Intermolecular Interaction between Anchoring Subunits Specify Subcellular Targeting and Function of RGS Proteins in Retina ON-Bipolar Neurons.

In vertebrate retina, light responses generated by the rod photoreceptors are transmitted to the second-order neurons, the ON-bipolar cells (ON-BC), and this communication is indispensible for vision in dim light. In ON-BCs, synaptic transmission is initiated by the metabotropic glutamate receptor, mGluR6, that signals via the G-protein Go to control opening of the effector ion channel, TRPM1. A key role in this process belongs to the GTPase Activating Protein (GAP) complex that catalyzes Go inactivation upon light-induced suppression of glutamate release in rod photoreceptors, thereby driving ON-BC depolarization to changes in synaptic input. The GAP complex has a striking molecular complexity. It contains two Regulator of G-protein Signaling (RGS) proteins RGS7 and RGS11 that directly act on Go and two adaptor subunits: RGS Anchor Protein (R9AP) and the orphan receptor, GPR179. Here we examined the organizational principles of the GAP complex in ON-BCs. Biochemical experiments revealed that RGS7 binds to a conserved site in GPR179 and that RGS11 in vivo forms a complex only with R9AP. R9AP and GPR179 are further integrated via direct protein-protein interactions involving their cytoplasmic domains. Elimination of GPR179 prevents postsynaptic accumulation of R9AP. Furthermore, concurrent knock-out of both R9AP and RGS7 does not reconfigure the GAP complex and completely abolishes synaptic transmission, resulting in a novel mouse model of night blindness. Based on these results, we propose a model of hierarchical assembly and function of the GAP complex that supports ON-BCs visual signaling.

A missense mutation in Grm6 reduces but does not eliminate mGluR6 expression or rod depolarizing bipolar cell function.

GRM6 encodes the metabotropic glutamate receptor 6 (mGluR6) used by retinal depolarizing bipolar cells (DBCs). Mutations in GRM6 lead to DBC dysfunction and underlie the human condition autosomal recessive complete congenital stationary night blindness. Mouse mutants for Grm6 are important models for this condition. Here we report a new Grm6 mutant, identified in an electroretinogram (ERG) screen of mice maintained at The Jackson Laboratory. The Grm6nob8 mouse has a reduced-amplitude b-wave component of the ERG, which reflects light-evoked DBC activity. Sequencing identified a missense mutation that converts a highly conserved methionine within the ligand binding domain to leucine (p.Met66Leu). Consistent with prior studies of Grm6 mutant mice, the laminar size and structure in the Grm6nob8 retina were comparable to control. The Grm6nob8 phenotype is distinguished from other Grm6 mutants that carry a null allele by a reduced but not absent ERG b-wave, decreased but present expression of mGluR6 at DBC dendritic tips, and mislocalization of mGluR6 to DBC somas. Consistent with a reduced but not absent b-wave, there were a subset of retinal ganglion cells whose responses to light onset have times to peak within the range of those in control retinas. These data indicate that the p.Met66Leu mutant mGluR6 is trafficked less than control. However, the mGluR6 that is localized to the DBC dendritic tips is able to initiate DBC signal transduction. The Grm6nob8 mouse extends the Grm6 allelic series and will be useful for elucidating the role of mGluR6 in DBC signal transduction and in human disease.NEW & NOTEWORTHY This article describes a mouse model of the human disease complete congenital stationary night blindness in which the mutation reduces but does not eliminate GRM6 expression and bipolar cell function, a distinct phenotype from that seen in other Grm6 mouse models.

Intravitreal delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of complete congenital stationary night blindness.

