Regulation of neuronal traits by a novel transcriptional complex.

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Compact myelin dictates the differential targeting of two sodium channel isoforms in the same axon.

Voltage-dependent sodium channels are uniformly distributed along unmyelinated axons, but are highly concentrated at nodes of Ranvier in myelinated axons. Here, we show that this pattern is associated with differential localization of distinct sodium channel alpha subunits to the unmyelinated and myelinated zones of the same retinal ganglion cell axons. In adult axons, Na(v)1.2 is localized to the unmyelinated zone, whereas Na(v)1.6 is specifically targeted to nodes. During development, Na(v)1.2 is expressed first and becomes clustered at immature nodes of Ranvier, but as myelination proceeds, Na(v)1.6 replaces Na(v)1.2 at nodes. In Shiverer mice, which lack compact myelin, Na(v)1.2 is found throughout adult axons, whereas little Na(v)1.6 is detected. Together, these data show that sodium channel isoforms are differentially targeted to distinct domains of the same axon in a process associated with formation of compact myelin.

Progesterone treatment abolishes exogenously expressed ionic currents in Xenopus oocytes.

Fully grown oocytes of Xenopus laevis undergo resumption of the meiotic cycle when treated with the steroid hormone progesterone. Previous studies have shown that meiotic maturation results in profound downregulation of specific endogenous membrane proteins in oocytes. To determine whether the maturation impacts the functional properties of exogenously expressed membrane proteins, we used cut-open recordings from Xenopus oocytes expressing several types of Na(+) and K(+) channels. Treatment of oocytes with progesterone resulted in a downregulation of heterologously expressed Na(+) and K(+) channels without a change in the kinetics of the currents. The time course of progesterone-induced ion channel inhibition was concentration dependent. Complete elimination of Na(+) currents temporally coincided with development of germinal vesicle breakdown, while elimination of K(+) currents was delayed by approximately 2 h. Coexpression of human beta(1)-subunit with rat skeletal muscle alpha-subunit in Xenopus oocytes did not prevent progesterone-induced downregulation of Na(+) channels. Addition of 8-bromo-cAMP to oocytes or injection of heparin before progesterone treatment prevented the loss of expressed currents. Pharmacological studies suggest that the inhibitory effects of progesterone on expressed Na(+) and K(+) channels occur downstream of the activation of cdc2 kinase. The loss of channels is correlated with a reduction in Na(+) channel immunofluorescence, pointing to a disappearance of the ion channel-forming proteins from the surface membrane.

Mechanisms of calcium oxalate crystal attachment to injured renal collecting duct cells.

BACKGROUND: Renal cell or tissue injury results in a loss of membrane lipid asymmetry and/or loss of cell polarity, and both events lead to changes on the surface of the cell membranes that enhance crystal attachment. We have proposed two distinct mechanisms of crystal attachment following membrane changes induced by various modes of injury. METHODS: Annexin V was used to determine whether phosphatidylserine (PS) exposure on the cell membrane surface plays a role in calcium oxalate monohydrate (COM) crystal attachment to cells that have lost their polarity as well as to cells that have lost their lipid asymmetry. We utilized two different experimental models of injury to renal epithelial cells in culture. The first model used calcium ionophore A23187 to induce a loss of lipid asymmetry, and the second model used EGTA to break down tight junctions and lose cell polarity. RESULTS: Inner medullary collecting duct cells that have lost lipid asymmetry demonstrated an increase in the number of cells that bound annexin V. However, when cells lost their polarity, they did not bind annexin V. In addition, the attachment of crystals to cells following a loss of cell polarity was not inhibited by annexin V. CONCLUSIONS: This study indicates that both individual cell injury (loss of lipid asymmetry) and generalized cell monolayer injury (loss of cell polarity) result in the presentation of different cell surfaces and that both forms of injury result in an increased affinity for crystal attachment. Both mechanisms could be important independently or collectively in the retention of microcrystals to renal collecting duct cells in urolithiasis.

Natural history of brainstem cavernous malformations.

