Adeno-associated virus (AAV)-mediated Cre recombinase expression in melanopsin ganglion cells without leaky expression in rod/cone photoreceptors.

BACKGROUND: Constituting about 5 % of mouse retinal ganglion cells (RGCs), intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin (gene symbol Opn4) and drive such photoresponses as pupil constriction, melatonin suppression, and circadian photoentrainment. Opn4Cre mice with Cre recombinase-expressing ipRGCs have enabled genetic manipulation of ipRGCs; unfortunately, while Cre expression within the inner retina is ipRGC-specific, leaky expression also occurs in some outer retinal photoreceptors, so Cre-induced alterations in the latter cells may confound certain studies of ipRGC function. Methods that express Cre in ipRGCs but not rods or cones are needed. NEW METHOD: We have constructed a recombinant serotype-2 adeno-associated virus, rAAV2-Opn4-Cre, with the improved Cre recombinase (iCre) gene under the control of a ∼3kbp Opn4 promoter sequence, and injected it intravitreally into mice to transduce inner retinal neurons while sparing the outer retina. RESULTS: We introduced rAAV2-Opn4-Cre into Cre reporter mice in which enhanced green fluorescent protein (EGFP) expression indicates Cre expression. Single-cell electrophysiological recordings and intracellular dye fills showed that 84 % of the EGFP+ cells were ipRGCs including M1-M6 types, while 16 % were conventional RGCs. COMPARISON WITH EXISTING METHODS: Whereas Opn4Cre mice express Cre in some rod/cone photoreceptors, intravitreally applied rAAV2-Opn4-Cre induces Cre only in the inner retina, albeit with leaky expression in some conventional RGCs. CONCLUSIONS: rAAV2-Opn4-Cre has overcome a significant limitation of Opn4Cre mice. We recommend usage scenarios where the Cre-expressing conventional RGCs should not pose a problem.

Amacrine Cells Forming Gap Junctions With Intrinsically Photosensitive Retinal Ganglion Cells: ipRGC Types, Neuromodulator Contents, and Connexin Isoform.

Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs) signal not only centrally to non-image-forming visual centers of the brain but also intraretinally to amacrine interneurons through gap junction electrical coupling, potentially modulating image-forming retinal processing. We aimed to determine (1) which ipRGC types couple with amacrine cells, (2) the neuromodulator contents of ipRGC-coupled amacrine cells, and (3) whether connexin36 (Cx36) contributes to ipRGC-amacrine coupling. Methods: Gap junction-permeable Neurobiotin tracer was injected into green fluorescent protein (GFP)-labeled ipRGCs in Opn4Cre/+; Z/EG mice to stain coupled amacrine cells, and immunohistochemistry was performed to reveal the neuromodulator contents of the Neurobiotin-stained amacrine cells. We also created Opn4Cre/+; Cx36flox/flox; Z/EG mice to knock out Cx36 in GFP-labeled ipRGCs and looked for changes in the number of ipRGC-coupled amacrine cells. Results: Seventy-three percent of ipRGCs, including all six types (M1-M6), were tracer-coupled with amacrine somas 5.7 to 16.5 µm in diameter but not with ganglion cells. Ninety-two percent of the ipRGC-coupled somas were in the ganglion cell layer and the rest in the inner nuclear layer. Some ipRGC-coupled amacrine cells were found to accumulate serotonin or to contain nitric oxide synthase or neuropeptide Y. Knocking out Cx36 in M2 and M4 dramatically reduced the number of coupled somas. Conclusions: Heterologous gap junction coupling with amacrine cells is widespread across mouse ipRGC types. ipRGC-coupled amacrine cells probably comprise multiple morphologic types and use multiple neuromodulators, suggesting that gap junctional ipRGC-to-amacrine signaling likely exerts diverse modulatory effects on retinal physiology. ipRGC-amacrine coupling is mediated partly, but not solely, by Cx36.

Prolonged Melanopsin-based Photoresponses Depend in Part on RPE65 and Cellular Retinaldehyde-binding Protein (CRALBP).