Adeno-associated virus (AAV) effectively targets therapeutic genes to photoreceptors, pigment epithelia, Müller glia and ganglion cells of the retina. To date, no one has shown the ability to correct, with gene replacement, an inherent defect in bipolar cells (BCs), the excitatory interneurons of the retina. Targeting BCs with gene replacement has been difficult primarily due to the relative inaccessibility of BCs to standard AAV vectors. This approach would be useful for restoration of vision in patients with complete congenital stationary night blindness (CSNB1), where signaling through the ON BCs is eliminated due to mutations in their G-protein-coupled cascade genes. For example, the majority of CSNB1 patients carry a mutation in nyctalopin (NYX), which encodes a protein essential for proper localization of the TRPM1 cation channel required for ON BC light-evoked depolarization. As a group, CSNB1 patients have a normal electroretinogram (ERG) a-wave, indicative of photoreceptor function, but lack a b-wave due to defects in ON BC signaling. Despite retinal dysfunction, the retinas of CSNB1 patients do not degenerate. The Nyx(nob) mouse model of CSNB1 faithfully mimics this phenotype. Here, we show that intravitreally injected, rationally designed AAV2(quadY-F+T-V) containing a novel 'Ple155' promoter drives either GFP or YFP_Nyx in postnatal Nyx(nob) mice. In treated Nyx(nob) retina, robust and targeted Nyx transgene expression in ON BCs partially restored the ERG b-wave and, at the cellular level, signaling in ON BCs. Our results support the potential for gene delivery to BCs and gene replacement therapy in human CSNB1.

Mutations in RAB39B cause X-linked intellectual disability and early-onset Parkinson disease with α-synuclein pathology.

Advances in understanding the etiology of Parkinson disease have been driven by the identification of causative mutations in families. Genetic analysis of an Australian family with three males displaying clinical features of early-onset parkinsonism and intellectual disability identified a ∼45 kb deletion resulting in the complete loss of RAB39B. We subsequently identified a missense mutation (c.503C>A [p.Thr168Lys]) in RAB39B in an unrelated Wisconsin kindred affected by a similar clinical phenotype. In silico and in vitro studies demonstrated that the mutation destabilized the protein, consistent with loss of function. In vitro small-hairpin-RNA-mediated knockdown of Rab39b resulted in a reduction in the density of α-synuclein immunoreactive puncta in dendritic processes of cultured neurons. In addition, in multiple cell models, we demonstrated that knockdown of Rab39b was associated with reduced steady-state levels of α-synuclein. Post mortem studies demonstrated that loss of RAB39B resulted in pathologically confirmed Parkinson disease. There was extensive dopaminergic neuron loss in the substantia nigra and widespread classic Lewy body pathology. Additional pathological features included cortical Lewy bodies, brain iron accumulation, tau immunoreactivity, and axonal spheroids. Overall, we have shown that loss-of-function mutations in RAB39B cause intellectual disability and pathologically confirmed early-onset Parkinson disease. The loss of RAB39B results in dysregulation of α-synuclein homeostasis and a spectrum of neuropathological features that implicate RAB39B in the pathogenesis of Parkinson disease and potentially other neurodegenerative disorders.

A novel transgenic mouse model of lysosomal storage disorder.

Knockout technology has proven useful for delineating functional roles of specific genes. Here we describe and provide an explanation for striking pathology that occurs in a subset of genetically engineered mice expressing a rat CaVß2a transgene under control of the cardiac α-myosin heavy chain promoter. Lesions were limited to mice homozygous for transgene and independent of native Cacnb2 genomic copy number. Gross findings included an atrophied pancreas; decreased adipose tissue; thickened, orange intestines; and enlarged liver, spleen, and abdominal lymph nodes. Immune cell infiltration and cell engulfment by macrophages were associated with loss of pancreatic acinar cells. Foamy macrophages diffusely infiltrated the small intestine's lamina propria, while similar macrophage aggregates packed liver and splenic red pulp sinusoids. Periodic acid-Schiff-positive, diastase-resistant, iron-negative, Oil Red O-positive, and autofluorescent cytoplasm was indicative of a lipid storage disorder. Electron microscopic analysis revealed liver sinusoids distended by clusters of macrophages containing intracellular myelin "swirls" and hepatocytes with enlarged lysosomes. Additionally, build up of cholesterol, cholesterol esters, and triglycerides, along with changes in liver metabolic enzyme levels, were consistent with a lipid processing defect. Because of this complex pathology, we examined the transgene insertion site. Multiple transgene copies inserted into chromosome 19; at this same site, an approximate 180,000 base pair deletion occurred, ablating cholesterol 25-hydroxylase and partially deleting lysosomal acid lipase and CD95 Loss of gene function can account for the altered lipid processing, along with hypertrophy of the immune system, which define this phenotype, and serendipitously provides a novel mouse model of lysosomal storage disorder.

