Stephan Tetenborg

Most electrical synapses in the mammalian central nervous system are composed of Connexin 36 (Cx36). Electrical synapses are functionally plastic, changing their degree of coupling based on the activity of the cell or connected cells, or based on activation of neurotransmitter or neurohormone receptors. Plasticity can reach an extreme in which electrical synapses become functionally silent, which is a normal operational condition for some circuits. Cx36 coupling is regulated by phosphorylation, which opens the channels. In retinal circuits, Cx36 is often maintained in a poorly phosphorylated, poorly coupled state. We reasoned that phosphomimetic mutants of Cx36 could remain constitutively open and maintain circuits in a well-coupled state that will interrogate the need for plasticity. We developed a constitutively open Cx36 mutant by systematically replacing phosphorylatable residues that regulate coupling with acidic residues. Single mutants of serine 315 significantly modified functional regulation of coupling in HeLa cells, but mutation of four residues was required to produce a mutant that was constitutively open. This mutant, Cx36-S110D, T111E, S293D, S315D, called Cx36-DEDD, displayed high coupling in control conditions and only modest changes under phosphorylating and dephosphorylating conditions. We developed a conditional knockin mouse that expresses Cx36-DEDD and cytoplasmic tdTomato in cells that expressed Cre recombinase. When crossed with Six3-Cre mice, Cx36-DEDD expressed widely in the retina including in photoreceptors, bipolar, amacrine and ganglion cells. Rod-cone electrical coupling displayed the maximum of its physiological dynamic range, and photopic visual acuity and contrast sensitivity were significantly reduced in Cx36-DEDD homozygous animals. We conclude that reduction of coupling in some retinal circuits is required for optimal daylight vision. Significance Statement: Two types of synapses, chemical and electrical, work together throughout the central nervous system to perform neurological functions. While it is widely understood for chemical synapses that plasticity, changing the strength of synaptic connections, plays critical roles in many processes, this is far less understood for electrical synapses. By developing an electrical synapse protein mutant that locks channels in an open state, we have investigated retinal circuits that retain functional electrical synapses but lack their latitude for plasticity. This perturbation significantly compromises visual acuity and contrast sensitivity in the daylight, revealing that electrical synapse plasticity is necessary to tune retinal functions for optimal performance. Thus, electrical synapse plasticity along with chemical synapse plasticity is required for neural function.

Electrical synapses containing Connexin 36 (Cx36) represent the main means for direct electrical communication among neurons in the mammalian nervous system. However, little is known about the protein complexes that constitute these synapses. In the present study, we applied different BioID strategies to screen the interactomes of Connexin 36 and its zebrafish orthologue Cx35.1 in retinal neurons. For in vivo proximity labeling in mice, we took advantage of the Cx36-EGFP strain and expressed a GFP-nanobody-TurboID fusion construct selectively in AII amacrine cells. For in vivo BioID in zebrafish, we generated a transgenic line expressing a Cx35.1-TurboID fusion under control of the Cx35.1 promoter. Both strategies allowed us to capture a plethora of molecules that were associated with electrical synapses and showed a high degree of evolutionary conservation in the proteomes of both species. Besides known interactors of Cx36 such as ZO-1 and ZO-2, we have identified more than 50 new proteins, such as scaffold proteins, adhesion molecules, and regulators of the cytoskeleton. Moreover, we determined the subcellular localization of these proteins in mouse retina and tested potential binding interactions with Cx36. Among these new interactors, we identified signal-induced proliferation associated 1 like 3 (Sipa1l3), a protein that has been implicated in cell junction formation and cell polarity, as a new scaffold of electrical synapses. Interestingly, Sipa1l3 was able to interact with ZO-1, ZO-2, and Cx36, suggesting a pivotal role in electrical synapse function. In summary, our study provides the first detailed view of the electrical synapse proteome in retinal neurons, which is likely to apply to electrical synapses elsewhere.