OBJECTIVE: To review the natural history and determine the rates of intra- and extralesional hemorrhaging of brainstem cavernous malformations (cavernomas) monitored by one neuro-ophthalmology service. METHODS: A record review of all patients with brainstem cavernomas who were evaluated by a neuroophthalmology service between 1987 and 1999 was performed. We recorded the clinical symptoms and Rankin disability grade at presentation, during the worst clinical episode, and at the last follow-up examination. Magnetic resonance imaging scans were reviewed for evidence of intralesional hemorrhage (a bleeding episode), edema, or venous anomalies, and the cavernoma size was assessed. RESULTS: Thirty-seven patients (age range, 6-73 yr; mean age at presentation, 37.5 yr) underwent a mean of 4.9 years of follow-up monitoring. At presentation, there were 27 bleeding events and 8 nonhemorrhagic events; 2 patients did not exhibit symptoms. Patients who were at least 35 years of age exhibited a lower risk of bleeding episodes (odds ratio, 0.15; 95% confidence interval, 0.1-0.4). Cavernomas of at least 10 mm were associated with a higher risk of bleeding episodes (odds ratio, 3.48; 95% confidence interval, 1.3-9.4). Thirty-nine bleeding episodes occurred in 31 patients, yielding a bleeding rate of 2.46%/yr. There were eight rebleeding episodes, yielding a rebleeding rate of 5.1%/yr. Three patients experienced extralesional bleeding episodes; all of these patients experienced rebleeding. Of the 39 follow-up magnetic resonance imaging scans, the cavernoma size was unchanged in 66.7%, smaller in 18%, and larger in 15%. At the last follow-up examination, the mean Rankin grade was 1.0 for all patients, 0.6 for the 25 nonsurgically treated patients, and 1.4 for the 12 surgically treated patients. CONCLUSION: Rebleeding is not more common among patients who first present with bleeding, and it often has little effect on the neurological status of patients. Significant morbidity attributable to a brainstem cavernoma occurred in 8% of patients during follow-up monitoring of medium duration.

Fibroblast growth factor receptor 3 induces gene expression primarily through Ras-independent signal transduction pathways.

Fibroblast growth factor receptors (FGFR) are widely expressed in many tissues and cell types, and the temporal expression of these receptors and their ligands play important roles in the control of development. There are four FGFR family members, FGFR-1-4, and understanding the ability of these receptors to transduce signals is central to understanding how they function in controlling differentiation and development. We have utilized signal transduction by FGF-1 in PC12 cells to compare the ability of FGFR-1 and FGFR-3 to elicit the neuronal phenotype. In PC12 cells FGFR-1 is much more potent in the induction of neurite outgrowth than FGFR-3. This correlated with the ability of FGFR-1 to induce robust and sustained activation of the Ras-dependent mitogen-activated protein kinase pathways. In contrast, FGFR-3 could not induce strong sustained Ras-dependent signals. In this study, we analyzed the ability of FGFR-3 to induce the expression of sodium channels, peripherin, and Thy-1 in PC12 cells because all three of these proteins are known to be induced via Ras-independent pathways. We determined that FGFR-3 was capable of inducing several Ras-independent gene expression pathways important to the neuronal phenotype to a level equivalent of that induced by FGFR-1. Thus, FGFR-3 elicits phenotypic changes primarily though activation of Ras-independent pathways in the absence of robust Ras-dependent signals.

Combinatorial roles of the nuclear receptor corepressor in transcription and development.

Transcriptional repression plays crucial roles in diverse aspects of metazoan development, implying critical regulatory roles for corepressors such as N-CoR and SMRT. Altered patterns of transcription in tissues and cells derived from N-CoR gene-deleted mice and the resulting block at specific points in CNS, erythrocyte, and thymocyte development indicated that N-CoR was a required component of short-term active repression by nuclear receptors and MAD and of a subset of long-term repression events mediated by REST/NRSF. Unexpectedly, N-CoR and a specific deacetylase were also required for transcriptional activation of one class of retinoic acid response element. Together, these findings suggest that specific combinations of corepressors and histone deacetylases mediate the gene-specific actions of DNA-bound repressors in development of multiple organ systems.

Diversity of voltage-gated sodium channels in the ascidian larval nervous system.

To gain insight into the origin of the molecular diversity of voltage-gated sodium channels (NaVs), a putative sodium channel gene (TuNa2) was cloned from the protochordate ascidian. TuNa2 showed two unusual features in its primary structure; (1) lysine in the P-region of the third repeat, a critical site determining ion selectivity, was changed to glutamic acid, predicting that the ionic permeability would not be rigidly sodium-selective (2) the III-IV linker, determinant of fast inactivation, was only weakly conserved. In contrast with a pan-neuronally expressed NaV (TuNa1), expression of TuNa2 was confined to subsets of neurons including motor neurons, suggesting that TuNa2 plays specialized roles in electrical activities unique to these neurons. Basic FGF, a neural inducer in the ascidian embryo, induces TuNa2 RNA expression in the ectodermal cells at lower doses than that required for TuNa1 gene expression. Thus, two types of NaV may play distinct roles and their gene expressions are controlled by distinct mechanisms.