CACNA1S expression in mouse retina: Novel isoforms and antibody cross-reactivity with GPR179.

Cacna1s encodes the α1S subunit (Cav1.1) of voltage-dependent calcium channels, and is required for normal skeletal and cardiac muscle function, where it couples with the ryanodine receptor to regulate muscle contraction. Recently CACNA1S was reported to be expressed on the tips of retinal depolarizing bipolar cells (DBCs) and colocalized with metabotropic glutamate receptor 6 (mGluR6), which is critical to DBC signal transduction. Further, in mGluR6 knockout mice, expression at this location is down regulated. We examined RNAseq data from mouse retina and found expression of a novel isoform of Cacna1s. To determine if CACNA1S was a functional component of the DBC signal transduction cascade, we performed immunohistochemistry to visualize its expression in several mouse lines that lack DBC function. Immunohistochemical staining with antibodies to CACNA1S show punctate labeling at the tips of DBCs in wild type (WT) retinas that are absent in Gpr179 nob5 mutant retinas and decreased in Grm6 -/- mouse retinas. CACNA1S and transient receptor potential cation channel, subfamily M, member 1 (TRPM1) staining also colocalized in WT retinas. Western blot analyses for CACNA1S of either retinal lysates or proteins after immunoprecipitation with the CACNA1S antibody failed to show the presence of bands expected for CACNA1S. Mass spectrometric analysis of CACNA1S immunoprecipitated proteins also failed to detect any peptides matching CACNA1S. Immunohistochemistry and western blotting after expression of GPR179 in HEK293T cells indicate that the CACNA1S antibody used here and in the retinal studies published to date, cross-reacts with GPR179. These data suggest caution should be exercised in conferring a role for CACNA1S in DBC signal transduction based solely on immunohistochemical staining.

GPR179 is required for high sensitivity of the mGluR6 signaling cascade in depolarizing bipolar cells.

Parallel visual pathways are initiated at the first retinal synapse by signaling between the rod and cone photoreceptors and two general classes of bipolar cells. For normal function, ON or depolarizing bipolar cells (DBCs) require the G-protein-coupled receptor, mGluR6, an intact G-protein-coupled cascade and the transient receptor potential melastatin 1 (TRPM1) cation channel. In addition, another seven transmembrane protein, GPR179, is required for DBC function and recruits the regulators of G-protein signaling (RGS) proteins, RGS7 and RGS11, to the dendritic tips of the DBCs. Here we use the Gpr179(nob5) mouse, which lacks GPR179 and has a no b-wave electroretinogram (ERG) phenotype, to demonstrate that despite the absence of both GPR179 and RGS7/RGS11, a small dark-adapted ERG b-wave remains and can be enhanced with long duration flashes. Consistent with the ERG, the mGluR6-mediated gating of TRPM1 can be evoked pharmacologically in Gpr179(nob5) and RGS7(-/-)/RGS11(-/-) rod BCs if strong stimulation conditions are used. In contrast, direct gating of TRPM1 by capsaicin in RGS7(-/-)/RGS11(-/-) and WT rod BCs is similar, but severely compromised in Gpr179(nob5) rod BCs. Noise and standing current analyses indicate that the remaining channels in Gpr179(nob5) and RGS7(-/-)/RGS11(-/-) rod BCs have a very low open probability. We propose that GPR179 along with RGS7 and RGS11 controls the ability of the mGluR6 cascade to gate TRPM1. In addition to its role in localizing RGS7 and RGS11 to the dendritic tips, GPR179 via a direct interaction with the TRPM1 channel alters its ability to be gated directly by capsaicin.

GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness.

