Biomedical subjects

Steven H Green

Publications and source records attributed to Steven H Green.

10 recordsLinked to original sources

PubMed2026

CASZ1 regulates the maturation of outer hair cells and is required for hearing in mice.

The transcription factor ATOH1 is a master regulator of mechanosensory hair-cell (HC) development in the ear. Here, we report that its target gene Casz1 regulates the maturation of outer HCs (OHCs). Genetic deletion of Casz1 during (but not after) cochlear development in the mouse caused: hearing loss; disorganization of mechanosensory stereocilia bundles in OHCs; reduced F-actin density in OHC cuticular plates; progressive OHC loss; and mild morphological alterations in inner HCs. This deletion also altered gene expression, delaying downregulation of genes expressed in immature OHCs, including the actin regulator-encoding gene Coro2a, and accelerating upregulation of genes expressed in mature OHCs. Deleting Coro2a in Casz1 mutant mice restored F-actin density in cuticular plates but increased stereocilia bundle disorganization and hearing thresholds, revealing that CORO2A provides an overall beneficial effect. Our data indicate that CASZ1 regulates transcriptional and morphological maturation of OHCs, and that CASZ1 in maturing HCs is necessary for hearing.

CASZ1↗

PubMed2006

Acid-sensing ion channel 1a is a postsynaptic proton receptor that affects the density of dendritic spines.

Extracellular proton concentrations in the brain may be an important signal for neuron function. Proton concentrations change both acutely when synaptic vesicles release their acidic contents into the synaptic cleft and chronically during ischemia and seizures. However, the brain receptors that detect protons and their physiologic importance remain uncertain. Using organotypic hippocampal slices and biolistic transfection, we found the acid-sensing ion channel 1a (ASIC1a), localized in dendritic spines where it functioned as a proton receptor. ASIC1a also affected the density of spines, the postsynaptic site of most excitatory synapses. Decreasing ASIC1a reduced the number of spines, whereas overexpressing ASIC1a had the opposite effect. Ca(2+)-mediated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) signaling was probably responsible, because acid evoked an ASIC1a-dependent elevation of spine intracellular Ca(2+) concentration, and reducing or increasing ASIC1a levels caused parallel changes in CaMKII phosphorylation in vivo. Moreover, inhibiting CaMKII prevented ASIC1a from increasing spine density. These data indicate that ASIC1a functions as a postsynaptic proton receptor that influences intracellular Ca(2+) concentration and CaMKII phosphorylation and thereby the density of dendritic spines. The results provide insight into how protons influence brain function and how they may contribute to pathophysiology.

Acid Sensing Ion Channels↗

PubMed2006

Constitutive neuregulin-1/ErbB signaling contributes to human vestibular schwannoma proliferation.

Vestibular schwannomas (VSs) are benign tumors that arise from the Schwann cells (SCs) lining the vestibular nerve. VS cells survive and proliferate far from neurons and axonally derived growth factors. We have previously shown that VSs produce the glial growth factor, neuregulin-1 (NRG1), and its receptors, ErbB2 and ErbB3. In the present work, we explore the contribution of constitutive NRG1:ErbB signaling to human VS cell proliferation. We confirm that human VSs, which express markers of immature and denervated SCs, also express endogenous NRG1 and activated ErbB2. We find that a blocking anti-NRG1 antibody and trastuzumab (Herceptin, HCN), a humanized anti-ErbB2 inhibitory monoclonal antibody, effectively inhibit NRG1 induced SC proliferation. Treatment of primary VS cultures with anti-NRG1 or HCN reduces cell proliferation in the absence of exogenous NRG1. Furthermore, conditioned medium from VS cell cultures contains NRG1 and stimulates SC proliferation in SC cultures, an effect that is inhibited by anti-NRG1 and HCN. These data suggest an autocrine pathway of VS growth stimulation involving NRG and ErbB receptors. Inhibition of constitutive NRG:ErbB signaling reduces VS cell proliferation in vitro and may have therapeutic potential for patients with VSs.

Animals↗

PubMed2005

Lentiviral transduction of murine oligodendrocytes in vivo.

