Anatomy and Neuroscience - Research Publications
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ItemNo Preview AvailableSOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha 1(II) collagen gene(AMER SOC MICROBIOLOGY, 1997-04)The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies.
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ItemDiacetylbis(N(4)-methylthiosemicarbazonato) Copper(II) (CuII(atsm)) Protects against Peroxynitrite-induced Nitrosative Damage and Prolongs Survival in Amyotrophic Lateral Sclerosis Mouse Model(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-12-23)Amyotrophic lateral sclerosis (ALS) is a progressive paralyzing disease characterized by tissue oxidative damage and motor neuron degeneration. This study investigated the in vivo effect of diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)), which is an orally bioavailable, blood-brain barrier-permeable complex. In vitro the compound inhibits the action of peroxynitrite on Cu,Zn-superoxide dismutase (SOD1) and subsequent nitration of cellular proteins. Oral treatment of transgenic SOD1G93A mice with CuII(atsm) at presymptomatic and symptomatic ages was performed. The mice were examined for improvement in lifespan and motor function, as well as histological and biochemical changes to key disease markers. Systemic treatment of SOD1G93A mice significantly delayed onset of paralysis and prolonged lifespan, even when administered to symptomatic animals. Consistent with the properties of this compound, treated mice had reduced protein nitration and carbonylation, as well as increased antioxidant activity in spinal cord. Treatment also significantly preserved motor neurons and attenuated astrocyte and microglial activation in mice. Furthermore, CuII(atsm) prevented the accumulation of abnormally phosphorylated and fragmented TAR DNA-binding protein-43 (TDP-43) in spinal cord, a protein pivotal to the development of ALS. CuII(atsm) therefore represents a potential new class of neuroprotective agents targeting multiple major disease pathways of motor neurons with therapeutic potential for ALS.
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ItemEarly Development of Electrical Excitability in the Mouse Enteric Nervous System(SOC NEUROSCIENCE, 2012-08-08)Neural activity is integral to the development of the enteric nervous system (ENS). A subpopulation of neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development (at E10.5 in the mouse). However, the electrical activity of these cells has not been previously characterized, and it is not known whether all cells expressing neuronal markers are capable of firing action potentials (APs). In this study, we examined the activity of "neuron"-like cells (expressing pan-neuronal markers or with neuronal morphology) in the gut of E11.5 and E12.5 mice using whole-cell patch-clamp electrophysiology and compared them to the activity of neonatal and adult enteric neurons. Around 30-40% of neuron-like cells at E11.5 and E12.5 fired APs, some of which were very similar to those of adult enteric neurons. All APs were sensitive to tetrodotoxin (TTX), indicating that they were driven by voltage-gated Na+ currents. Expression of mRNA encoding several voltage-gated Na+ channels by the E11.5 gut was detected using RT-PCR. The density of voltage-gated Na+ currents increased from E11.5 to neonates. Immature active responses, mediated in part by TTX- and lidocaine-insensitive channels, were observed in most cells at E11.5 and E12.5, but not in P0/P1 or adult neurons. However, some cells expressing neuronal markers at E11.5 or E12.5 did not exhibit an active response to depolarization. Spontaneous depolarizations resembling excitatory postsynaptic potentials were observed at E12.5. The ENS is one of the earliest parts of the developing nervous system to exhibit mature forms of electrical activity.
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ItemN-Glycosylation Determines Ionic Permeability and Desensitization of the TRPV1 Capsaicin Receptor(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012-06-22)The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.
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ItemCellular Up-regulation of Nedd4 Family Interacting Protein 1 (Ndfip1) using Low Levels of Bioactive Cobalt Complexes(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-03-11)The delivery of metal ions using cell membrane-permeable metal complexes represents a method for activating cellular pathways. Here, we report the synthesis and characterization of new [Co(III)(salen)(acac)] complexes capable of up-regulating the ubiquitin ligase adaptor protein Ndfip1. Ndfip1 is a neuroprotective protein that is up-regulated in the brain after injury and functions in combination with Nedd4 ligases to ubiquitinate harmful proteins for removal. We previously showed that Ndfip1 can be increased in human neurons using CoCl(2) that is toxic at high concentration. Here we demonstrate a similar effect can be achieved by low concentrations of synthetic Co(III) complexes that are non-toxic and designed to be activated following cellular entry. Activation is achieved by intracellular reduction of Co(III) to Co(II) leading to release of Co(II) ions for Ndfip1 up-regulation. The cellular benefit of Ndfip1 up-regulation by Co(III) complexes includes demonstrable protection against cell death in SH-SY5Y cells during stress. In vivo, focal delivery of Co(III) complexes into the adult mouse brain was observed to up-regulate Ndfip1 in neurons. These results demonstrate that a cellular response pathway can be advantageously manipulated by chemical modification of metal complexes, and represents a significant step of harnessing low concentration metal complexes for therapeutic benefit.
