STAT Signaling Modifies Ascl1 Chromatin Binding and Limits Neural Regeneration from Muller Glia in Adult Mouse Retina.

Müller glia (MG) serve as sources for retinal regeneration in non-mammalian vertebrates. We find that this process can be induced in mouse MG, after injury, by transgenic expression of the proneural transcription factor Ascl1 and the HDAC inhibitor TSA. However, new neurons are generated only from a subset of MG. Identifying factors that limit Ascl1-mediated MG reprogramming could make this process more efficient. In this study, we test whether injury-induced STAT activation hampers the ability of Ascl1 to reprogram MG into retinal neurons. Single-cell RNA-seq shows that progenitor-like cells derived from Ascl1-expressing MG have a higher level of STAT signaling than do those cells that become neurons. Ascl1-ChIPseq and ATAC-seq show that STAT potentially directs Ascl1 to developmentally inappropriate targets. Using a STAT inhibitor, in combination with our previously described reprogramming paradigm, we found a large increase in the ability of MG to generate neurons.

Small molecule Photoregulin3 prevents retinal degeneration in the RhoP23H mouse model of retinitis pigmentosa.

Regulation of rod gene expression has emerged as a potential therapeutic strategy to treat retinal degenerative diseases like retinitis pigmentosa (RP). We previously reported on a small molecule modulator of the rod transcription factor Nr2e3, Photoregulin1 (PR1), that regulates the expression of photoreceptor-specific genes. Although PR1 slows the progression of retinal degeneration in models of RP in vitro, in vivo analyses were not possible with PR1. We now report a structurally unrelated compound, Photoregulin3 (PR3) that also inhibits rod photoreceptor gene expression, potentially though Nr2e3 modulation. To determine the effectiveness of PR3 as a potential therapy for RP, we treated RhoP23H mice with PR3 and assessed retinal structure and function. PR3-treated RhoP23H mice showed significant structural and functional photoreceptor rescue compared with vehicle-treated littermate control mice. These results provide further support that pharmacological modulation of rod gene expression provides a potential strategy for the treatment of RP.

Potential of Small Molecule-Mediated Reprogramming of Rod Photoreceptors to Treat Retinitis Pigmentosa.

Purpose: Mutations in rod photoreceptor genes can cause retinitis pigmentosa (RP). Rod gene expression is regulated by the nuclear hormone receptor, Nr2e3. Genetic deletion of Nr2e3 reprograms rods into cells that resemble cone photoreceptors, and might therefore prevent their death from some forms of RP. There are no identified ligands for Nr2e3; however, reverse agonists might mimic the genetic rescue effect and may be therapeutically useful for the treatment of RP. Methods: We screened for small molecule modulators of Nr2e3 using primary retinal cell cultures and characterized the most potent, which we have named photoregulin1 (PR1), in vitro and in vivo. We also tested the ability of PR1 to slow the progression of photoreceptor degeneration in two common mouse models of autosomal dominant RP, the RhoP23H and the Pde6brd1 mutations. Results: In developing retina, PR1 causes a decrease in rod gene expression and an increase in S opsin+ cones. Photoregulin1 continues to inhibit rod gene expression in adult mice. When applied to two mouse models of RP, PR1 slows the degeneration of photoreceptors. Conclusions: Chemical compounds identified as modulators of Nr2e3 activity may be useful for the treatment of RP through their effects on expression of disease-causing mutant genes.

EphB2 signaling regulates lesion-induced axon sprouting but not critical period length in the postnatal auditory brainstem.

BACKGROUND: Studies of developmental plasticity may provide insight into plasticity during adulthood, when neural circuitry is less responsive to losses or changes in input. In the mammalian auditory brainstem, globular bushy cell axons of the ventral cochlear nucleus (VCN) innervate the contralateral medial nucleus of the trapezoid body (MNTB) principal neurons. VCN axonal terminations in MNTB, known as calyces of Held, are very large and specialized for high-fidelity transmission of auditory information. Following unilateral deafferentation during postnatal development, VCN axons from the intact side form connections with novel targets, including the ipsilateral MNTB. EphB signaling has been shown to play a role in this process during the first postnatal week, but mechanisms involved in this reorganization during later developmental periods remain unknown. RESULTS: We found that EphB2 signaling reduces the number of induced ipsilateral projections to the MNTB after unilateral VCN removal at postnatal day seven (P7), but not after removal of the VCN on one side at P10, after the closure of the critical period for lesion-induced innervation of the ipsilateral MNTB. CONCLUSIONS: Results from this study indicate that molecular mechanisms involved in the development of circuitry may also play a part in rewiring after deafferentation during development, but do not appear to regulate the length of critical periods for plasticity.