Lentiviral vectors are used widely to direct efficient gene transfer in vivo. We examined cell-specific expression in adult murine white matter after stereotaxic microinjection of four lentiviral constructs. We synthesized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviruses with combinations of two promoters, cytomegalovirus (CMV) or myelin basic protein (MBP), and two reporter sequences, cytosolic enhanced green fluorescent protein (eGFP) or a plasma membrane-targeted eGFP (human lymphocyte-specific protein tyrosine kinase [Lck]-eGFP). For all constructs, intracerebral injections to lateral corpus callosum resulted in widespread GFP expression in forebrain white matter glial cells. Intense cellular GFP fluorescence was observed within 3 days after injection and lasted for at least 28 days. The CMV promoter directed GFP expression in multiple glial cell types, whereas the MBP promoter targeted GFP specifically to oligodendrocytes. Expression of the membrane-targeted Lck-eGFP construct distinctly labeled individual myelinating processes of oligodendrocytes. Lentiviral constructs expressing eGFP or Lck-eGFP under the MBP promoter provide excellent visualization of oligodendrocyte morphology in intact white matter, and may prove valuable for delivering additional genes of interest to oligodendrocytes in vivo.

Animals↗

PubMed2005

Synaptic activity and F-actin coordinately regulate CaMKIIalpha localization to dendritic postsynaptic sites in developing hippocampal slices.

We examined the timing and mechanisms of CaMKIIalpha recruitment to nascent synapses of developing rat hippocampal pyramidal neurons in slice culture. Time-lapse confocal imaging shows that GFP-CaMKIIalpha in transfected neurons accumulates in spines as they are forming, and loss of CaMKIIalpha coincides with spine destabilization. Immunolabeling shows that endogenous CaMKIIalpha is concentrated at postsynaptic sites in spines under ambient slice culture conditions, and this is not disrupted by short-term (3 h) synaptic activity blockade or Latrunculin-induced F-actin depolymerization. However, the combination of activity blockade and F-actin depolymerization significantly reduces synaptic CaMKIIalpha. Conversely, postsynaptic activation induces synaptic recruitment of CaMKIIalpha even in the presence of F-actin depolymerizing drugs. Thus, synaptic-activity-dependent mechanisms and (synaptic activity-independent) F-actin-based mechanisms are individually sufficient and act in parallel to localize CaMKIIalpha to the dendritic spine compartment. Moreover, the timing of CaMKIIalpha recruitment to developing spines suggests a role for CaMKIIalpha in spine assembly and maintenance.

Actins↗

PubMed2005

Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures.

Protoplasmic astrocytes in mammalian CNS tissues in vivo have a highly complex 3D morphology, but in dissociated cell cultures they often assume a flattened, fibroblast-like morphology bearing only a few, simple processes. By fluorescent labeling and confocal reconstruction we show that many astrocytes in organotypic hippocampal slice cultures exhibit a more native complex cytoarchitecture. Although astrocytes at the surface of slice cultures show a reactive form with several thick glial fibrillary acidic protein (GFAP)-positive processes, astrocytes situated in deeper portions of tissue slices retain a highly complex 3D morphology with many fine spine- or veil-like protrusions. Dozens of astrocytes can be labeled in single slice cultures by gene gun-mediated ballistic delivery of gold or tungsten particles carrying cDNAs (Biolistics), lipophilic dyes (DiOlistics), or fluorescent intracellular calcium indicators (Calistics). Expression of a membrane-targeted form of eGFP (Lck-GFP) is superior to soluble eGFP for resolving fine astrocytic processes. Time-lapse confocal imaging of Lck-GFP transfected astrocytes or "calistically" labeled astrocytes show structural remodeling and calcium transients, respectively. This approach provides an in vitro system for investigating the functional architecture, development and dynamic remodeling of astrocytes and their relationships to neurons and glia in live mammalian brain tissues.

Animals↗

PubMed2005

Regulation of hippocampal synapse remodeling by epileptiform activity.

We examined the regulation of dendritic spines and synapses by epileptiform activity (EA) in rat hippocampal slice cultures. EA, which was induced by a GABA(A) receptor inhibitor, gabazine, reduced pyramidal neuron spine density by approximately 50% after 48 h and also caused an increase in the average length of remaining spines. To directly determine the effects of EA on synapses, we used fluorescent protein-tagged PSD95, which marks postsynaptic densities. EA induced a net loss of synapses on spines but not shafts; conversely, activity blockade (TTX) induced a loss of shaft synapses. Time-lapse confocal imaging in live tissue slices revealed that EA (1) shifts the balance of synapse gain and loss in dendrites leading to a net loss of spine synapses and (2) induces the formation of new filopodia-like dendritic structures having abnormally slow motility. These results identify EA-induced changes in the density and distribution of synaptic structures on dendrites.

Animals↗

PubMed2004

Forskolin, 8-Br-3',5'-cyclic adenosine 5'-monophosphate, and catalytic protein kinase A expression in the nucleus increase radioiodide uptake and sodium/iodide symporter protein levels in RET/PTC1-expressing cells.

