dysbiotic gut microbiota, increased translocation of microbial products such as lipopolysaccharide, and inflammation has been hypothesized to cause immune system dysfunction in early HIV infection. However, despite evidence of a chronic inflammatory phenotype in patients on antiretroviral therapy (ART), the role of oral microbiota in systemic immune activation and the relationship between oral and gut bacterial and fungal diversity have not been explored. Our study suggests a crucial role for oral bacterial and fungal communities in long-term systemic immune activation in patients on ART, expanding the current paradigm focused on gut bacteria. Our results indicate that interventions targeting both inflammation and microbial diversity are needed to mitigate oral inflammation-related comorbidities, particularly in HIV-positive patients. More broadly, these findings can bolster general models of microbiome-mediated chronic systemic immune activation and aid the development of precise microbiota-targeted interventions to reverse chronic inflammation.

mTOR acts as a pivotal signaling hub for neural crest cells during craniofacial development.

mTOR is a highly conserved serine/threonine protein kinase that is critical for diverse cellular processes in both developmental and physiological settings. mTOR interacts with a set of molecules including Raptor and Rictor to form two distinct functional complexes, namely the mTORC1 and mTORC2. Here, we used novel genetic models to investigate functions of the mTOR pathway for cranial neural crest cells (NCCs), which are a temporary type of cells arising from the ectoderm layer and migrate to the pharyngeal arches participating craniofacial development. mTOR deletion elicited a proliferation deficit and excessive apoptosis of post-migratory NCCs, leading to growth arrest of the facial primordia along with midline orofacial clefts. Furthermore, NCC differentiation was impaired. Thus, NCC derivatives, such as skeletons, vasculatures and neural tissues were either rudimentary or malformed. We further demonstrate that disruption of mTOR caused P53 hyperactivity and cell cycle arrest in cranial NCCs, and lowering P53 activity by one copy reduction attenuated the severity of craniofacial phenotype in NCC-mTOR knockout mice. Remarkably, NCC-Rptor disruption caused a spectrum of defects mirroring that of the NCC-mTOR deletion, whereas NCC-Rictor disruption only caused a mild craniofacial phenotype compared to the mTOR and Rptor conditional knockout models. Altogether, our data demonstrate that mTOR functions mediated by mTORC1 are indispensable for multiple processes of NCC development including proliferation, survival, and differentiation during craniofacial morphogenesis and organogenesis, and P53 hyperactivity in part accounts for the defective craniofacial development in NCC-mTOR knockout mice.

Profiling of Stem/Progenitor Cell Regulatory Genes of the Synovial Joint by Genome-Wide RNA-Seq Analysis.

Synovial joints suffer from arthritis and trauma that may be severely debilitative. Despite robust investigations in the roles of individual genes in synovial joint development and arthritis, little is known about global profiles of genes that regulate stem/progenitor cells of a synovial joint. The temporomandibular joint is a poorly understood synovial arthrosis with few clinical treatment options. Here, we isolated the articular and mature zones of the mandibular condyle by laser capture microdissection, performed genome-wide profiling, and analyzed molecular signaling pathways relevant to stem/progenitor cell functions. A total of 804 genes were differentially expressed between the articular and mature zones. Pathway analyses revealed 29 enriched signaling pathways, including the PI3K-Akt, Wnt, and Toll-like receptor signaling pathways that may regulate stem/progenitor cell homeostasis and differentiation into the chondrocyte lineage. Upstream regulator analyses further predicted potential upstream key regulators such as Xbp1, Nupr1, and Hif1a, and associated underlying mechanism networks were described. Among the multiple candidates of growth and transcriptional factors that may regulate stem/progenitor cells, we immunolocalized Sox9, Ihh, Frzb, Dkk1, Lgr5, and TGFß3 in the articular and mature zones. These findings provide a comprehensive genetic mapping of growth and transcriptional genes in the articular and mature zones of a synovial joint condyle. Differentially expressed genes may play crucial roles in the regulation of stem/progenitor cells in development, homeostasis, and tissue regeneration.

IGF1 Promotes Adipogenesis by a Lineage Bias of Endogenous Adipose Stem/Progenitor Cells.