Although chemical and electrical synapses function fundamentally differently, they evidently share common design principles. Like neurotransmitter receptors, Connexin 36 (Cx36) containing gap junction channels, key constituents of electrical synapses, are anchored to scaffolding proteins that stabilize the connexin at the synapse. One of the most prominent proteins that has been described in this context is the Zonula occludens protein 1 (ZO-1). ZO-1 interacts with Cx36 via one of its three PDZ domains. This interaction is inherently weak and was suggested to facilitate the dynamic regulation of electrical synapses. In the present study, we have combined Gaussian accelerated molecular dynamics simulations and binding assays to identify the exact residues in the PDZ binding motif of Cx36 that are necessary to sustain these low affinity interactions. Among the different Cx36 mutations we have generated, we discovered a single substitution at position 319 within the PDZ binding motif that massively increases binding in different experimental settings. In addition to this site, we found that acidic residues adjacent to the PDZ binding motif (PBM) in Cx36 and its fish orthologues are evolutionarily tuned to weaken PDZ interactions as well. We were able to enhance PDZ1 binding drastically by substituting these residues with hydrophobic or positively charged amino acids. Finally, we demonstrate that the weak PDZ1/Cx36 interaction is sensitive to CaMKII mediated phosphorylation of Cx36, suggesting that ZO-1 unbinding may be a necessary event to potentiate electrical synapses. In summary, our study provides a detailed analysis of different mechanisms that can be exploited to modify the interaction between two key components of an electrical synapse: Cx36 and ZO-1.

Retinitis Pigmentosa is a chronic retinal degenerative disease characterized by the gradual loss of rod, and later, cone photoreceptors until the individual is completely blind. Regeneration of photoreceptors from endogenous progenitor cells is a possible therapeutic approach, but mammals do not do this naturally. Mammalian models can be induced to generate retinal progenitors from Müller glial cells, but there has been limited success in rod photoreceptor specific regeneration. Unlike mammals, zebrafish have the natural ability to regenerate neurons after injury or disease and can provide insight into the molecular mechanisms of regeneration. In this study, we used a zebrafish model of Retinitis Pigmentosa to investigate the class of progenitors responsible for rod photoreceptor regeneration in the context of chronic disease. Using bioinformatic analyses of single-cell RNA sequencing datasets, we identified master regulator genes responsible for proliferation of retinal progenitors, differentiation of progenitors into rod photoreceptors, and maturation of the new rod photoreceptors. Using transient knockdown of gene expression in adult regenerating retina we determined that e2f1, e2f2, e2f3 and aurkb are critical for proliferation of progenitors, and prdm1a is critical for differentiation of progenitors into rod photoreceptors. This study provides a list of master regulators responsible for the specific regeneration of rod photoreceptors during chronic retinal degeneration.

Retinitis Pigmentosa is a chronic retinal degenerative disease characterized by the gradual loss of rod, and later, cone photoreceptors until the individual is completely blind. Regeneration of photoreceptors from endogenous progenitor cells is a possible therapeutic approach, but mammals do not do this naturally. Mammalian models can be induced to generate retinal progenitors from Müller glial cells, but there has been limited success in rod photoreceptor specific regeneration. Unlike mammals, zebrafish have the natural ability to regenerate neurons after injury or disease and can provide insight into the molecular mechanisms of regeneration. In this study, we used a zebrafish model of Retinitis Pigmentosa to investigate the class of progenitors responsible for rod photoreceptor regeneration in the context of chronic disease. Using bioinformatic analyses of single-cell RNA sequencing datasets, we identified master regulator genes responsible for proliferation of retinal progenitors, differentiation of progenitors into rod photoreceptors, and maturation of the new rod photoreceptors. Using transient knockdown of gene expression in adult regenerating retina we determined that e2f1 , e2f2 , e2f3 and aurkb are critical for proliferation of progenitors, and prdm1a is critical for differentiation of progenitors into rod photoreceptors. This study provides a list of master regulators responsible for the specific regeneration of rod photoreceptors during chronic retinal degeneration. Impact Statement: Identification of master regulating genes that drive the proliferation of progenitor cells and their differentiation specifically into rod photoreceptors provides insight that can be used to develop regenerative therapies for retinal degenerative diseases.