Profiling neurotransmitter receptor expression in the Ambystoma mexicanum brain.

Ability to regenerate limbs and central nervous system (CNS) is unique to few vertebrates, most notably the axolotl (Ambystoma sp.). However, despite the fact the neurotransmitter receptors are involved in axonal regeneration, little is known regarding its expression profile. In this project, RT-PCR and qPCR were performed to gain insight into the neurotransmitter receptors present in Ambystoma. Its functional ability was studied by expressing axolotl receptors in Xenopus laevis oocytes by either injection of mRNA or by direct microtransplantation of brain membranes. Oocytes injected with axolotl mRNA expressed ionotropic receptors activated by GABA, aspartate+glycine and kainate, as well as metabotropic receptors activated by acetylcholine and glutamate. Interestingly, we did not see responses following the application of serotonin. Membranes from the axolotl brain were efficiently microtransplanted into Xenopus oocytes and two types of native GABA receptors that differed in the temporal course of their responses and affinities to GABA were observed. Results of this study are necessary for further characterization of axolotl neurotransmitter receptors and may be useful for guiding experiments aimed at understanding activity-dependant limb and CNS regeneration.

EphB signaling regulates target innervation in the developing and deafferented auditory brainstem.

Precision in auditory brainstem connectivity underlies sound localization. Cochlear activity is transmitted to the ventral cochlear nucleus (VCN) in the mammalian brainstem via the auditory nerve. VCN globular bushy cells project to the contralateral medial nucleus of the trapezoid body (MNTB), where specialized axons terminals, the calyces of Held, encapsulate MNTB principal neurons. The VCN-MNTB pathway is an essential component of the circuitry used to compute interaural intensity differences that are used for localizing sounds. When input from one ear is removed during early postnatal development, auditory brainstem circuitry displays robust anatomical plasticity. The molecular mechanisms that control the development of auditory brainstem circuitry and the developmental plasticity of these pathways are poorly understood. In this study we examined the role of EphB signaling in the development of the VCN-MNTB projection and in the reorganization of this pathway after unilateral deafferentation. We found that EphB2 and EphB3 reverse signaling are critical for the normal development of the projection from VCN to MNTB, but that successful circuit assembly most likely relies upon the coordinated function of many EphB proteins. We have also found that ephrin-B reverse signaling repels induced projections to the ipsilateral MNTB after unilateral deafferentation, suggesting that similar mechanisms regulate these two processes.

Formation and maturation of the calyx of Held.

Sound localization requires precise and specialized neural circuitry. A prominent and well-studied specialization is found in the mammalian auditory brainstem. Globular bushy cells of the ventral cochlear nucleus (VCN) project contralaterally to neurons of the medial nucleus of the trapezoid body (MNTB), where their large axons terminate on cell bodies of MNTB principal neurons, forming the calyces of Held. The VCN-MNTB pathway is necessary for the accurate computation of interaural intensity and time differences; MNTB neurons provide inhibitory input to the lateral superior olive, which compares levels of excitation from the ipsilateral ear to levels of tonotopically matched inhibition from the contralateral ear, and to the medial superior olive, where precise inhibition from MNTB neurons tunes the delays of binaural excitation. Here we review the morphological and physiological aspects of the development of the VCN-MNTB pathway and its calyceal termination, along with potential mechanisms that give rise to its precision. During embryonic development, VCN axons grow towards the midline, cross the midline into the region of the presumptive MNTB and then form collateral branches that will terminate in calyces of Held. In rodents, immature calyces of Held appear in MNTB during the first few days of postnatal life. These calyces mature morphologically and physiologically over the next three postnatal weeks, enabling fast, high fidelity transmission in the VCN-MNTB pathway.

Ephrin-B reverse signaling is required for formation of strictly contralateral auditory brainstem pathways.