Adipogenesis is essential for soft tissue reconstruction following trauma or tumor resection. We demonstrate that CD31(-)/34(+)/146(-) cells, a subpopulation of the stromal vascular fraction (SVF) of human adipose tissue, were robustly adipogenic. Insulin growth factor-1 (IGF1) promoted a lineage bias towards CD31(-)/34(+)/146(-) cells at the expense of CD31(-)/34(+)/146(+) cells. IGF1 was microencapsulated in poly(lactic-co-glycolic acid) scaffolds and implanted in the inguinal fat pad of C57Bl6 mice. Control-released IGF1 induced remarkable adipogenesis in vivo by recruiting endogenous cells. In comparison with the CD31(-)/34(+)/146(+) cells, CD31(-)/34(+)/146(-) cells had a weaker Wnt/ß-catenin signal. IGF1 attenuated Wnt/ß-catenin signaling by activating Axin2/PPARγ pathways in SVF cells, suggesting IGF1 promotes CD31(-)/34(+)/146(-) bias through tuning Wnt signal. PPARγ response element (PPRE) in Axin2 promoter was crucial for Axin2 upregulation, suggesting that PPARγ transcriptionally activates Axin2. Together, these findings illustrate an Axin2/PPARγ axis in adipogenesis that is particularly attributable to a lineage bias towards CD31(-)/34(+)/146(-) cells, with implications in adipose regeneration.

A positive feedback mechanism that regulates expression of miR-9 during neurogenesis.

MiR-9, a neuron-specific miRNA, is an important regulator of neurogenesis. In this study we identify how miR-9 is regulated during early differentiation from a neural stem-like cell. We utilized two immortalized rat precursor clones, one committed to neurogenesis (L2.2) and another capable of producing both neurons and non-neuronal cells (L2.3), to reproducibly study early neurogenesis. Exogenous miR-9 is capable of increasing neurogenesis from L2.3 cells. Only one of three genomic loci capable of encoding miR-9 was regulated during neurogenesis and the promoter region of this locus contains sufficient functional elements to drive expression of a luciferase reporter in a developmentally regulated pattern. Furthermore, among a large number of potential regulatory sites encoded in this sequence, Mef2 stood out because of its known pro-neuronal role. Of four Mef2 paralogs, we found only Mef2C mRNA was regulated during neurogenesis. Removal of predicted Mef2 binding sites or knockdown of Mef2C expression reduced miR-9-2 promoter activity. Finally, the mRNA encoding the Mef2C binding partner HDAC4 was shown to be targeted by miR-9. Since HDAC4 protein could be co-immunoprecipitated with Mef2C protein or with genomic Mef2 binding sequences, we conclude that miR-9 regulation is mediated, at least in part, by Mef2C binding but that expressed miR-9 has the capacity to reduce inhibitory HDAC4, stabilizing its own expression in a positive feedback mechanism.

Epigenome analysis of pluripotent stem cells.

Mis-regulation of gene expression due to epigenetic abnormalities has been linked with complex genetic disorders, psychiatric illness, and cancer. In addition, the dynamic epigenetic changes that occur in pluripotent stem cells are believed to impact regulatory networks essential for proper lineage development. Chromatin immunoprecipitation (ChIP) is a technique used to enrich genomic fragments using antibodies against specific chromatin modifications, such as DNA-binding proteins or modified histones. Until recently, many ChIP protocols required large numbers of cells for each immunoprecipitation. This severely limited analysis of rare cell populations or post-mitotic, differentiated cell lines. Here, we describe a low cell number ChIP protocol with next generation sequencing and analysis that has the potential to uncover novel epigenetic regulatory pathways that were previously difficult or impossible to obtain.

Glutamate dehydrogenase 1 and SIRT4 regulate glial development.

Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by an activation mutation of glutamate dehydrogenase 1 (GDH1), a mitochondrial enzyme responsible for the reversible interconversion between glutamate and α-ketoglutarate. The syndrome presents clinically with hyperammonemia, significant episodic hypoglycemia, seizures, and frequent incidences of developmental and learning defects. Clinical research has implicated that although some of the developmental and neurological defects may be attributed to hypoglycemia, some characteristics cannot be ascribed to low glucose and as hyperammonemia is generally mild and asymptomatic, there exists the possibility that altered GDH1 activity within the brain leads to some clinical changes. GDH1 is allosterically regulated by many factors, and has been shown to be inhibited by the ADP-ribosyltransferase sirtuin 4 (SIRT4), a mitochondrially localized sirtuin. Here we show that SIRT4 is localized to mitochondria within the brain. SIRT4 is highly expressed in glial cells, specifically astrocytes, in the postnatal brain and in radial glia during embryogenesis. Furthermore, SIRT4 protein decreases in expression during development. We show that factors known to allosterically regulate GDH1 alter gliogenesis in CTX8 cells, a novel radial glial cell line. We find that SIRT4 and GDH1 overexpression play antagonistic roles in regulating gliogenesis and that a mutant variant of GDH1 found in HI/HA patients accelerates the development of glia from cultured radial glia cells.