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ItemPurinergic mechanisms in the control of gastrointestinal motility(SPRINGER, 2008-09)For many years, ATP and adenosine have been implicated in movement regulation of the gastrointestinal tract. They act through three major receptor subtypes: adenosine or P1 receptors, P2X receptors and P2Y receptors. Each of these major receptor types can be subdivided into several different classes and is widely distributed amongst various neurons, muscle types, glia and interstitial cells that regulate intestinal functions. Several key roles for the different receptors and their endogenous ligands have been identified in physiological and pharmacological studies. For example, adenosine acting at A(1) receptors appears to inhibit intestinal motility in various pathological conditions. Similarly, ATP acting at P2Y receptors is an important component of inhibitory neuromuscular transmission, acting as a cotransmitter with nitric oxide. ATP acting at P2X and P2Y(1) receptors is important for synaptic transmission in simple descending excitatory and inhibitory reflex pathways. Some P2Y receptor subtypes prefer uridine nucleotides over purine nucleotides. Thus, roles for UTP and UDP as enteric transmitters in place of ATP cannot be excluded. ATP also appears to be important for sensory transduction, especially in chemosensitive pathways that initiate local inhibitory reflexes. Despite this evidence, data are lacking about the roles of either adenosine or ATP in more complex motility patterns such as segmentation or the interdigestive migrating motor complex. Clarification of roles for purinergic transmission in these common, but understudied, motility patterns will depend on the use of subtype-specific antagonists that in some cases have not yet been developed.
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ItemExpression of muscarinic receptor subtypes in tree shrew ocular tissues and their regulation during the development of myopia(MOLECULAR VISION, 2009-03-02)PURPOSE: Muscarinic receptors are known to regulate several important physiologic processes in the eye. Antagonists to these receptors such as atropine and pirenzepine are effective at stopping the excessive ocular growth that results in myopia. However, their site of action is unknown. This study details ocular muscarinic subtype expression within a well documented model of eye growth and investigates their expression during early stages of myopia induction. METHODS: Total RNA was isolated from tree shrew corneal, iris/ciliary body, retinal, choroidal, and scleral tissue samples and was reverse transcribed. Using tree shrew-specific primers to the five muscarinic acetylcholine receptor subtypes (CHRM1-CHRM5), products were amplified using polymerase chain reaction (PCR) and their identity confirmed using automated sequencing. The expression of the receptor proteins (M1-M5) were also explored in the retina, choroid, and sclera using immunohistochemistry. Myopia was induced in the tree shrew for one or five days using monocular deprivation of pattern vision, and the expression of the receptor subtypes was assessed in the retina, choroid, and sclera using real-time PCR. RESULTS: All five muscarinic receptor subtypes were expressed in the iris/ciliary body, retina, choroid, and sclera while gene products corresponding to CHRM1, CHRM3, CHRM4, and CHRM5 were present in the corneal samples. The gene expression data were confirmed by immunohistochemistry with the M1-M5 proteins detected in the retina, choroid, and sclera. After one or five days of myopia development, muscarinic receptor gene expression remained unaltered in the retinal, choroidal, and scleral tissue samples. CONCLUSIONS: This study provides a comprehensive profile of muscarinic receptor gene and protein expression in tree shrew ocular tissues with all receptor subtypes found in tissues implicated in the control of eye growth. Despite the efficacy of muscarinic antagonists at inhibiting myopia development, the genes of the muscarinic receptor subtypes are neither regulated early in myopia (before measurable axial elongation) nor after significant structural change.
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ItemEphA4 (Sek1) receptor tyrosine kinase is required for the development of the corticospinal tract(NATL ACAD SCIENCES, 1998-10-27)Members of the Eph family of tyrosine kinase receptors have been implicated in the regulation of developmental processes and, in particular, axon guidance in the developing nervous system. The function of the EphA4 (Sek1) receptor was explored through creation of a null mutant mouse. Mice with a null mutation in the EphA4 gene are viable and fertile but have a gross motor dysfunction, which is evidenced by a loss of coordination of limb movement and a resultant hopping, kangaroo-like gait. Consistent with the observed phenotype, anatomical studies and anterograde tracing experiments reveal major disruptions of the corticospinal tract within the medulla and spinal cord in the null mutant animals. These results demonstrate a critical role for EphA4 in establishing the corticospinal projection.