Specificity in the projections from the mammalian ventral cochlear nucleus (VCN) is essential for sound localization. Globular bushy cells project from the VCN to the medial nucleus of the trapezoid body (MNTB) on the contralateral, but not the ipsilateral, side of the brainstem, terminating in large synaptic endings known as calyces of Held. The precision in this pathway is critical for the computation of interaural intensity differences, which are used in sound localization. The mechanisms underlying the development of this projection are not completely understood. In this study, we tested the role of Eph receptor tyrosine kinases and their ephrin ligands in limiting the VCN-MNTB projection to the contralateral side. We found that mice with null mutations in EphB2 and EphB3 had normal contralateral VCN-MNTB projections, yet these projections also had significant numbers of aberrant collateral branches in the ipsilateral MNTB. These aberrant branches ended in calyceal terminations in MNTB. Similar ipsilateral projections were seen in mice with mutations in ephrin-B2. In both of these mouse lines, ipsilateral projections formed concurrently with normal contralateral projections and were not eliminated later in development. However, mice with mutations that affected only the intracellular domain of EphB2 had normal, strictly contralateral VCN-MNTB projections. Expression studies showed that EphB2 is expressed in VCN axons and ephrin-B2 is expressed in MNTB. Together, these data suggest that EphB2-ephrin-B2 reverse signaling is required to prevent the formation of ipsilateral VCN-MNTB projections and that this signaling operates non-cell autonomously.

Auditory brainstem neural activation patterns are altered in EphA4- and ephrin-B2-deficient mice.

Auditory processing requires proper formation of tonotopically ordered projections. We have evaluated the role of an Eph receptor tyrosine kinase and an ephrin ligand in the development of these frequency maps. We demonstrated expression of EphA4 and ephrin-B2 in auditory nuclei and found expression gradients along the frequency axis in neonates. We tested the roles of EphA4 and ephrin-B2 in development of auditory projections by evaluating whether mutations result in altered patterns of expression of the immediate early gene c-fos after exposure to pure tone stimuli. We evaluated two nuclei, the dorsal cochlear nucleus (DCN) and the medial nucleus of the trapezoid body (MNTB), which project in two distinct auditory pathways. The mean number of c-fos-positive neurons in EphA4(-/-) DCN after 8-kHz pure tone stimulation was 42% lower than in wild-type DCN. Along the dorsoventral, tonotopic axis of DCN, the mean position of c-fos-positive neurons was similar for mutant and wild-type mice, but the spread of these neurons along the tonotopic axis was 35% greater for ephrin-B2(lacZ/+) mice than for wild-type mice. We also examined these parameters in MNTB after exposure to 40-kHz pure tones. Both EphA4(-/-) and ephrin-B2(lacZ/+) mice had significantly fewer c-fos-positive cells than wild-type littermates. The labeled band of cells was narrower and laterally shifted in EphA4(-/-) mice compared with wild-type mice. These differences in cell number and distribution suggest that EphA4 and ephrin-B2 signaling influence auditory activation patterns.

Mitogen-associated protein kinase- and protein kinase A-dependent regulation of rhodopsin promoter expression in zebrafish rod photoreceptor cells.

Mitogen-associated protein kinase (MAPK)- and protein kinase A (PKA)-dependent signal transductions play important roles in the regulation of gene expression. Both MAPK and PKA pathways can be activated by light exposure. In this study, we investigated the effect of light on MAPK and PKA signal transduction and their roles in the regulation of rhodopsin promoter expression by using transgenic zebrafish [Tg(rhod::GFP)]. The Tg(rhod::GFP) fish express short half-life GFP that is under the transcriptional control of the zebrafish rhodopsin promoter and can therefore be used for in vivo studies of rhodopsin gene transcription in live cells. Blue light plays a role in the regulation of rhodopsin promoter expression via an MAPK-mediated signal transduction cascade. Blue light excites cryptochromes (CRY), which activate the downstream PKC-dependent MAPK signal pathway. White light, on the other hand, regulates rhodopsin promoter expression via a G-protein-coupled cAMP-dependent PKA pathway. White light promotes dopamine release in the retina, which activates dopamine receptors and the downstream PKA pathway. Blocking MAPK signaling diminishes the blue light-induced increases in rhodopsin promoter expression, but this treatment has no effect on white light-mediated rhodopsin promoter expression. Conversely, blocking the PKA pathway diminishes the white light-induced rhodopsin promoter expression but does not affect rhodopsin promoter expression regulated by blue light. Together, the data suggest that MAPK and PKA regulate rhodopsin transcription through parallel signal transduction pathways.