Nuclear accumulation of HDAC4 in ATM deficiency promotes neurodegeneration in ataxia telangiectasia.

Ataxia telangiectasia is a neurodegenerative disease caused by mutation of the Atm gene. Here we report that ataxia telangiectasia mutated (ATM) deficiency causes nuclear accumulation of histone deacetylase 4 (HDAC4) in neurons and promotes neurodegeneration. Nuclear HDAC4 binds to chromatin, as well as to myocyte enhancer factor 2A (MEF2A) and cAMP-responsive element binding protein (CREB), leading to histone deacetylation and altered neuronal gene expression. Blocking either HDAC4 activity or its nuclear accumulation blunts these neurodegenerative changes and rescues several behavioral abnormalities of ATM-deficient mice. Full rescue of the neurodegeneration, however, also requires the presence of HDAC4 in the cytoplasm, suggesting that the ataxia telangiectasia phenotype results both from a loss of cytoplasmic HDAC4 as well as its nuclear accumulation. To remain cytoplasmic, HDAC4 must be phosphorylated. The activity of the HDAC4 phosphatase, protein phosphatase 2A (PP2A), is downregulated by ATM-mediated phosphorylation. In ATM deficiency, enhanced PP2A activity leads to HDAC4 dephosphorylation and the nuclear accumulation of HDAC4. Our results define a crucial role of the cellular localization of HDAC4 in the events leading to ataxia telangiectasia neurodegeneration.

Isolation of a novel rat neural progenitor clone that expresses Dlx family transcription factors and gives rise to functional GABAergic neurons in culture.

Gamma-aminobutyric acid (GABA) ergic interneurons are lost in conditions including epilepsy and central nervous system injury, but there are few culture models available to study their function. Toward the goal of obtaining renewable sources of GABAergic neurons, we used the molecular profile of a functionally incomplete GABAergic precursor clone to screen 17 new clones isolated from GFP(+) rat E14.5 cortex and ganglionic eminence (GE) that were generated by viral introduction of v-myc. The clones grow as neurospheres in medium with FGF2, and after withdrawal of FGF2, they exhibit varying patterns of differentiation. Transcriptional profiling and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) indicated that one clone (GE6) expresses high levels of mRNAs encoding Dlx1, 2, 5, and 6, glutamate decarboxylases, and presynaptic proteins including neuropeptide Y and somatostatin. Protein expression confirmed that GE6 is a progenitor with restricted differentiation giving rise mostly to neurons with GABAergic markers. In cocultures with hippocampal neurons, GE6 neurons became electrically excitable and received both inhibitory and excitatory synapses. After withdrawal of FGF2 in cultures of GE6 alone, neurons matured to express ßIII-tubulin, and staining for synaptophysin and vesicular GABA transporter were robust after 1-2 weeks of differentiation. GE6 neurons also became electrically excitable and displayed synaptic activity, but synaptic currents were carried by chloride and were blocked by bicuculline. The results suggest that the GE6 clone, which is ventrally derived from the GE, resembles GABAergic interneuron progenitors that migrate into the developing forebrain. This is the first report of a relatively stable fetal clone that can be differentiated into GABAergic interneurons with functional synapses.

Microscale plasma-initiated patterning of electrospun polymer scaffolds.

Microscale plasma-initiated patterning (µPIP) is a novel micropatterning technique used to create biomolecular micropatterns on polymer surfaces. The patterning method uses a polydimethylsiloxane (PDMS) stamp to selectively protect regions of an underlying substrate from oxygen plasma treatment resulting in hydrophobic and hydrophilic regions. Preferential adsorption of the biomolecules onto either the plasma-exposed (hydrophilic) or plasma-protected (hydrophobic) regions leads to the biomolecular micropatterns. In the current work, laminin-1 was applied to an electrospun polyamide nanofibrillar matrix following plasma treatment. Radial glial clones (neural precursors) selectively adhered to these patterned matrices following the contours of proteins on the surface. This work demonstrates that textured surfaces, such as nanofibrillar scaffolds, can be micropatterned to provide external chemical cues for cellular organization.

Efficient, high-throughput transfection of human embryonic stem cells.

INTRODUCTION: Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications. METHODS: We investigated the ability of two commercially available electroporation systems, the Nucleofection® 96-well Shuttle® System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency. RESULTS: Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However, the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection® 96-well Shuttle® System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency, producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally, we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference. CONCLUSIONS: Our results indicate that these electroporation methods provide a reliable, efficient, and high-throughput approach to the genetic manipulation of hESC.