PURPOSE: Intrinsically photosensitive retinal ganglion cells (ipRGCs) contain the photopigment melanopsin and can signal light continuously for many hours. Melanopsin is excited when its chromophore 11-cis-retinal absorbs a photon and becomes all-trans-retinal, which must be reisomerized to 11-cis-retinal to regenerate photoexcitable melanopsin. Due to the great distance separating ipRGCs from the retinal pigment epithelium (RPE) whose retinoid cycle produces 11-cis-retinal, ipRGCs had been assumed to regenerate all melanopsin molecules autonomously. Surprisingly, we previously found that pharmacologically inhibiting the retinoid cycle rendered melanopsin-based responses to prolonged illumination less sustained, suggesting that the RPE may supply retinoids to help ipRGCs regenerate melanopsin during extended photostimulation. However, the specificity of those drugs is unclear. Here, we reexamined the role of the retinoid cycle, and tested whether the RPE-to-ipRGC transport of retinoids utilizes cellular retinaldehyde-binding protein (CRALBP), present throughout the RPE and Müller glia. METHODS: To measure melanopsin-mediated photoresponses in isolation, all animals were 8- to 12-month-old rod/cone-degenerate mice. We genetically knocked out RPE-specific 65 kDa protein (RPE65), a critical enzyme in the retinoid cycle. We also knocked out the CRALBP gene rlbp1 mainly in Foxg1-expressing Müller cells. We obtained multielectrode-array recordings from ipRGCs in a novel RPE-attached mouse retina preparation, and imaged pupillary light reflexes in vivo. RESULTS: Melanopsin-based ipRGC responses to prolonged light became less tonic in both knockout lines, and pupillary light reflexes were also less sustained in RPE65-knockout than control mice. CONCLUSIONS: These results confirm that ipRGCs rely partly on the retinoid cycle to continuously regenerate melanopsin during prolonged photostimulation, and suggest that CRALBP in Müller glia likely transports 11-cis-retinal from the RPE to ipRGCs - this is the first proposed functional role for CRALBP in the inner retina.

Mechanisms creating transient and sustained photoresponses in mammalian retinal ganglion cells.

Retinal neurons use sustained and transient light responses to encode visual stimuli of different frequency ranges, but the underlying mechanisms remain poorly understood. In particular, although earlier studies in retinal ganglion cells (RGCs) proposed seven potential mechanisms, all seven have since been disputed, and it remains unknown whether different RGC types use different mechanisms or how many mechanisms are used by each type. Here, we conduct a comprehensive survey in mice and rats of 12 candidate mechanisms that could conceivably produce tonic rod/cone-driven ON responses in intrinsically photosensitive RGCs (ipRGCs) and transient ON responses in three types of direction-selective RGCs (TRHR+, Hoxd10+ ON, and Hoxd10+ ON-OFF cells). We find that the tonic kinetics of ipRGCs arises from their substantially above-threshold resting potentials, input from sustained ON bipolar cells, absence of amacrine cell inhibition of presynaptic ON bipolar cells, and mGluR7-mediated maintenance of light-evoked glutamatergic input. All three types of direction-selective RGCs receive input from transient ON bipolar cells, and each type uses additional strategies to promote photoresponse transience: presynaptic inhibition and dopaminergic modulation for TRHR+ cells, center/surround antagonism and relatively negative resting potentials for Hoxd10+ ON cells, and presynaptic inhibition for Hoxd10+ ON-OFF cells. We find that the sustained nature of ipRGCs' rod/cone-driven responses depends neither on melanopsin nor on N-methyl-d-aspartate (NMDA) receptors, whereas the transience of the direction-selective cells' responses is influenced neither by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor desensitization nor by glutamate uptake. For all cells, we further rule out spike frequency adaptation and intracellular Ca2+ as determinants of photoresponse kinetics. In conclusion, different RGC types use diverse mechanisms to produce sustained or transient light responses. Parenthetically, we find evidence in both mice and rats that the kinetics of light-induced mGluR6 deactivation determines whether an ON bipolar cell responds tonically or transiently to light.