The co-repressor mSin3A is a functional component of the REST-CoREST repressor complex.

The repressor REST/NRSF restricts expression of a large set of genes to neurons by suppressing their expression in non-neural tissues. We find that REST repression involves two distinct repressor proteins. One of these, CoREST, interacts with the COOH-terminal repressor domain of REST (Andres, M. E., Burger, C., Peral-Rubio, M. J., Battaglioli, E., Anderson, M. E., Grimes, J., Dallmanm J., Ballas, N. , and Mandel, G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9873-9878). Here we show that the co-repressor mSin3A also interacts with REST. The REST-mSin3A association involves the NH(2)-terminal repressor domain of REST and the paired amphipathic helix 2 domain of mSin3A. REST forms complexes with endogenous mSin3A in mammalian cells, and both mSin3A and CoREST interact with REST in intact mammalian cells. REST repression is blocked in yeast lacking Sin3 and rescued in its presence. In mammalian cells, repression by REST is reduced when binding to mSin3A is inhibited. In mouse embryos, the distribution of mSin3A and REST transcripts is largely coincident. The pattern of CoREST gene expression is more restricted, suggesting that mSin3A is required constitutively for REST repression, whereas CoREST is recruited for more specialized repressor functions.

Distinction among neuronal subtypes of voltage-activated sodium channels by mu-conotoxin PIIIA.

The functional properties of most sodium channels are too similar to permit identification of specific sodium channel types underlying macroscopic current. Such discrimination would be particularly advantageous in the nervous system in which different sodium channel family isoforms are coexpressed in the same cell. To test whether members of the mu-conotoxin family can discriminate among known neuronal sodium channel types, we examined six toxins for their ability to block different types of heterologously expressed sodium channels. PIIIA mu-conotoxin blocked rat brain type II/IIA (rBII/IIA) and skeletal muscle sodium current at concentrations that resulted in only slight inhibition of rat peripheral nerve (rPN1) sodium current. Recordings from variant lines of PC12 cells, which selectively express either rBII/IIA or rPN1 channel subtypes, verified that the differential block by PIIIA also applied to native sodium current. The sensitivity to block by PIIIA toxin was then used to discriminate between rBII/IIA and rPN1 sodium currents in NGF-treated PC12 cells in which both mRNAs are induced. During the first 24 hr of NGF-treatment, PN1 sodium channels accounted for over 90% of the sodium current. However, over the ensuing 48 hr period, a sharp rise in the proportion of rBII/IIA sodium current occurred, confirming the idea, based on previous mRNA measurements, that two distinct sodium channel types appear sequentially during neuronal differentiation of PC12 cells.

Voltage-dependent sodium channel function is regulated through membrane mechanics.

Cut-open recordings from Xenopus oocytes expressing either nerve (PN1) or skeletal muscle (SkM1) Na(+) channel alpha subunits revealed slow inactivation onset and recovery kinetics of inward current. In contrast, recordings using the macropatch configuration resulted in an immediate negative shift in the voltage-dependence of inactivation and activation, as well as time-dependent shifts in kinetics when compared to cut-open recordings. Specifically, a slow transition from predominantly slow onset and recovery to exclusively fast onset and fast recovery from inactivation occurred. The shift to fast inactivation was accelerated by patch excision and by agents that disrupted microtubule formation. Application of positive pressure to cell-attached macropatch electrodes prevented the shift in kinetics, while negative pressure led to an abrupt shift to fast inactivation. Simultaneous electrophysiological recording and video imaging of the cell-attached patch membrane revealed that the pressure-induced shift to fast inactivation coincided with rupture of sites of membrane attachment to cytoskeleton. These findings raise the possibility that the negative shift in voltage-dependence and the fast kinetics observed normally for endogenous Na(+) channels involve mechanical destabilization. Our observation that the beta1 subunit causes similar changes in function of the Na(+) channel alpha subunit suggests that beta1 may act through interaction with cytoskeleton.

CoREST: a functional corepressor required for regulation of neural-specific gene expression.