Complete congenital stationary night blindness (cCSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impairment of night vision, absence of the electroretinogram (ERG) b-wave, and variable degrees of involvement of other visual functions. We report here that mutations in GPR179, encoding an orphan G protein receptor, underlie a form of autosomal-recessive cCSNB. The Gpr179(nob5/nob5) mouse model was initially discovered by the absence of the ERG b-wave, a component that reflects depolarizing bipolar cell (DBC) function. We performed genetic mapping, followed by next-generation sequencing of the critical region and detected a large transposon-like DNA insertion in Gpr179. The involvement of GPR179 in DBC function was confirmed in zebrafish and humans. Functional knockdown of gpr179 in zebrafish led to a marked reduction in the amplitude of the ERG b-wave. Candidate gene analysis of GPR179 in DNA extracted from patients with cCSNB identified GPR179-inactivating mutations in two patients. We developed an antibody against mouse GPR179, which robustly labeled DBC dendritic terminals in wild-type mice. This labeling colocalized with the expression of GRM6 and was absent in Gpr179(nob5/nob5) mutant mice. Our results demonstrate that GPR179 plays a critical role in DBC signal transduction and expands our understanding of the mechanisms that mediate normal rod vision.

Identification of a new mutant allele, Grm6(nob7), for complete congenital stationary night blindness.

Electroretinogram (ERG) studies identified a new mouse line with a normal a-wave but lacking the b-wave component. The ERG phenotype of this new allele, nob7, matched closely that of mouse mutants for Grm6, Lrit3, Trpm1, and Nyx, which encode for proteins expressed in depolarizing bipolar cells (DBCs). To identify the underlying mutation, we first crossed nob7 mice with Grm6 nob3 mutants and measured the ERGs in offspring. All the offspring lacked the b-wave, indicating that nob7 is a new allele for Grm6: Grm6 nob7 . Sequence analyses of Grm6 nob7 cDNAs identified a 28 base pair insertion between exons 8 and 9, which would result in a frameshift mutation in the open reading frame that encodes the metabotropic glutamate receptor 6 (Grm6). Sequencing both the cDNA and genomic DNA from exon 8 and intron 8, respectively, from the Grm6 nob7 mouse revealed a G to A transition at the last position in exon 8. This mutation disrupts splicing and the normal exon 8 is extended by 28 base pairs, because splicing occurs 28 base pairs downstream at a cryptic splice donor. Consistent with the impact of the resulting frameshift mutation, there is a loss of mGluR6 protein (encoded by Grm6) from the dendritic tips of DBCs in the Grm6 nob7 retina. These results indicate that Grm6 nob7 is a new model of the complete form of congenital stationary night blindness, a human condition that has been linked to mutations of GRM6.

Mutations in zebrafish lrp2 result in adult-onset ocular pathogenesis that models myopia and other risk factors for glaucoma.

The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

rnaset2 mutant zebrafish model familial cystic leukoencephalopathy and reveal a role for RNase T2 in degrading ribosomal RNA.

T2-family acidic endoribonucleases are represented in all genomes. A physiological role for RNase T2 has yet to be defined for metazoa. RNASET2 mutation in humans is linked with a leukoencephalopathy that arises in infancy characterized by cortical cysts and multifocal white matter lesions. We now show localization of RNASET2 within lysosomes. Further, we demonstrate that loss of rnaset2 in mutant zebrafish results in accumulation of undigested rRNA within lysosomes within neurons of the brain. Further, by using high field intensity magnetic resonance microimaging, we reveal white matter lesions in these animals comparable to those observed in RNASET2-deficient infants. This correlates with accumulation of Amyloid precursor protein and astrocytes at sites of neurodegeneration. Thus we conclude that familial cystic leukoencephalopathy is a lysosomal storage disorder in which rRNA is the best candidate for the noxious storage material.

Cone Synaptic function is modulated by the leucine rich repeat (LRR) adhesion molecule LRFN2.

