An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and its activity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.egregation of visual signals into ON and OFF pathways originates in BCs, the second-order neurons in the retina (1, 2). ON and OFF BCs express metabotropic glutamate receptors, mGluR6, and ionotropic glutamate receptors (iGluRs), respectively, on their dendrites (3-5). Reduction of glutamate released from photoreceptors by light stimulation depolarizes ON BCs and hyperpolarizes OFF BCs (6-8) mediated through respective glutamate receptors. The mGluR6 couples to a heterotrimeric G protein complex, Go (9, 10). Signals require Goα, which ultimately closes a downstream nonselective cation channel in ON BCs (6, 9, 11-13). However, this transduction cation channel in ON BCs has not been identified, despite intensive investigation.In our screen to identify functionally important molecules in the retina, we found that TRPM1 is predominantly expressed in retinal BCs. Most members of the TRP superfamily, which are found in a variety of sense organs, are non-voltage-gated cation channels (14-16). The founding member of the TRP family was discovered as a key component of the light response in Drosophila photoreceptors (17). TRPM1, also known as melastatin, was the first member of the melanoma-related transient receptor potential (TRPM) subfamily to be discovered (18,19). TRPM1 is alternatively spliced, resulting in the production of a long form (TRPM1-L) and a short N-terminal form devoid of transmembrane segments (TRPM1-S) (18,20). Although mouse TRPM1-S was previously identified as melastatin, mouse TRPM1-L has not been identified (18). The distinct physiological and biological functions of TRPM1 still remain elusive, although some recent evidences including us suggested that TRPM1 might contribute to retinal BC function (21-23). Here, we show that TRPM1-L is the transduction cation channel of retinal ON BCs in the downstream of mGluR6 cascade.

MicroRNA-124a (miR-124a) is the most abundant microRNA expressed in the vertebrate CNS. Despite past investigations into the role of miR-124a, inconsistent results have left the in vivo function of miR-124a unclear. We examined the in vivo function of miR-124a by targeted disruption of Rncr3 (retinal non-coding RNA 3), the dominant source of miR-124a. Rncr3(-/-) mice exhibited abnormalities in the CNS, including small brain size, axonal mis-sprouting of dentate gyrus granule cells and retinal cone cell death. We found that Lhx2 is an in vivo target mRNA of miR-124a. We also observed that LHX2 downregulation by miR-124a is required for the prevention of apoptosis in the developing retina and proper axonal development of hippocampal neurons. These results suggest that miR-124a is essential for the maturation and survival of dentate gyrus neurons and retinal cones, as it represses Lhx2 translation.

We previously reported that Otx2 is essential for photoreceptor cell fate determination; however, the functional role of Otx2 in postnatal retinal development is still unclear although it has been reported to be expressed in retinal bipolar cells and photoreceptors at postnatal stages. In this study, we first examined the roles of Otx2 in the terminal differentiation of photoreceptors by analyzing Otx2; Crx double-knockout mice. In Otx2 ؉/؊ ; Crx ؊/؊ retinas, photoreceptor degeneration and downregulation of photoreceptor-specific genes were much more prominent than in Crx ؊/؊ retinas, suggesting that Otx2 has a role in the terminal differentiation of the photoreceptors. Moreover, bipolar cells decreased in the Otx2 ؉/؊ ; Crx ؊/؊ retina, suggesting that Otx2 is also involved in retinal bipolar-cell development. To further investigate the role of Otx2 in bipolar-cell development, we generated a postnatal bipolar-cellspecific Otx2 conditional-knockout mouse line. Immunohistochemical analysis of this line showed that the expression of protein kinase C, a marker of mature bipolar cells, was significantly downregulated in the retina. Electroretinograms revealed that the electrophysiological function of retinal bipolar cells was impaired as a result of Otx2 ablation. These data suggest that Otx2 plays a functional role in the maturation of retinal photoreceptor and bipolar cells.The vertebrate neural retina is comprised of six types of neurons and one type of glial cell, all derived from one population of multipotent progenitors (38,39,41). Transcription factors such as homeobox and basic helix-loop-helix factors have been known to play pivotal roles in the specification and development of retinal cell subtypes. Among the Otx-like homeobox genes, Otx2 and Crx play critical roles in retinal photoreceptor development. The expression of Otx2 covers most of the forebrain and midbrain neuroepithelium, including the eye domain, during development (32). Complete elimination of Otx2 functions in mice by gene targeting results in the absence of the forebrain and embryonic lethality (1,3,27). In a previous study, we have shown that Otx2 is essential and sufficient for the cell fate determination of retinal photoreceptors (29). Crx, on the other hand, is reported to be expressed abundantly in retinal photoreceptors and pinealocytes and also weakly in retinal bipolar cells (12,19). It has also been reported that Crx regulates various photoreceptor-specific genes (12,19,20). Mutations of human CRX are associated with three types of photoreceptor diseases: cone-rod dystrophy 2, retinitis pigmentosa, and Leber's congenital amaurosis (15,16,33,36). A gene-targeting study has revealed that Crx is essential for the terminal differentiation of photoreceptors and normal circadian entrainment (20). Thus, Otx2 and Crx have distinct roles in retinal photoreceptor development although their expression patterns in the retina overlap to some extent. However, they are structurally related transcription factors and can bind to a common DNA-bindin...

