SIGNALING MECHANISMS THAT CONTROL GAP JUNCTIONAL COUPLING BETWEEN RETINAL NEURONS

Gap junctions between neurons form the structural substrate for electrical synapses. Connexin 36 (Cx36, and its non-mammalian ortholog connexin 35) is the major neuronal gap junction protein in the central nervous system (CNS), and contributes to several important neuronal functions including neuronal synchronization, signal averaging, network oscillations, and motor learning. Connexin 36 is strongly expressed in the retina, where it is an obligatory component of the high-sensitivity rod photoreceptor pathway. A fundamental requirement of the retina is to adapt to broadly varying inputs in order to maintain a dynamic range of signaling output. Modulation of the strength of electrical coupling between networks of retinal neurons, including the Cx36-coupled AII amacrine cell in the primary rod circuit, is a hallmark of retinal luminance adaptation. However, very little is known about the mechanisms regulating dynamic modulation of Cx36-mediated coupling. The primary goal of this work was to understand how cellular signaling mechanisms regulate coupling through Cx36 gap junctions. We began by developing and characterizing phospho-specific antibodies against key regulatory phosphorylation sites on Cx36. Using these tools we showed that phosphorylation of Cx35 in fish models varies with light adaptation state, and is modulated by acute changes in background illumination. We next turned our focus to the well-studied and readily identifiable AII amacrine cell in mammalian retina. Using this model we showed that increased phosphorylation of Cx36 is directly related to increased coupling through these gap junctions, and that the dopamine-stimulated uncoupling of the AII network is mediated by dephosphorylation of Cx36 via protein kinase A-stimulated protein phosphatase 2A activity. We then showed that increased phosphorylation of Cx36 on the AII amacrine network is driven by depolarization of presynaptic ON-type bipolar cells as well as background light increments. This increase in phosphorylation is mediated by activation of extrasynaptic NMDA receptors associated with Cx36 gap junctions on AII amacrine cells and by Ca2+-calmodulin-dependent protein kinase II activation. Finally, these studies indicated that coupling is regulated locally at individual gap junction plaques. This work provides a framework for future study of regulation of Cx36-mediated coupling, in which increased phosphorylation of Cx36 indicates increased neuronal coupling.

Dopamine-stimulated dephosphorylation of connexin 36 mediates AII amacrine cell uncoupling.

Gap junction proteins form the substrate for electrical coupling between neurons. These electrical synapses are widespread in the CNS and serve a variety of important functions. In the retina, connexin 36 (Cx36) gap junctions couple AII amacrine cells and are a requisite component of the high-sensitivity rod photoreceptor pathway. AII amacrine cell coupling strength is dynamically regulated by background light intensity, and uncoupling is thought to be mediated by dopamine signaling via D(1)-like receptors. One proposed mechanism for this uncoupling involves dopamine-stimulated phosphorylation of Cx36 at regulatory sites, mediated by protein kinase A. Here we provide evidence against this hypothesis and demonstrate a direct relationship between Cx36 phosphorylation and AII amacrine cell coupling strength. Dopamine receptor-driven uncoupling of the AII network results from protein kinase A activation of protein phosphatase 2A and subsequent dephosphorylation of Cx36. Protein phosphatase 1 activity negatively regulates this pathway. We also find that Cx36 gap junctions can exist in widely different phosphorylation states within a single neuron, implying that coupling is controlled at the level of individual gap junctions by locally assembled signaling complexes. This kind of synapse-by-synapse plasticity allows for precise control of neuronal coupling, as well as cell-type-specific responses dependent on the identity of the signaling complexes assembled.