Several genes encoding proteins critical to the neuronal phenotype, such as the brain type II sodium channel gene, are expressed to high levels only in neurons. This cell specificity is due, in part, to long-term repression in nonneural cells mediated by the repressor protein REST/NRSF (RE1 silencing transcription factor/neural-restrictive silencing factor). We show here that CoREST, a newly identified human protein, functions as a corepressor for REST. A single zinc finger motif in REST is required for CoREST interaction. Mutations of the motif that disrupt binding also abrogate repression. When fused to a Gal4 DNA-binding domain, CoREST functions as a repressor. CoREST is present in cell lines that express REST, and the proteins are found in the same immunocomplex. CoREST contains two SANT (SW13/ADA2/NCoR/TFIIIB B) domains, a structural feature of the nuclear receptor and silencing mediator for retinoid and thyroid human receptors (SMRT)-extended corepressors that mediate inducible repression by steroid hormone receptors. Together, REST and CoREST mediate repression of the type II sodium channel promoter in nonneural cells, and the REST/CoREST complex may mediate long-term repression essential to maintenance of cell identity.

Activation and repression in the nervous system.

The mechanisms underlying transcriptional activation and repression have become much clearer. Recent evidence suggests that transcription factors that do not bind DNA directly, the co-activators and co-repressors, mediate a large number of cell signaling events. Their association with histone acetylases, to mediate activation, or deacetylases, to mediate repression, provide a model for explaining how gene expression is regulated.

A single zinc finger motif in the silencing factor REST represses the neural-specific type II sodium channel promoter.

The type II voltage-dependent sodium channel is present in neuronal cells, where it mediates the propagation of nerve impulses. Restricted expression of the type II sodium channel gene to neurons is due, at least in part, to binding of the repressor protein REST (also termed NRSF or XBR) to the RE1 (also called NRSE) sequence in the type II sodium channel gene. Previous studies have shown that a domain in REST containing eight GL1-Krüppel zinc finger motifs mediates DNA binding. Deletional and GAL4-fusion gene analyses now reveal repressor domains that lie outside of the DNA-binding domain in both the amino and carboxyl termini of REST. Mutational analysis further identifies a single zinc finger motif in the carboxyl-terminal domain as being essential for repressing type II sodium channel reporter genes. These studies reveal two domains in REST that may mediate interactions with other proteins involved in restricting expression of a large set of genes to the vertebrate nervous system.

Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons.

Membrane excitability in different tissues is due, in large part, to the selective expression of distinct genes encoding the voltage-dependent sodium channel. Although the predominant sodium channels in brain, skeletal muscle, and cardiac muscle have been identified, the major sodium channel types responsible for excitability within the peripheral nervous system have remained elusive. We now describe the deduced primary structure of a sodium channel, peripheral nerve type 1 (PN1), which is expressed at high levels throughout the peripheral nervous system and is targeted to nerve terminals of cultured dorsal root ganglion neurons. Studies using cultured PC12 cells indicate that both expression and targeting of PN1 is induced by treatment of the cells with nerve growth factor. The preferential localization suggests that the PN1 sodium channel plays a specific role in nerve excitability.

Regulation of sodium channel gene expression by transcriptional silencing.

Fast electrical signaling in the nervous system is mediated by action potentials. The type II Na channel often is responsible for the generation of action potentials in the mammalian central nervous system. The 5' flanking sequence of the type II gene has been cloned and characterized. The presence of a 28-bp DNA element in the regulatory region is required for restricting the expression of type II reporter genes to neuronal cells (PC12). The transcription factor (REST) that binds to this silencer element in the type II gene and mediates its neuron-specific expression has been identified. In cell lines and mouse embryos, REST ist detected only in cell types that fail to express this sodium channel gene. Furthermore, cotransfection assays of PC12 cells with type II reporter genes and a recombinant REST cDNA results in silencing of the type II promoter. We propose that expression of this sodium channel reflects a 'default' pathway in neurons which is blocked by the presence of REST in nonneuronal cells and perhaps in some cells in the adult peripheral nervous system. A rapidly increasing number of genes containing an RE1-like sequence has been reported (SCG10, synapsin, Ng-CAM and DBH) suggesting that a similar silencing mechanism underlies neuron-specific expression of these genes.

Identification of sodium channel subtypes induced in cultured retinal pigment epithelium cells.

Recent electrophysiological experiments have shown that retinal pigment epithelium (RPE) cells begin to produce neuronal-type voltage-dependent sodium currents when placed in dissociated cell culture. In this study, the sodium channel types induced in cultured rat RPE cells were identified. Sodium channel mRNAs encoding two distinct alpha subunits were detected in the cultured RPE cells, brain type II/IIA, and a novel rat mRNA which we have termed RET1. These two sodium channel types may correspond to the TTX-sensitive and TTX-insensitive components of sodium current reported previously in cultured rat RPE cells.