Functional differentiation of a clone resembling embryonic cortical interneuron progenitors.

We have generated clones (L2.3 and RG3.6) of neural progenitors with radial glial properties from rat E14.5 cortex that differentiate into astrocytes, neurons, and oligodendrocytes. Here, we describe a different clone (L2.2) that gives rise exclusively to neurons, but not to glia. Neuronal differentiation of L2.2 cells was inhibited by bone morphogenic protein 2 (BMP2) and enhanced by Sonic Hedgehog (SHH) similar to cortical interneuron progenitors. Compared with L2.3, differentiating L2.2 cells expressed significantly higher levels of mRNAs for glutamate decarboxylases (GADs), DLX transcription factors, calretinin, calbindin, neuropeptide Y (NPY), and somatostatin. Increased levels of DLX-2, GADs, and calretinin proteins were confirmed upon differentiation. L2.2 cells differentiated into neurons that fired action potentials in vitro, and their electrophysiological differentiation was accelerated and more complete when cocultured with developing astroglial cells but not with conditioned medium from these cells. The combined results suggest that clone L2.2 resembles GABAergic interneuron progenitors in the developing forebrain.

Differentiating human multipotent mesenchymal stromal cells regulate microRNAs: prediction of microRNA regulation by PDGF during osteogenesis.

OBJECTIVE: Human multipotent mesenchymal stromal cells (MSC) have the potential to differentiate into multiple cell types, although little is known about factors that control their fate. Differentiation-specific microRNAs may play a key role in stem cell self-renewal and differentiation. We propose that specific intracellular signaling pathways modulate gene expression during differentiation by regulating microRNA expression. MATERIALS AND METHODS: Illumina mRNA and NCode microRNA expression analyses were performed on MSC and their differentiated progeny. A combination of bioinformatic prediction and pathway inhibition was used to identify microRNAs associated with platelet-derived growth factor (PDGF) signaling. RESULTS: The pattern of microRNA expression in MSC is distinct from that in pluripotent stem cells, such as human embryonic stem cells. Specific populations of microRNAs are regulated in MSC during differentiation targeted toward specific cell types. Complementary mRNA expression analysis increases the pool of markers characteristic of MSC or differentiated progeny. To identify microRNA expression patterns affected by signaling pathways, we examined the PDGF pathway found to be regulated during osteogenesis by microarray studies. A set of microRNAs bioinformatically predicted to respond to PDGF signaling was experimentally confirmed by direct PDGF inhibition. CONCLUSION: Our results demonstrate that a subset of microRNAs regulated during osteogenic differentiation of MSCs is responsive to perturbation of the PDGF pathway. This approach not only identifies characteristic classes of differentiation-specific mRNAs and microRNAs, but begins to link regulated molecules with specific cellular pathways.

Activated Notch1 maintains the phenotype of radial glial cells and promotes their adhesion to laminin by upregulating nidogen.

Radial glia are neural stem cells that exist only transiently during central nervous system (CNS) development, where they serve as scaffolds for neuronal migration. Their instability makes them difficult to study, and therefore we have isolated stabilized radial glial clones from E14.5 cortical progenitors (e.g., L2.3) after expression of v-myc. Activated Notch1 intracellular region (actNotch1) promotes radial glia in the embryonic mouse forebrain (Gaiano et al., (2000), and when it was introduced into E14.5 cortical progenitors or radial glial clone L2.3, the cells exhibited enhanced radial morphology and increased expression of the radial glial marker BLBP. A representative clone of L2.3 cells expressing actNotch1 called NL2.3-4 migrated more extensively than L2.3 cells in culture and in white matter of the adult rat spinal cord. Microarray and RT-PCR comparisons of mRNAs expressed in these closely related clones showed extensive similarities, but differed significantly for certain mRNAs including several cell adhesion molecules. Cell adhesion assays demonstrated significantly enhanced adhesion to laminin of NL2.3-4 by comparison to L2.3 cells. The laminin binding protein nidogen was the most highly induced adhesion molecule in NL2.3-4, and immunological analyses indicated that radial glia synthesize and secrete nidogen. Adhesion of NL2.3-4 cells to laminin was inhibited by anti-nidogen antibodies and required the nidogen binding region in laminin, indicating that nidogen promotes cell adhesion to laminin. The combined results indicate that persistent expression of activated Notch1 maintains the phenotype of radial glial cells, inhibits their differentiation, and promotes their adhesion and migration on a laminin/nidogen complex.