David L. Paul - US grants

Intercellular communication mediated by gap junctions is essential for the synchronized spread of excitation between myocardial cells. I propose to study the biochemistry and biophysics of the cardiac gap junction channel. I will use cDNA for rat liver gap junction protein, which I have already cloned and characterized, as a probe to isolate cDNA for rat cardiac gap junction protein. The primary sequence of the heart gap junction will be determined and compared to liver. Antibodies specific to defined protein domains will be produced and used to determine, by immunocytochemical methods, the transmembrane orientation of the channel protein. The channel forming ability of the protein will be studied by using the cloned cDNA to produce molecularly pure heart gap junction protein in vitro. The protein will be incorporated into membrane vesicles by translation of synthetic mRNA in the presence of dog pancreatic microsomes and then fused into planar lipid bilayers where the ion selectivity and gating properties of the channel can be precisely determined. I will attempt to develop a system where the requirements for the establishment of gap junction-mediated intercellular communication can be examined. Xenopus oocytes will be injected with synthetic RNA. The synthesis, processing, membrane insertion and assembly of gap junction protein will be examined. The ability of pairs of similarly injected oocytes to form functional gap junctions will be assessed. These studies will provide a molecular basis for the understanding of synchronization between heart cells, and the pathogenesis in synchronization patterns which accompany cardiac ischemic and reperfusion episodes.

Intracellular communication mediated by gap junctions is thought to underli a number of crucial cell behaviors. These include the spread of electrical and hormonal signals among populations of cells and regulation of growth an development. DNA cloning indicates that there are a number of related gap junction proteins, which we generically call connexins. 1) The functional properties of gap junctions formed from many of the identified connexins have not been described. We will use an in vitro expression system consisting of pairs of voltage clamped Xenopus oocytes to study those properties. Hybrid proteins will be functionally expressed to identify protein domains that control different behavior. 2) The identity of gap junction proteins in the lens remains unclear. There are reports suggestin that a 26 kD protein (MP26) and a 70 kD protein (MP70 are gap junction structural proteins. In addition, we have cloned CDNA for what may be another gap junction protein in lens. We will make antibodies to this new protein and determine its distribution. We will clone cDNA for MP70 to determine its relationship to MP26 and other possible gap junction proteins 3) We have identified a gap junction protein whose mRNA is present only in Xenopus early embryos. We will determine the spatial and temporal expression of the protein and attempt to modulate its expression in early Xenopus embryos. 4) We will clone cDNAs coding for gap junction proteins in Drosophila. The powerful techniques available for the manipulation of gene expression in this organism will permit close examination of possible roles of gap junctional communication in growth and development.

Intracellular communication mediated by gap junctions is thought to underli a number of crucial cell behaviors. These include the spread of electrical and hormonal signals among populations of cells and regulation of growth an development. DNA cloning indicates that there are a number of related gap junction proteins, which we generically call connexins. 1) The functional properties of gap junctions formed from many of the identified connexins have not been described. We will use an in vitro expression system consisting of pairs of voltage clamped Xenopus oocytes to study those properties. Hybrid proteins will be functionally expressed to identify protein domains that control different behavior. 2) The identity of gap junction proteins in the lens remains unclear. There are reports suggestin that a 26 kD protein (MP26) and a 70 kD protein (MP70 are gap junction structural proteins. In addition, we have cloned CDNA for what may be another gap junction protein in lens. We will make antibodies to this new protein and determine its distribution. We will clone cDNA for MP70 to determine its relationship to MP26 and other possible gap junction proteins 3) We have identified a gap junction protein whose mRNA is present only in Xenopus early embryos. We will determine the spatial and temporal expression of the protein and attempt to modulate its expression in early Xenopus embryos. 4) We will clone cDNAs coding for gap junction proteins in Drosophila. The powerful techniques available for the manipulation of gene expression in this organism will permit close examination of possible roles of gap junctional communication in growth and development.

DESCRIPTION (provided by applicant): Previously, we showed that mutations in the gene encoding connexin32 (Cx32) caused a demyelinating peripheral neuropathy called Charcot-Marie-Tooth disease (CMTX). Consistent with this finding, Schwann cells contain Cx32 and regulate its expression like a myelin-related gene. Thus, maintenance of myelin in the human peripheral nervous system requires connexin expression. However, oligodendrocytes also express and regulate Cx32 like a myelin gene and yet central abnormalities are rare in CMTX patients. Since one explanation for this discrepancy would be redundant expression of other connexins, we searched for connexins in myelinating glia. We found two novel connexins, Cx29 and Cx47. All three connexins can be found in oligodendrocytes and Schwann cells. Cx29 and Cx32, however, are present in non-overlapping subsets of spinal cord oligodendrocytes and,while they are both present in Schwann cells, their subcellular distributions are strikingly different. Single knockouts of either Cx32 or Cx47 myelinate relatively normally and have no functional deficits. In contrast, double knockouts develop severe central demyelination and die during the 6th postnatal week of life. Surprisingly, these animals display only subtle abnormalities in peripheral myelin. Together, our studies suggest that connexins are critical for both central and peripheral myelination but that different connexins may have different functions within myelinating glia. We propose to define the separate and interacting roles of connexins in myelination using a combination of immunocytochemistry, targeted gene ablation and functional analysis of connexin channel activity.