: Electrical synapses containing Connexin 36 (Cx36) represent the main means for direct electrical communication among neurons in the mammalian nervous system. However, little is known about the protein complexes that constitute these synapses. In the present study, we applied different BioID strategies to screen the interactomes of Connexin 36 and its zebrafish orthologue Cx35b in retinal neurons. For in vivo proximity labeling in mice, we took advantage of the Cx36-EGFP strain and expressed a GFP-nanobody-TurboID fusion construct selectively in AII amacrine cells. For in vivo BioID in zebrafish, we generated a transgenic line expressing a Cx35b-TurboID fusion under control of the Cx35b promoter. Both strategies allowed us to capture a plethora of molecules that were associated with electrical synapses and showed a high degree of evolutionary conservation in the proteomes of both species. Besides known interactors of Cx36 such as ZO-1 and ZO-2 we have identified more than 50 new proteins, such as scaffold proteins, adhesion molecules and regulators of the cytoskeleton. Moreover, we determined the subcellular localization of these proteins in mouse retina and tested potential binding interactions with Cx36. Amongst these new interactors, we identified signal induced proliferation associated 1 like 3 (Sipa1l3), a protein that has been implicated in cell junction formation and cell polarity, as a new scaffold of electrical synapses. Interestingly, Sipa1l3 was able to interact with ZO-1, ZO-2 and Cx36, suggesting a pivotal role in electrical synapse function. In summary, our study provides the first detailed view of the electrical synapse proteome in retinal neurons, which is likely to apply to electrical synapses elsewhere.

Gap junctions formed by the major neuronal connexin Cx36 function as electrical synapses in the nervous system and provide unique functions such as synchronizing neuron activities or supporting network oscillations. Although the physiological significance of electrical synapses for neuronal networks is well established, little is known about the pathways that regulate the transport of its main component: Cx36. Here we have used HEK293T cells as an expression system in combination with siRNA and BioID screens to study the transition of Cx36 from the ER to the cis Golgi. Our data indicate that the C-terminal tip of Cx36 is a key factor in this process, mediating binding interactions with two distinct components in the early secretory pathway: the COPII complex and the Golgi stacking protein Grasp55. The C-terminal amino acid valine serves as an ER export signal to recruit COPII cargo receptors Sec24A/B/C at ER exit sites, whereas the PDZ binding motif “SAYV” mediates an interaction with Grasp55. These two interactions have opposing effects in their respective compartments. While Sec24 subunits carry Cx36 out of the ER, Grasp55 stabilizes Cx36 in the Golgi as shown in over expression experiments. These early regulatory steps of Cx36 are expected to be essential for the formation, function, regulation and plasticity of electrical synapses in the developing and mature nervous system.

Gap junctions formed by the major neuronal connexin Cx36 function as electrical synapses in the nervous system and provide unique functions such as synchronizing activities or network oscillations. Although the physiological significance of electrical synapses for neuronal networks is well established, little is known about the pathways that regulate the transport of its main component: Cx36. Here we have used HEK293T cells as an expression system in combination with siRNA and BioID screens to study the transition of Cx36 from the ER to the cis Golgi. Our data indicate that the C-terminal tip of Cx36 is a key factor in this process, mediating binding interactions with two distinct components in the early secretory pathway: the COPII complex and the Golgi stacking protein Grasp55. The C-terminal amino acid valine serves as an ER export signal to recruit COPII cargo receptors Sec24A/B/C at ER exit sites, whereas the PDZ binding motif “SAYV” mediates an interaction with Grasp55. These two interactions have opposing effects in their respective compartments. While Sec24 subunits carry Cx36 out of the ER, Grasp55 stabilizes Cx36 in the Golgi as shown in over expression experiments. These early regulatory steps of Cx36 are expected to be essential for the formation, function, regulation and plasticity of electrical synapses in the developing and mature nervous system.

Overexpression of proteins in transiently transfected cells is a simple way to study basic transport mechanisms and the underlying protein-protein interactions. While expression systems have obvious drawbacks compared to in vivo experiments, they allow a quick assessment of more conserved functions, for instance, ER export or sorting of proteins in the Golgi. In a previous study, our group described the formation of ER-derived removal vesicles for the gap junction protein Cx36 in transfected HEK293T cells. These removal vesicles, termed "whorls" because of their concentric structure, were formed by Cx36 channels that failed to escape the ER. In this article, we describe an imaging protocol that can be used to determine these ER retention defects for Cx36 expressed in cultured cells. The protocol we provide here employs regular confocal microscopy, which allows for sufficient resolution to reveal the characteristic shape of ER whorls.