TURNOVER RATE OF THE NEURONAL CONNEXIN CX36 IN HELA CELLS

Electrical synapses formed of the gap junction protein Cx36 show a great deal of functional plasticity, much dependent on changes in phosphorylation state of the connexin. However, gap junction turnover may also be important for regulating cell-cell communication, and turnover rates of Cx36 have not been studied. Connexins have relatively fast turnover rates, with short half-lives measured to be 1.5 to 3.5 hours in pulse-chase analyses of connexins (Cx26 and Cx43) in tissue culture cells and whole organs. We utilized HaloTag technology to study the turnover rate of Cx36 in transiently transfected HeLa cells. The HaloTag protein forms irreversible covalent bonds with chloroalkane ligands, allowing pulse-chase experiments to be performed very specifically. The HaloTag open reading frame was inserted into an internal site in the C-terminus of Cx36 designed not to disrupt the regulatory phosphorylation sites and not to block the C-terminal PDZ interaction motif. Functional properties of Cx36-Halo were assessed by Neurobiotin tracer coupling, live cell imaging, and immunostaining. For the pulse-chase study, transiently transfected HeLa cells were pulse labeled with Oregon Green (OG) HaloTag ligand and chase labeled at various times with tetramethylrhodamine (TMR) HaloTag ligand. Cx36-Halo formed large junctional plaques at sites of contact between transfected HeLa cells and was also contained in a large number of intracellular vesicles. The Cx36-Halo transfected HeLa cells supported Neurobiotin tracer coupling that was regulated by activation and inhibition of PKA in the same manner as wild-type Cx36 transfected cells. In the pulse-chase study, junctional protein labeled with the pulse ligand (OG) was gradually replaced by newly synthesized Cx36 labeled with the chase ligand (TMR). The half-life for turnover of protein in junctional plaques was 2.8 hours. Treatment of the pulse-labeled cells with Brefeldin A (BFA) prevented the addition of new connexins to junctional plaques, suggesting that the assembly of Cx36 into gap junctions involves the traditional ER-Golgi-TGN-plasma membrane pathway. In conclusion, Cx36-Halo is functional and has a turnover rate in HeLa cells similar to that of other connexins that have been studied. This turnover rate is likely too slow to contribute substantially to short-term changes in coupling of neurons driven by transmitters such as dopamine, which take minutes to achieve. However, turnover may contribute to longer-term changes in coupling.

Protein-Protein Interactions That Regulate Neurotransmitter Release from Retinal Ribbon Synapses

Protein-Protein Interactions That Regulate Neurotransmitter Release from Retinal Ribbon Synapses Photoreceptors and bipolar cells in the retina form specialized chemical synapses called ribbon synapses. This type of synapse differs physiologically from “conventional” chemical synapses. While “conventional” synapses exocytose neurotransmitter-filled vesicles in an all-or-none fashion in response to an action potential, a retinal ribbon synapse can release neurotransmitter tonically (sustained) in response to graded changes in membrane potential or phasically (transient) in response to a large change in membrane potential. Synaptic vesicle exocytosis is a tightly controlled process involving many protein-protein interactions. Therefore, it is likely that the dissimilarity in the release properties of retinal ribbon synapses and conventional synapses is the result of molecular differences between the two synapse types. Consistent with this idea, previous studies have demonstrated that ribbon synapses in the retina do not contain the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system. In contrast, ribbon synapses of the mammalian retina contain the related isoform, syntaxin 3B. Given that SNARE proteins play an important role in neurotransmitter release in conventional synapses, the purpose of this study was to characterize syntaxin 3B in order to elucidate what role this protein plays in neurotransmitter release from retinal ribbon synapses. Using molecular and biochemical techniques, it was demonstrated that syntaxin 3B is a binding partner of several presynaptic proteins that play a important role in synaptic vesicle exocytosis from retinal ribbon synapses and it is an evolutionarily conserved protein.

A model of the roles of essential kinases in the induction and expression of late long-term potentiation.

The induction of late long-term potentiation (L-LTP) involves complex interactions among second-messenger cascades. To gain insights into these interactions, a mathematical model was developed for L-LTP induction in the CA1 region of the hippocampus. The differential equation-based model represents actions of protein kinase A (PKA), MAP kinase (MAPK), and CaM kinase II (CAMKII) in the vicinity of the synapse, and activation of transcription by CaM kinase IV (CAMKIV) and MAPK. L-LTP is represented by increases in a synaptic weight. Simulations suggest that steep, supralinear stimulus-response relationships between stimuli (e.g., elevations in [Ca(2+)]) and kinase activation are essential for translating brief stimuli into long-lasting gene activation and synaptic weight increases. Convergence of multiple kinase activities to induce L-LTP helps to generate a threshold whereby the amount of L-LTP varies steeply with the number of brief (tetanic) electrical stimuli. The model simulates tetanic, -burst, pairing-induced, and chemical L-LTP, as well as L-LTP due to synaptic tagging. The model also simulates inhibition of L-LTP by inhibition of MAPK, CAMKII, PKA, or CAMKIV. The model predicts results of experiments to delineate mechanisms underlying L-LTP induction and expression. For example, the cAMP antagonist RpcAMPs, which inhibits L-LTP induction, is predicted to inhibit ERK activation. The model also appears useful to clarify similarities and differences between hippocampal L-LTP and long-term synaptic strengthening in other systems.