Daylight vision is mediated by cone photoreceptors in vertebrates, which synapse with bipolar cells (BCs) and horizontal (HCs) cells. This cone synapse is functionally and anatomically complex, connecting to 8 types of depolarizing BCs (DBCs) and 5 types of hyperpolarizing BCs (HBCs) in mice. The dendrites of DBCs and HCs cells make invaginating ribbon synapses with the cone axon terminal, while HBCs form flat synapses with the cone pedicles. The molecular architecture that underpins this organization is relatively poorly understood. To identify new proteins involved in synapse formation and function we used an unbiased proteomic approach and identified LRFN2 (leucine-rich repeat and fibronectin III domain-containing 2) as a component of the DBC signaling complex. LRFN2 is selectively expressed at cone terminals and co-localizes with PNA, and other DBC signalplex members. In LRFN2 deficient mice, the synaptic markers: LRIT3, ELFN2, mGluR6, TRPM1 and GPR179 are properly localized. Similarly, LRFN2 expression and localization is not dependent on these synaptic proteins. In the absence of LRFN2 the cone-mediated photopic electroretinogram b-wave amplitude is reduced at the brightest flash intensities. These data demonstrate that LRFN2 absence compromises normal synaptic transmission between cones and cone DBCs.Significance Statement Signaling between cone photoreceptors and the downstream bipolar cells is critical to normal vision. Cones synapse with 13 different types of bipolar cells forming an invaginating ribbon synapses with 8 types, and flat synapses with 5 types, to form one of the most complex synapses in the brain. In this report a new protein, LRFN2 (leucine-rich repeat and fibronectin III domain-containing 2), was identified that is expressed at the cone synapse. Using Lrfn2 knockout mice we show LRFN2 is required for the normal cone signaling.

Presence of the Gpr179(nob5) allele in a C3H-derived transgenic mouse.

PURPOSE: To identify the mutation responsible for an abnormal electroretinogram (ERG) in a transgenic mouse line (tg21) overexpressing erythropoietin (Epo). The tg21 line was generated on a mixed (C3H; C57BL/6) background and lacked the b-wave component of the ERG. This no-b-wave (nob) ERG is seen in other mouse models with depolarizing bipolar cell (DBC) dysfunction and in patients with the complete form of congenital stationary night blindness (cCSNB). We determined the basis for the nob ERG phenotype and screened C3H mice for the mutation to evaluate whether this finding is important for the vision research community. METHODS: ERGs were used to examine retinal function. The retinal structure of the transgenic mice was investigated using histology and immunohistochemistry. Inverse PCR was performed to identify the insertion site of the Epo transgene in the mouse genome. Affected mice were backcrossed to follow the inheritance pattern of the nob ERG phenotype. Quantitative real-time PCR (qRT PCR), Sanger sequencing, and immunohistochemistry were used to identify the mutation causing the defect. Additional C3H sublines were screened for the detected mutation. RESULTS: Retinal histology and blood vessel structure were not disturbed, and no loss of DBCs was observed in the tg21 nob mice. The mutation causing the nob ERG phenotype is inherited independently of the tg21 transgene. The qRT PCR experiments revealed that the nob ERG phenotype reflected a mutation in Gpr179, a gene involved in DBC signal transduction. PCR analysis confirmed the presence of the Gpr179(nob5) insertional mutation in intron 1 of Gpr179. Screening for mutations in other C3H-derived lines revealed that C3H.Pde6b(+) mice carry the Gpr179 (nob5) allele whereas C3H/HeH mice do not. CONCLUSIONS: We identified the presence of the Gpr179(nob5) mutation causing DBC dysfunction in a C3H-derived transgenic mouse line. The nob phenotype is not related to the presence of the transgene. The Gpr179(nob5) allele can be added to the list of background alleles that impact retinal function in commonly used mouse lines. By providing primers to distinguish between Gpr179 mutant and wild-type alleles, this study allows investigators to monitor for the presence of the Gpr179(nob5) mutation in other mouse lines derived from C3H.

LRIT3 expression in cone photoreceptors restores post-synaptic bipolar cell signalplex assembly and partial function in Lrit3 -/- mice.