PIK3C2B inhibition improves function and prolongs survival in myotubular myopathy animal models.

Myotubular myopathy (MTM) is a devastating pediatric neuromuscular disorder of phosphoinositide (PIP) metabolism resulting from mutations of the PIP phosphatase MTM1 for which there are no treatments. We have previously shown phosphatidylinositol-3-phosphate (PI3P) accumulation in animal models of MTM. Here, we tested the hypothesis that lowering PI3P levels may prevent or reverse the MTM disease process. To test this, we targeted class II and III PI3 kinases (PI3Ks) in an MTM1-deficient mouse model. Muscle-specific ablation of Pik3c2b, but not Pik3c3, resulted in complete prevention of the MTM phenotype, and postsymptomatic targeting promoted a striking rescue of disease. We confirmed this genetic interaction in zebrafish, and additionally showed that certain PI3K inhibitors prevented development of the zebrafish mtm phenotype. Finally, the PI3K inhibitor wortmannin improved motor function and prolonged lifespan of the Mtm1-deficient mice. In all, we have identified Pik3c2b as a genetic modifier of Mtm1 mutation and demonstrated that PIK3C2B inhibition is a potential treatment strategy for MTM. In addition, we set the groundwork for similar reciprocal inhibition approaches for treating other PIP metabolic disorders and highlight the importance of modifier gene pathways as therapeutic targets.

All spiking, sustained ON displaced amacrine cells receive gap-junction input from melanopsin ganglion cells.

Retinal neurons exhibit sustained versus transient light responses, which are thought to encode low- and high-frequency stimuli, respectively. This dichotomy has been recognized since the earliest intracellular recordings from the 1960s, but the underlying mechanisms are not yet fully understood. We report that in the ganglion cell layer of rat retinas, all spiking amacrine interneurons with sustained ON photoresponses receive gap-junction input from intrinsically photosensitive retinal ganglion cells (ipRGCs), recently discovered photoreceptors that specialize in prolonged irradiance detection. This input presumably allows ipRGCs to regulate the secretion of neuromodulators from these interneurons. We have identified three morphological varieties of such ipRGC-driven displaced amacrine cells: (1) monostratified cells with dendrites terminating exclusively in sublamina S5 of the inner plexiform layer, (2) bistratified cells with dendrites in both S1 and S5, and (3) polyaxonal cells with dendrites and axons stratifying in S5. Most of these amacrine cells are wide field, although some are medium field. The three classes respond to light differently, suggesting that they probably perform diverse functions. These results demonstrate that ipRGCs are a major source of tonic visual information within the retina and exert widespread intraretinal influence. They also add to recent evidence that ganglion cells signal not only to the brain.

Characterizing and modeling the intrinsic light response of rat ganglion-cell photoreceptors.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate both image-forming vision and non-image-forming visual responses such as pupillary constriction and circadian photoentrainment. Five types of ipRGCs, named M1-M5, have been discovered in rodents. To further investigate their photoresponse properties, we made multielectrode array spike recordings from rat ipRGCs, classified them into M1, M2/M4, and M3/M5 clusters, and measured their intrinsic, melanopsin-based responses to single and flickering light pulses. Results showed that ipRGC spiking can track flickers up to ∼0.2 Hz in frequency and that flicker intervals between 5 and 14 s evoke the most spikes. We also learned that melanopsin's integration time is intensity and cluster dependent. Using these data, we constructed a mathematical model for each cluster's intrinsic photoresponse. We found that the data for the M1 cluster are best fit by a model that assumes a large photoresponse, causing the cell to enter depolarization block. Our models also led us to hypothesize that the M2/M4 and M3/M5 clusters experience comparable photoexcitation but that the M3/M5 cascade decays significantly faster than the M2/M4 cascade, resulting in different response waveforms between these clusters. These mathematical models will help predict how each ipRGC cluster might respond to stimuli of any waveform and could inform the invention of lighting technologies that promote health through melanopsin stimulation.

Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures.

Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na(+) and K(+) currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.

The rat retina has five types of ganglion-cell photoreceptors.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are inner retinal photoreceptors that mediate non-image-forming visual functions, e.g. pupillary constriction, regulation of pineal melatonin release, and circadian photoentrainment. Five types of ipRGCs were recently discovered in mouse, but whether they exist in other mammals remained unknown. We report that the rat also has five types of ipRGCs, whose morphologies match those of mouse ipRGCs; this is the first demonstration of all five cell types in a non-mouse species. Through immunostaining and λmax measurements, we showed that melanopsin is likely the photopigment of all rat ipRGCs. The various cell types exhibited diverse spontaneous spike rates, with the M1 type spiking the least and M4 spiking the most, just like we had observed for their mouse counterparts. Also similar to mouse, all ipRGCs in rat generated not only sluggish intrinsic photoresponses but also fast, synaptically driven ones. However, we noticed two significant differences between these species. First, whereas we learned previously that all mouse ipRGCs had equally sustained synaptic light responses, rat M1 cells' synaptic photoresponses were far more transient than those of M2-M5. Since M1 cells provide all input to the circadian clock, this rat-versus-mouse discrepancy could explain the difference in photoentrainment threshold between mouse and other species. Second, rat ipRGCs' melanopsin-based spiking photoresponses could be classified into three varieties, but only two were discerned for mouse ipRGCs. This correlation of spiking photoresponses with cell types will help researchers classify ipRGCs in multielectrode-array (MEA) spike recordings.

Conditional knockout of pik3c3 causes a murine muscular dystrophy.

Abnormalities in phosphoinositide metabolism are an emerging theme in human neurodegenerative disease. Myotubular myopathy is a prototypical disorder of phosphoinositide dysregulation that is characterized by profound muscle pathology and weakness and that is caused by mutations in MTM1, which encodes a phosphatase that targets 3-position phosphoinositides, including phosphatidylinositol 3-phosphate. Although the association between MTM1 and muscle disease has become increasingly clarified, the normal role(s) of phosphatidylinositol 3-phosphate metabolism in muscle development and homeostasis remain poorly understood. To begin to address the function of phosphatidylinositol 3-phosphate in skeletal muscle, we focused on the primary kinase responsible for its production, and created a muscle-specific conditional knockout of the class III phosphatidylinositol 3-kinase, Pik3c3. Muscle-specific deletion of Pik3c3 did not disturb embryogenesis or early postnatal development, but resulted in progressive disease characterized by reduced activity and death by 2 months of age. Histopathological analysis demonstrated changes consistent with a murine muscular dystrophy. Examination for cellular mechanism(s) responsible for the dystrophic phenotype revealed significant alterations in the autophagolysosomal pathway with mislocation of known dystrophy proteins to the lysosomal compartment. In all, we present the first analysis of Pik3c3 in skeletal muscle, and report a novel association between deletion of Pik3c3 and muscular dystrophy.

Murine Fig4 is dispensable for muscle development but required for muscle function.