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ItemAssociation of genetic variation with systolic and diastolic blood pressure among African Americans: the Candidate Gene Association Resource study(OXFORD UNIV PRESS, 2011-06-01)The prevalence of hypertension in African Americans (AAs) is higher than in other US groups; yet, few have performed genome-wide association studies (GWASs) in AA. Among people of European descent, GWASs have identified genetic variants at 13 loci that are associated with blood pressure. It is unknown if these variants confer susceptibility in people of African ancestry. Here, we examined genome-wide and candidate gene associations with systolic blood pressure (SBP) and diastolic blood pressure (DBP) using the Candidate Gene Association Resource (CARe) consortium consisting of 8591 AAs. Genotypes included genome-wide single-nucleotide polymorphism (SNP) data utilizing the Affymetrix 6.0 array with imputation to 2.5 million HapMap SNPs and candidate gene SNP data utilizing a 50K cardiovascular gene-centric array (ITMAT-Broad-CARe [IBC] array). For Affymetrix data, the strongest signal for DBP was rs10474346 (P= 3.6 × 10(-8)) located near GPR98 and ARRDC3. For SBP, the strongest signal was rs2258119 in C21orf91 (P= 4.7 × 10(-8)). The top IBC association for SBP was rs2012318 (P= 6.4 × 10(-6)) near SLC25A42 and for DBP was rs2523586 (P= 1.3 × 10(-6)) near HLA-B. None of the top variants replicated in additional AA (n = 11 882) or European-American (n = 69 899) cohorts. We replicated previously reported European-American blood pressure SNPs in our AA samples (SH2B3, P= 0.009; TBX3-TBX5, P= 0.03; and CSK-ULK3, P= 0.0004). These genetic loci represent the best evidence of genetic influences on SBP and DBP in AAs to date. More broadly, this work supports that notion that blood pressure among AAs is a trait with genetic underpinnings but also with significant complexity.
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ItemNo Preview AvailableIdentification of New and Diverse Inducers of Fetal Hemoglobin with High Throughput Screening (HTS)(American Society of Hematology, 2010-11-19)Pharmacologic augmentation of fetal hemoglobin (HbF, γ-globin) production, to replace diminished β-globin chains in the β-thalassemias and to inhibit HbS polymerization in sickle cell disease, is a definitive therapeutic modality. Despite long-term efforts, regulatory approval has been obtained for only one chemotherapeutic agent. Pharmacologic reactivation of high-level HbF expression with non-cytotoxic, tolerable therapeutics is still an unmet medical need for this global health burden. To investigate potential therapeutic libraries for unrecognized HbF inducers, we developed a high-throughput screening (HTS) program to interrogate diverse chemical libraries, including a library of FDA-approved and clinical stage drugs. This program has identified unexpected new and highly potent HbF-inducing drugs, some of which are already in clinical use for other medical indications and have established safety profiles. A human cell-based assay which was previously used in low throughput assays, utilizing a 1.4-kilobase (kb) KpnI-BglII fragment of the HS2 of the locus control region (LCR) linked to the γ-globin gene promoter and the enhanced green fluorescent protein (EGFP) reporter gene, was adapted for high throughput screening and employed as the primary screen. Cytotoxic activity was assayed in a simultaneous counter screen. A number of hits were identified as being more potent than positive controls (such as butyrate). Several hits were immediately eliminated from further development as potential hemoglobinopathy therapeutics because of cytotoxicity (e.g., Idarubicin) or undesirable off-target effects, but nonetheless validated the HTS itself and were validated in secondary confirmatory assays as highly-potent HbF-inducers. The HTS assay identified 8 FDA-approved drugs as potent inducers of γ-globin gene expression, with activity at 1–2 logs lower concentrations (1000-fold higher potency) than prior generation therapeutic candidates. The γ-globin-specificity of hits was determined in a secondary assay employing a stably-transfected dual-luciferase reporter construct containing the LCR and the β-globin promoter linked to renilla luciferase and the Aγ-globin promoter linked to firefly luciferase (μLCRβprRlucAγprFluc cassette). Clinical-stage or clinically-approved agents, including Ambroxol at 1 μM, Desloratadine at 1 μM, Resveratrol at 10 μM, Benserazide at 5 μM, the HDAC inhibitor MS-275 at 5 μM, and an established bioactive, NSC-95397, at 1 μM were all significantly more active in this assay than Butyrate at 2000 μM, with MS-275 and Resveratrol being the most active. These drugs were then assayed for their ability to induce γ-globin mRNA expression in cultured primary human erythroid progenitors, at concentrations which are pharmacologically achievable in humans. Drugs significantly more active in γ -globin mRNA induction than the positive control (2-fold induction) in this system included Ambroxol (3-fold), Desloratadine (up to 6-fold), Resveratrol (up to 3-fold), Benserazide (up to 5-fold), and MS-275 (up to 3.7-fold). Two agents were subsequently studied in anemic baboons, and demonstrated in vivo induction of γ-globin mRNA, HbF, and F-reticulocytes. Unexpectedly, rises in total hemoglobin (>1 gm/dL) also occurred with 2 agents. Thus, a panel of structurally- and functionally-unrelated compounds demonstrate greater HbF-inducing activity, with up to 1000-fold higher potency, than current HbF-inducers which have significant activity in clinical trials. Some of the drugs identified by HTS have entirely benign safety profiles. These candidates could be clinically evaluated rapidly and at significantly less cost than new chemical entities, which require extensive toxicology, manufacturing, and clinical evaluation. These findings demonstrate the utility of a high-throughput screening program based on γ-globin gene promoter induction.