Complete congenital stationary night blindness (cCSNB) is a heterogeneous disorder characterized by poor dim-light vision, myopia, and nystagmus that is caused by mutations in genes critical for signal transmission between photoreceptors and depolarizing bipolar cells (DBCs). One such gene, LRIT3, is required for assembly of the post-synaptic signaling complex (signalplex) at the dendritic tips of DBCs, although the number of signalplex components impacted is greater in cone DBCs (all components) than in rod bipolar cells (only TRPM1 and Nyctalopin). Here we show that rAAV-mediated expression of LRIT3 in cones results in robust rescue of cone DBC signalplex components and partially restores downstream visual function, as measured by the light-adapted electroretinogram (ERG) b-wave and electrophysiological recordings of bipolar cells (BCs) and RGCs. These data show that LRIT3 successfully restores partial function to cone DBCs most likely in a trans-synaptic manner, potentially paving the way for therapeutic intervention in LRIT3-associated cCSNB.

A role for nyctalopin, a small leucine-rich repeat protein, in localizing the TRP melastatin 1 channel to retinal depolarizing bipolar cell dendrites.

Expression of channels to specific neuronal sites can critically impact their function and regulation. Currently, the molecular mechanisms underlying this targeting and intracellular trafficking of transient receptor potential (TRP) channels remain poorly understood, and identifying proteins involved in these processes will provide insight into underlying mechanisms. Vision is dependent on the normal function of retinal depolarizing bipolar cells (DBCs), which couple a metabotropic glutamate receptor 6 to the TRP melastatin 1 (TRPM1) channel to transmit signals from photoreceptors. We report that the extracellular membrane-attached protein nyctalopin is required for the normal expression of TRPM1 on the dendrites of DBCs in mus musculus. Biochemical and genetic data indicate that nyctalopin and TRPM1 interact directly, suggesting that nyctalopin is acting as an accessory TRP channel subunit critical for proper channel localization to the synapse.

Autoantibodies in melanoma-associated retinopathy target TRPM1 cation channels of retinal ON bipolar cells.

Melanoma-associated retinopathy (MAR) is characterized by night blindness, photopsias, and a selective reduction of the electroretinogram b-wave. In certain cases, the serum contains autoantibodies that react with ON bipolar cells, but the target of these autoantibodies has not been identified. Here we show that the primary target of autoantibodies produced in MAR patients with reduced b-wave is the TRPM1 cation channel, the newly identified transduction channel in ON bipolar cells. Sera from two well characterized MAR patients, but not from a control subject, stained human embryonic kidney cells transfected with the TRPM1 gene, and Western blots probed with these MAR sera showed the expected band size (∼180 kDa). Staining of mouse and primate retina with MAR sera revealed immunoreactivity in all types of ON bipolar cells. Similar to staining for TRPM1, staining with the MAR sera was strong in dendritic tips and somas and was weak or absent in axon terminals. This staining colocalized with GFP in Grm6-GFP transgenic mice, where GFP is expressed in all and only ON bipolar cells, and also colocalized with Gα(o), a marker for all types of ON bipolar cells. The staining in ON bipolar cells was confirmed to be specific to TRPM1 because MAR serum did not stain these cells in a Trpm1(-/-) mouse. Evidence suggests that the recognized epitope is likely intracellular, and the sera can be internalized by retinal cells. We conclude that the vision of at least some patients with MAR is compromised due to autoantibody-mediated inactivation of the TRPM1 channel.

Depolarizing bipolar cell dysfunction due to a Trpm1 point mutation.