The molecular mechanisms underlying cell fate determination from common progenitors in the vertebrate CNS remain elusive. We previously reported that the OTX2 homeoprotein regulates retinal photoreceptor cell fate determination. While Otx2 transactivation is a pivotal process for photoreceptor cell fate determination, its transactivation mechanism in the retina is unknown. Here, we identified an evolutionarily conserved Otx2 enhancer of ϳ500 bp, named embryonic enhancer locus for photoreceptor Otx2 transcription (EELPOT), which can recapitulate initial Otx2 expression in the embryonic mouse retina. We found that the RAX homeoprotein interacts with EELPOT to transactivate Otx2, mainly in the final cell cycle of retinal progenitors. Conditional inactivation of Rax results in downregulation of Otx2 expression in vivo. We also showed that NOTCH-HES signaling negatively regulates EELPOT to suppress Otx2 expression. These results suggest that the integrated activity of cell-intrinsic and -extrinsic factors on EELPOT underlies the molecular basis of photoreceptor cell fate determination in the embryonic retina.

Amacrine interneurons, which are highly diversified in morphological, neurochemical, and physiological features, play crucial roles in visual information processing in the retina. However, the specification mechanisms and functions in vision for each amacrine subtype are not well understood. We found that the Prdm13 transcriptional regulator is specifically expressed in developing and mature amacrine cells in the mouse retina. Most Prdm13-positive amacrine cells are Calbindin-and Calretinin-positive GABAergic or glycinergic neurons. Absence of Prdm13 significantly reduces GABAergic and glycinergic amacrines, resulting in a specific defect of the S2/S3 border neurite bundle in the inner plexiform layer. Forced expression of Prdm13 distinctively induces GABAergic and glycinergic amacrine cells but not cholinergic amacrine cells, whereas Ptf1a, an upstream transcriptional regulator of Prdm13, induces all of these subtypes. Moreover, Prdm13-deficient mice showed abnormally elevated spatial, temporal, and contrast sensitivities in vision. Together, these results show that Prdm13 regulates development of a subset of amacrine cells, which newly defines an amacrine subtype to negatively modulate visual sensitivities. Our current study provides new insights into mechanisms of the diversification of amacrine cells and their function in vision.

T cells reorganize their metabolic profiles after being activated, but the systemic metabolic effect of sustained activation of the immune system has remained unexplored. Here we report that augmented T cell responses in Pdcd1 mice, which lack the inhibitory receptor PD-1, induced a metabolic serum signature characterized by depletion of amino acids. We found that the depletion of amino acids in serum was due to the accumulation of amino acids in activated Pdcd1 T cells in the lymph nodes. A systemic decrease in tryptophan and tyrosine led to substantial deficiency in the neurotransmitters serotonin and dopamine in the brain, which resulted in behavioral changes dominated by anxiety-like behavior and exacerbated fear responses. Together these data indicate that excessive activation of T cells causes a systemic metabolomic shift with consequences that extend beyond the immune system.