Syntaxin 3B is essential for the exocytosis of synaptic vesicles in ribbon synapses of the retina.

Ribbon synapses of the vertebrate retina are specialized synapses that release neurotransmitter by synaptic vesicle exocytosis in a manner that is proportional to the level of depolarization of the cell. This release property is different from conventional neurons, in which the release of neurotransmitter occurs as a short-lived burst triggered by an action potential. Synaptic vesicle exocytosis is a calcium regulated process that is dependent on a set of interacting synaptic proteins that form the so-called SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex. Syntaxin 3B has been identified as a specialized SNARE molecule in ribbon synapses of the rodent retina. However, the best physiologically-characterized neuron that forms ribbon-style synapses is the rod-dominant or Mb1 bipolar cell of the goldfish retina. We report here the molecular characterization of syntaxin 3B from the goldfish retina. Using a combination of reverse transcription (RT) polymerase chain reaction (PCR) and immunostaining with a specific antibody, we show that syntaxin 3B is highly enriched in the plasma membrane of bipolar cell synaptic terminals of the goldfish retina. Using membrane capacitance measurements we demonstrate that a peptide derived from goldfish syntaxin 3B inhibits synaptic vesicle exocytosis. These experiments demonstrate that syntaxin 3B is an important factor for synaptic vesicle exocytosis in ribbon synapses of the vertebrate retina.

Syntaxin 3b is a t-SNARE specific for ribbon synapses of the retina.

Previous studies have demonstrated that ribbon synapses in the retina do not contain the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system. In contrast, ribbon synapses of the retina contain the related isoform syntaxin 3. In addition to its localization in ribbon synapses, syntaxin 3 is also found in nonneuronal cells, where it has been implicated in the trafficking of transport vesicles to the apical plasma membrane of polarized cells. The syntaxin 3 gene codes for four different splice forms, syntaxins 3A, 3B, 3C, and 3D. We demonstrate here by using analysis of EST databases, RT-PCR, in situ hybridization, and Northern blot analysis that cells in the mouse retina express only syntaxin 3B. In contrast, nonneuronal tissues, such as kidney, express only syntaxin 3A. The two major syntaxin isoforms (3A and 3B) have an identical N-terminal domain but differ in the C-terminal half of the SNARE domain and the C-terminal transmembrane domain. These two domains are thought to be directly involved in synaptic vesicle fusion. The interaction of syntaxin 1A and syntaxin 3B with other synaptic proteins was examined. We found that both proteins bind Munc18/N-sec1 with similar affinity. In contrast, syntaxin 3B had a much lower binding affinity for the t-SNARE SNAP25 compared with syntaxin 1A. By using an in vitro fusion assay, we could demonstrate that vesicles containing syntaxin 3B and SNAP25 could fuse with vesicles containing synaptobrevin2/VAMP2, demonstrating that syntaxin 3B can function as a t-SNARE.