Mutations in TRPM1 are found in humans with an autosomal recessive form of complete congenital stationary night blindness (cCSNB). The Trpm1(-/-) mouse has been an important animal model for this condition. Here we report a new mouse mutant, tvrm27, identified in a chemical mutagenesis screen. Genetic mapping of the no b-wave electroretinogram (ERG) phenotype of tvrm27 localized the mutation to a chromosomal region that included Trpm1. Complementation testing with Trpm1(-/-) mice confirmed a mutation in Trpm1. Sequencing identified a nucleotide change in exon 23, converting a highly conserved alanine within the pore domain to threonine (p.A1068T). Consistent with prior studies of Trpm1(-/-) mice, no anatomical changes were noted in the Trpm1(tvrm27/tvrm27) retina. The Trpm1(tvrm27/tvrm27) phenotype is distinguished from that of Trpm1(-/-) by the retention of TRPM1 expression on the dendritic tips of depolarizing bipolar cells (DBCs). While ERG b-wave amplitudes of Trpm1(+/-) heterozygotes are comparable to wild type, those of Trpm1(+/tvrm27) mice are reduced by 32%. A similar reduction in the response of Trpm1(+/tvrm27) DBCs to LY341495 or capsaicin is evident in whole cell recordings. These data indicate that the p.A1068T mutant TRPM1 acts as a dominant negative with respect to TRPM1 channel function. Furthermore, these data indicate that the number of functional TRPM1 channels at the DBC dendritic tips is a key factor in defining DBC response amplitude. The Trpm1(tvrm27/tvrm27) mutant will be useful for elucidating the role of TRPM1 in DBC signal transduction, for determining how Trpm1 mutations impact central visual processing, and for evaluating experimental therapies for cCSNB.

Mutations in TRPM1 are a common cause of complete congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impaired night vision and variable decreased visual acuity. We report here that six out of eight female probands with autosomal-recessive complete CSNB (cCSNB) had mutations in TRPM1, a retinal transient receptor potential (TRP) cation channel gene. These data suggest that TRMP1 mutations are a major cause of autosomal-recessive CSNB in individuals of European ancestry. We localized TRPM1 in human retina to the ON bipolar cell dendrites in the outer plexifom layer. Our results suggest that in humans, TRPM1 is the channel gated by the mGluR6 (GRM6) signaling cascade, which results in the light-evoked response of ON bipolar cells. Finally, we showed that detailed electroretinography is an effective way to discriminate among patients with mutations in either TRPM1 or GRM6, another autosomal-recessive cCSNB disease gene. These results add to the growing importance of the diverse group of TRP channels in human disease and also provide new insights into retinal circuitry.

Loss of Lrp2 in zebrafish disrupts pronephric tubular clearance but not forebrain development.

Low-density lipoprotein receptor-related protein 2 (LRP2) is a multifunctional cell surface receptor conserved from nematodes to humans. In mammals, it acts as regulator of sonic hedgehog and bone morphogenetic protein pathways in patterning of the embryonic forebrain and as a clearance receptor in the adult kidney. Little is known about activities of this LRP in other phyla. Here, we extend the functional elucidation of LRP2 to zebrafish as a model organism of receptor (dys)function. We demonstrate that expression of Lrp2 in embryonic and larval fish recapitulates the patterns seen in mammalian brain and kidney. Furthermore, we studied the consequence of receptor deficiencies in lrp2 and in lrp2b, a homologue unique to fish, using ENU mutagenesis or morpholino knockdown. While receptor-deficient zebrafish suffer from overt renal resorption deficiency, their brain development proceeds normally, suggesting evolutionary conservation of receptor functions in pronephric duct clearance but not in patterning of the teleost forebrain.

Gain control by sparse, ultra-slow glycinergic synapses.

In the retina, ON starburst amacrine cells (SACs) play a crucial role in the direction-selective circuit, but the sources of inhibition that shape their response properties remain unclear. Previous studies demonstrate that ∼95% of their inhibitory synapses are GABAergic, yet we find that the light-evoked inhibitory currents measured in SACs are predominantly glycinergic. Glycinergic inhibition is extremely slow, relying on non-canonical glycine receptors containing α4 subunits, and is driven by both the ON and OFF retinal pathways. These attributes enable glycine inputs to summate and effectively control the output gain of SACs, expanding the range over which they compute direction. Serial electron microscopic reconstructions reveal three specific types of ON and OFF narrow-field amacrine cells as the presumptive sources of glycinergic inhibition. Together, these results establish an unexpected role for specific glycinergic amacrine cells in the retinal computation of stimulus direction by SACs.