BackgroundParaneoplastic retinopathy (PR), including cancer-associated retinopathy (CAR) and melanoma-associated retinopathy (MAR), is a progressive retinal disease caused by antibodies generated against neoplasms not associated with the eye. While several autoantibodies against retinal antigens have been identified, there has been no known autoantibody reacting specifically against bipolar cell antigens in the sera of patients with PR. We previously reported that the transient receptor potential cation channel, subfamily M, member 1 (TRPM1) is specifically expressed in retinal ON bipolar cells and functions as a component of ON bipolar cell transduction channels. In addition, this and other groups have reported that human TRPM1 mutations are associated with the complete form of congenital stationary night blindness. The purpose of the current study is to investigate whether there are autoantibodies against TRPM1 in the sera of PR patients exhibiting ON bipolar cell dysfunction.Methodology/Principal FindingsWe performed Western blot analysis to identify an autoantibody against TRPM1 in the serum of a patient with lung CAR. The electroretinograms of this patient showed a severely reduced ON response with normal OFF response, indicating that the defect is in the signal transmission between photoreceptors and ON bipolar cells. We also investigated the sera of 26 patients with MAR for autoantibodies against TRPM1 because MAR patients are known to exhibit retinal ON bipolar cell dysfunction. Two of the patients were found to have autoantibodies against TRPM1 in their sera.Conclusion/SignificanceOur study reveals TRPM1 to be one of the autoantigens targeted by autoantibodies in at least some patients with CAR or MAR associated with retinal ON bipolar cell dysfunction.

A number of homeodomain transcription factors, which play significant roles in retinal development, have been identified in vertebrates (1-4). Rax is a homeodomain transcription factor that is essential for various processes in vertebrate retinal development (5). The Rax gene was first identified as a paired-type homeobox gene expressed in the optic vesicle and the presumptive diencephalon area in the early mouse embryo (6, 7). Rax is evolutionarily well conserved from Drosophila melanogaster to humans. Rax is highly expressed in retinal progenitor cells (RPCs), and its expression in the retina gradually decreases as RPCs become postmitotic and begin to differentiate. Rax-null mutant mice exhibit a reduction of brain size and an absence of the optic vesicle (5, 7). Mutations in human RAX are associated with anophthalmia and microphthalmia (8, 9). Rax overexpression promotes the proliferation of RPCs in frogs and zebra fish (7,(10)(11)(12)(13). In addition to the function in RPCs, Rax plays significant roles in the development of photoreceptor cells and Müller glial cells (14)(15)(16)(17)(18)(19).Rax paralog genes have been identified in various vertebrate species (20)(21)(22). In Xenopus laevis, two Rax genes (xRx and xRx-L/xRx2) have been identified (7, 21), and in zebra fish, three Rax genes (zRx1 to zRx3) have been isolated (7). Interestingly, the expression pattern of zebra fish Rx3 showed more similarity to that of frog and mouse Rax genes than to that of the zebra fish Rx1 and Rx2 genes (23). In chicks, two Rax genes (cRax and cRaxL/ cRax2) have been identified (20). The chick Rax2 gene is expressed in both retinal progenitor cells and early-developing photoreceptors, while chick Rax is predominantly expressed in retinal progenitor cells. It was also reported that chick Rax2 is implicated in cone photoreceptor differentiation and that the expression of a putative dominant negative allele of a chick Rax2 gene caused a significant reduction in the level of expression of cone photoreceptor genes (20). Human RAX2/QRX, which is expressed in the outer nuclear layer (ONL) and inner nuclear layer (INL) of the adult human retina, was identified to be a PCE-1-binding protein by acting synergistically with CRX and NRL to modulate the expression of photoreceptor genes. Monkey, cow, and dog genomes also contain two Rax genes. On the other hand, the Rax2 gene is absent from mouse and rat genomes (22). This raises the question of whether mouse Rax plays an essential role in photoreceptor development during postnatal stages like human Rax2 does.In the current study, we investigated a functional role for Rax in postnatal mouse retinas, which contain a single Rax gene. We report that mouse Rax modulates the expression of photoreceptor genes in the postnatal retina by interacting with Crx. Conditional ablation of Rax in postnatal photoreceptors led to a significant decrease in the level of expression of rod and cone genes and to cone photoreceptor cell death, suggesting that Rax is essential for the maturation of rods and co...