Confocal analysis of synaptic connectivity of the rod pathway in the rabbit retina

The retina is a specialized neuronal structure that transforms the optical image into electrical signals which are transmitted to the brain via the optic nerve. As part of the strategy to cover a stimulus range as broad as 10 log units, from dim starlight to bright sunlight, retinal circuits are broadly divided into rod and cone pathways, responsible for dark and light-adapted vision, respectively. ^ In this dissertation, confocal microscopy and immunocytochemical methods were combined to study the synaptic connectivity of the rod pathway from the level of individual synapses to whole populations of neurons. The study was focused on synaptic interactions at the rod bipolar terminal. The purpose is to understand the synaptic structure of the dyad synapse made by rod bipolar terminals, including the synaptic components and connections, and their physiological functions in the rod pathway. In addition, some additional components and connections of the rod pathway were also studied in these experiments. The major results can be summarized as following: At the dyad synapse of rod bipolar terminals, three postsynaptic components—processes of All amacrine cells and the varicosities of S1 or S2 amacrine cells express different glutamate receptor subunits, which may underlie the functional diversity of these postsynaptic neurons. A reciprocal feedback system is formed by rod bipolar terminals and S1/S2 amacrine cells. Analysis showed these two wide-field GABA amacrine cells have stereotyped synaptic connections with the appropriate morphology and distribution to perform specific functions. In addition, S1 and S2 cells have different coupling patterns and, in general, there is no coupling between the two types. Besides the classic rod pathway though rod bipolar cells and All amacrine cells, the finding of direct connections between certain types of OFF cone bipolar cells and rods indicates the presence of an alternative rod pathway in the rabbit retina. ^ In summary, this dissertation presents a detailed view of the connection and receptors at rod bipolar terminals. Based on the morphology, distribution and coupling, different functional roles were identified for S1 and S2 amacrine cells. Finally, an alternative to the classic rod pathway was found in the rabbit retina. ^

Mechanism of molecular regulation of nuclear -encoded mitochondrial gene expression by electrical stimulation in the cultured neonatal rat cardiac myocytes

To answer the question whether increased energy demand resulting from myocyte hypertrophy and enhanced $\beta$-myosin heavy chain mRNA, contractile protein synthesis and assembly leads to mitochondrial proliferation and differentiation, we set up an electrical stimulation model of cultured neonatal rat cardiac myocytes. We describe, as a result of increased contractile activity, increased mitochondrial profiles, cytochrome oxidase mRNA, and activity, as well as a switch in mitochondrial carnitine palmitoyltransferase-I (CPT-I) from the liver to muscle isoform. We investigate physiological pathways that lead to accumulation of gene transcripts for nuclear encoded mitochondrial proteins in the heart. Cardiomyocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation (c-fos, c-jun, junB, nuclear respiratory factor 1 (Nrf-1)), mitochondrial proliferation (cytochrome c (Cyt c), cytochrome oxidase), and mitochondrial differentiation (carnitine palmitonyltransferase I (CPT-I) isoforms) were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25-3 hr) and followed by c-jun (0.5-3 hr), junB (0.5-6 hr), NRF-1 (1-12 hr), Cyt c (12-72 hr), cytochrome c oxidase (12-72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA. Electrical stimulation increased c-fos, $\beta$-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element (CRE), and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the Nrf-1 and CRE sites inhibited the induction by electrical stimulation or by transfection of c-jun into non-paced cardiac myocytes whereas mutation of the Sp-1 site maintained or increased the fold induction. This is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c. Overexpression of c-jun by transfection also activates the Nrf-1 and Cyt c mRNA sequentially. Electrical stimulation of cardiac myocytes activates the c-Jun-N-terminal kinase so that the fold-activation of the cyt c promoter is increased by pacing when either c-jun or c-fos/c-jun are cotransfected. We have identified physical association of Nrf-1 protein with the Nrf-1 enhancer element and of c-Jun with the CRE binding sites on the Cyt c promoter. This is the first demonstration that induction of Nrf-1 and c-Jun by pacing of cardiac myocytes directly mediates Cyt c gene expression and mitochondrial proliferation in response to hypertrophic stimuli in the heart.^ Subsequent to gene activation pathways that lead to mitochondrial proliferation, we observed an isoform switch in CPT-I from the liver to muscle mRNA. We have found that the half-life for the muscle CPT-I is not affected by electrical stimulation, but electrical decrease the T1/2 in the liver CPT-I by greater than 50%. This suggests that the liver CPT-I switch to muscle isoform is due to (1) a decrease in T1/2 of liver CPT-I and (2) activation of muscle CPT-Itranscripts by electrical stimulation. (Abstract shortened by UMI.) ^