DESCRIPTION (provided by applicant): A compelling body of evidence indicates that signals from rod photoreceptors use multiple pathways to reach ganglion cells. In the canonical primary pathway, rod signals gain access to downstream cone circuitry through the AII amacrines which form electrical synapses (gap junctions) with ON cone bipolar and glycinergic synapses with OFF cone bipolar. In the secondary pathway, electrical synapses between rods and cones provide a direct entry for rod signals into cone circuits. However, a credible direct demonstration of rod-cone coupling has been made only in monkey. Because technical issues prevent a conventional approach to measurement of rod-cone coupling in the mouse (i.e. injections of junction-permeant tracers using microelectrodes), we developed a novel method to evaluated coupling. The method is based on transgenic, cell-specific expression of a transporter, the movement of the transported molecule through gap junctions to neighboring cells and the detection of that molecule with specific antibodies. This method eliminates the need for any physical manipulation of the live cells. Despite the widely held assumption that rod-cone coupling underlies the secondary pathway, we found no evidence of rod-cone coupling in the mouse. Thus, we propose a set of experiments to determine the generality of the canonical secondary pathway model by testing rod-cone coupling in the rabbit and to evaluate cone-cone and possible rod-rod coupling in mouse and rabbit retinas. In contrast, our data strongly support the basic tenets of the rod primary pathway. However, while the prevailing model postulates that AII amacrines express Cx36 and cone ON bipolar express Cx45, forming a 'heterotypic'electrical synapse, our data indicate more complexity. We propose there are two types of glycinergic amacrine cells, those expressing Cx45 and those expressing Cx36 and that each forms homotypic junctions with a subset of cone bipolar cells expressing the same connexin. We hypothesize that the expression of incompatible connexins is mechanism to allow segregation of amacrine- cone bipolar interactions according to cell subtype. We will determine the types of cone bipolar involved in rod primary pathway signaling and which connexins they employ. In addition, we will determine if different retinal connexins can functionally interact. PUBLIC HEALTH RELEVANCE: Our studies address fundamental questions about the neural circuitry employed by rod photoreceptors, which contribute to retinal responses over a range of light inputs from near total darkness to bright moonlight. Disorders of the neural retina are a primary cause of human blindness and a rational pursuit of therapeutic strategies requires a full understand of mammalian retinal circuitry.

DESCRIPTION (provided by applicant): Project Summary: Aim #1. The function of Cx43 in the ciliary epithelium will be studied in three mouse models. In the nestin-cre /Cx43flox/flox line, Cx43 is selectively deleted from the pigmented (PE) layer of the ciliary epithelium resulting in a decrease in aqueous humor secretion and backflow of plasma proteins into the aqueous compartment. In the GjaUrt line, Cx43 immunostaining in the ciliary epithelium is globally reduced and also shows backflow of plasma proteins. The pax6alpha-cre/Cx43flox/flox line shows a loss of Cx43 from the non-pigmented epithelial (NPE) cells and a decrease in intraocular pressure. The three models will be studied in parallel using light and electron microscopy, immunohistochemistry, dye transfer methods and measurement of mouse intraocular pressure. Aim #2. Epithelial cells in CxSOnull lenses exhibit a striking reduction in mitotic index during the first postnatal week. Since replacement of Cx50 with Cx46 does not restore the normal growth rate, Cx50 must provide a unique functionality relating to epithelial cell proliferation. We will test two hypotheses regarding that function. The first is that Cx50 channels exhibit unique properties of selectivity, permitting the exchange of regulatory signals between cells. The second hypothesis is that Cx50, but not Cx46, regulates mitotic progression by C-terminal domain interactions with cytoplasmic proteins. To distinguish between channel properties and signaling involving connexin cytoplasmic domains, we will employ "channel-dead" Cx50 mutants that traffic normally to the plasma membrane and assemble into gap junctional plaques. One mutant with these properties is already characterized and others will be identified. The mutants will be introduced into CxSOnull mice as transgenes using a novel lentivirus approach. This strategy insures an ability to test multiple mutant genes very quickly. If these mutants rescue lens growth, this will strongly implicate the cytoplasmic domains of Cx50 as critical for the regulation of mitotic rate. If not, the properties of the intercellular channel are key and a rescue of mitotic rate will be tested by the introduction of tailless Cx50 mutants. Relevance: These studies will investigate the functional roles of gap junctions in the formation of aqueous humor and lens growth in the eye. They will permit a better understanding of the pathologies involved in glaucoma and cataract.