RET/PTC1, a thyroid-specific oncogene, has been reported to down-regulate sodium/iodide symporter (NIS) expression and function in vitro and in vivo. Recently, RET/PTC1 has been shown to interfere with TSH signaling at multiple levels in thyroid cells. The objective of this study was to investigate whether RET/PTC1-mediated NIS reduction can be rescued by activating cAMP-protein kinase A (PKA) pathways. We showed that both forskolin and 8-Br-cAMP increase radioiodide uptake and NIS protein in RET/PTC1-expressing cells to the same extent as the parental PC Cl 3 cells. We found that RET/PTC1 decreases nuclear localization of catalytic PKA, and forskolin treatment was able to counteract this RET/PTC1 effect. Furthermore, transient expression of catalytic PKA in the nucleus increased radioiodide uptake and NIS protein in RET/PTC1-expressing cells. Taken together, these studies suggest that RET/PTC1 down-regulates NIS expression by interrupting TSH/cAMP signaling, and this RET/PTC1 effect can be reversed by activating cAMP-PKA pathways.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2003

Ca2+/calmodulin-dependent protein kinases II and IV both promote survival but differ in their effects on axon growth in spiral ganglion neurons.

Spiral ganglion neuron (SGN) survival in vitro can be maintained by neurotrophins, permeant cAMP analogs, and depolarization in an additive manner, with depolarization being the most efficacious. Therefore, we used cultured SGNs to determine the mechanism by which depolarization promotes neuronal survival. Our data implicate Ca(2+)/calmodulin-dependent protein kinase (CaMK) activity by showing that it is induced by depolarization, that CaMK activity is necessary for at least part of the survival-promoting effect of depolarization, and that CaMKII or CamKIV activity suffices to support neuronal survival in the absence of other trophic stimuli. First, that depolarization of SGNs activates CaMKs is evidenced by observation of increased CaMKII phosphorylation and of CaMK-dependent CREB phosphorylation. Second, the requirement for CaMKs is shown by a reduction of SGN survival under depolarizing conditions in the presence of CaMK inhibitors. Third, transfection of COOH-terminal-truncated (lacking regulatory domain), constitutively active CaMKII or CaMKIV, but not of normal, full-length CAMKs, promotes SGN survival in the absence of other trophic stimuli, indicating that CaMK activity is sufficient to promote survival. The survival-promoting effect of truncated CaMKs is additive with that of depolarization, neurotrophins, or cyclic AMP. Although both CaMKII and CaMKIV activities converge in promoting survival, their actions on axon growth are markedly different: Transfection of truncated CaMKII, but not of truncated CaMKIV, into SGNs prevents axon outgrowth.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2003

An extranuclear locus of cAMP-dependent protein kinase action is necessary and sufficient for promotion of spiral ganglion neuronal survival by cAMP.

We showed previously that cAMP is a survival-promoting stimulus for cultured postnatal rat spiral ganglion neurons (SGNs) and that depolarization promotes SGN survival in part via recruitment of cAMP signaling. We here investigate the subcellular locus of cAMP prosurvival signaling. Transfection of GPKI, a green fluorescent protein (GFP)-tagged cAMP-dependent protein kinase (PKA) inhibitor, inhibits the ability of the permeant cAMP analog cpt-cAMP [8-(4-chlorophenylthio)-cAMP] to promote survival, indicating that PKA activity is necessary. Transfection of GFP-tagged PKA (GPKA) is sufficient to promote SGN survival, but restriction of GPKA to the nucleus by addition of a nuclear localization signal (GPKAnls) almost completely abrogates its prosurvival effect. In contrast, GPKA targeted to the extranuclear cytoplasm by addition of a nuclear export signal (GPKAnes) promotes SGN survival as effectively as does GPKA. Moreover, GPKI targeted to the nucleus lacks inhibitory effect on SGN survival attributable to cpt-cAMP or depolarization. These data indicate an extranuclear target of PKA for promotion of neuronal survival. Consistent with this, we find that dominant-inhibitory CREB mutants inhibit the prosurvival effect of depolarization but not that of cpt-cAMP. SGN survival is compromised by overexpression of the proapoptotic regulator Bad, previously shown to be phosphorylated in the cytoplasm by PKA. This Bad-induced apoptosis is prevented by cpt-cAMP or by cotransfection of GPKA or of GPKAnes but not of GPKAnls. Thus, cAMP prevents SGN death through a cytoplasmic as opposed to nuclear action, and inactivation of Bad proapoptotic function is a mechanism by which PKA can prevent neuronal death.

Animals↗