BACKGROUND: Phosphatidylinositol phosphates (PIPs) are low-abundance phospholipids that participate in a range of cellular processes, including cell migration and membrane traffic. PIP levels and subcellular distribution are regulated by a series of lipid kinases and phosphatases. In skeletal muscle, PIPs and their enzymatic regulators serve critically important functions exemplified by mutations of the PIP phosphatase MTM1 in myotubular myopathy (MTM), a severe muscle disease characterized by impaired muscle structure and abnormal excitation-contraction coupling. FIG4 functions as a PIP phosphatase that participates in both the synthesis and breakdown of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Mutation of FIG4 results in a severe neurodegenerative disorder in mice and a progressive peripheral polyneuropathy in humans. The effect of FIG4 mutation on skeletal muscle has yet to be examined. METHODS: Herein we characterize the impact of FIG4 on skeletal muscle development and function using the spontaneously occurring mouse mutant pale tremor (plt), a mouse line with a loss of function mutation in Fig4. RESULTS: In plt mice, we characterized abnormalities in skeletal muscle, including reduced muscle size and specific force generation. We also uncovered ultrastructural abnormalities and increased programmed cell death. Conversely, we detected no structural or functional abnormalities to suggest impairment of excitation-contraction coupling, a process previously shown to be influenced by PI(3,5)P2 levels. Conditional rescue of Fig4 mutation in neurons prevented overt muscle weakness and the development of obvious muscle abnormalities, suggesting that the changes observed in the plt mice were primarily related to denervation of skeletal muscle. On the basis of the ability of reduced FIG4 levels to rescue aspects of Mtmr2-dependent neuropathy, we evaluated the effect of Fig4 haploinsufficiency on the myopathy of Mtm1-knockout mice. Male mice with a compound Fig4+/-/Mtm1-/Y genotype displayed no improvements in muscle histology, muscle size or overall survival, indicating that FIG4 reduction does not ameliorate the Mtm1-knockout phenotype. CONCLUSIONS: Overall, these data indicate that loss of Fig4 impairs skeletal muscle function but does not significantly affect its structural development.

Application of Cre-loxP recombination for lineage tracing of adult zebrafish retinal stem cells.

The Cre-loxP recombination system is widely used as a genetic tool to achieve conditional gene expression and for lineage tracing. Though extensively used in mice, this technology has only recently been applied to zebrafish. Here we describe Cre-loxP methodology for conditional expression of transgenes in zebrafish and their use in lineage tracing Müller glia as they undergo cellular reprogramming and proliferation to repair damaged retinal circuitry following mechanical injury. This methodology can be used for conditional gene expression and lineage tracing at any stage of development and in any cell type.

Conditional gene expression and lineage tracing of tuba1a expressing cells during zebrafish development and retina regeneration.

The tuba1a gene encodes a neural-specific α-tubulin isoform whose expression is restricted to the developing and regenerating nervous system. By using zebrafish as a model system for studying CNS regeneration, we recently showed that retinal injury induces tuba1a gene expression in Müller glia that reentered the cell cycle. However, because of the transient nature of tuba1a gene expression during development and regeneration, it was not possible to trace the lineage of the tuba1a-expressing cells with a reporter directly under the control of the tuba1a promoter. To overcome this limitation, we generated tuba1a:CreER(T2) and ß-actin2:loxP-mCherrry-loxP-GFP double transgenic fish that allowed us to label tuba1a-expressing cells conditionally and permanently via ligand-induced recombination. During development, recombination revealed transient tuba1a expression in not only neural progenitors but also cells that contribute to skeletal muscle, heart, and intestine. In the adult, recombination revealed tuba1a expression in brain, olfactory neurons, and sensory cells of the lateral line, but not in the retina. After retinal injury, recombination showed tuba1a expression in Müller glia that had reentered the cell cycle, and lineage tracing indicated that these cells are responsible for regenerating retinal neurons and glia. These results suggest that tuba1a-expressing progenitors contribute to multiple cell lineages during development and that tuba1a-expressing Müller glia are retinal progenitors in the adult.

Building biological memory by linking positive feedback loops.

A common topology found in many bistable genetic systems is two interacting positive feedback loops. Here we explore how this relatively simple topology can allow bistability over a large range of cellular conditions. On the basis of theoretical arguments, we predict that nonlinear interactions between two positive feedback loops can produce an ultrasensitive response that increases the range of cellular conditions at which bistability is observed. This prediction was experimentally tested by constructing a synthetic genetic circuit in Escherichia coli containing two well-characterized positive feedback loops, linked in a coherent fashion. The concerted action of both positive feedback loops resulted in bistable behavior over a broad range of inducer concentrations; when either of the feedback loops was removed, the range of inducer concentrations at which the system exhibited bistability was decreased by an order of magnitude. Furthermore, bistability of the system could be tuned by altering growth conditions that regulate the contribution of one of the feedback loops. Our theoretical and experimental work shows how linked positive feedback loops may produce the robust bistable responses required in cellular networks that regulate development, the cell cycle, and many other cellular responses.