Nyctalopin is required for localization of the TRPM1 channel to the retinal depolarizing bipolar cell dendrites.

Mutations in the NYX gene, encoding the novel protein nyctalopin, cause human congenital stationary night blindness type 1 (CSNB1). CSNB1 is an X-linked recessive condition and is identified by a loss of the electroretinogram (ERG) b-wave while the a-wave remains intact (Miyake et al., 1986). The b-wave in normal patients is generated by the depolarization of one class of bipolar cells, which receive signals from the photoreceptors. This indicates that CSNB1 is a loss of signaling from the photo receptors to the depolarizing bipolar cells. Normal stimulation of photoreceptors by light causes the photoreceptors to hyperpolarize and reduce the amount of neurotransmitter, glutamate, released into the synaptic cleft. The decrease in glutamate causes Grm6, a metabotropic glutamate receptor, at the postsynaptic depolarizing bipolar cell dendrites to reduce its activity. This signal is relayed by a second messenger system to result in the opening of a non-selective cation channel, which depolarizes one class of bipolar cells. The signaling to the non-selective cation channel in depolarizing bipolar cells is disrupted when nyctalopin is absent. Recent data shows that the non-selective cation channel is the transient receptor potential subfamily M member 1 (TRPM1) channel (Shen et al., 2009; Morgans et al., 2009; Koike et al., 2009). The focus of my studies has been to understand the function of nyctalopin in the depolarizing bipolar cells. I characterized TRPM1 expression in the retina, investigated the interdependence between nyctalopin, Grm6 and TRPM1 and determined the interactions between nyctalopin and Grm6 or TRPM1. There are five retinal splice variants of TRPM1. Variants containing all six transmembrane domains have a developmental expression profile similar to bipolar cells. Immunohistochemical analysis shows TRPM1 is localized to BC somas and dendritic tips where it co-localizes with YFP-nyctalopin. Nyctalopin interacts with the TRPMl channel is required for proper localization of the TRPMl channel to the bipolar cell dendritic tips

Disrupting the ciliary gradient of active Arl3 affects rod photoreceptor nuclear migration

The small GTPase Arl3 is important for the enrichment of lipidated proteins to primary cilia, including the outer segment of photoreceptors. Human mutations in the small GTPase Arl3 cause both autosomal recessive and dominant inherited retinal dystrophies. We discovered that dominant mutations result in increased active G-protein-Arl3-D67V has constitutive activity and Arl3-Y90C is fast cycling-and their expression in mouse rods resulted in a displaced nuclear phenotype due to an aberrant Arl3-GTP gradient. Using multiple strategies, we go on to show that removing or restoring the Arl3-GTP gradient within the cilium is sufficient to rescue the nuclear migration defect. Together, our results reveal that an Arl3 ciliary gradient is involved in proper positioning of photoreceptor nuclei during retinal development

Loss of Arf4 causes severe degeneration of the exocrine pancreas but not cystic kidney disease or retinal degeneration

Arf4 is proposed to be a critical regulator of membrane protein trafficking in early secretory pathway. More recently, Arf4 was also implicated in regulating ciliary trafficking, however, this has not been comprehensively tested in vivo. To directly address Arf4\u27s role in ciliary transport, we deleted Arf4 specifically in either rod photoreceptor cells, kidney, or globally during the early postnatal period. Arf4 deletion in photoreceptors did not cause protein mislocalization or retinal degeneration, as expected if Arf4 played a role in protein transport to the ciliary outer segment. Likewise, Arf4 deletion in kidney did not cause cystic disease, as expected if Arf4 were involved in general ciliary trafficking. In contrast, global Arf4 deletion in the early postnatal period resulted in growth restriction, severe pancreatic degeneration and early death. These findings are consistent with Arf4 playing a critical role in endomembrane trafficking, particularly in the pancreas, but not in ciliary function

661W photoreceptor cell line as a cell model for studying retinal ciliopathies

Copyright © 2019 Wheway, Nazlamova, Turner and Cross. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. The retina contains several ciliated cell types, including the retinal pigment epithelium (RPE) and photoreceptor cells. The photoreceptor cilium is one of the most highly modified sensory cilia in the human body. The outer segment of the photoreceptor is a highly elaborate primary cilium, containing stacks or folds of membrane where the photopigment molecules are located. Perhaps unsurprisingly, defects in cilia often lead to retinal phenotypes, either as part of syndromic conditions involving other organs, or in isolation in the so-called retinal ciliopathies. The study of retinal ciliopathies has been limited by a lack of retinal cell lines. RPE1 retinal pigment epithelial cell line is commonly used in such studies, but the existence of a photoreceptor cell line has largely been neglected in the retinal ciliopathy field. 661W cone photoreceptor cells, derived from mouse, have been widely used as a model for studying macular degeneration, but not described as a model for studying retinal ciliopathies such as retinitis pigmentosa. Here, we characterize the 661W cell line as a model for studying retinal ciliopathies. We fully characterize the expression profile of these cells, using whole transcriptome RNA sequencing, and provide this data on Gene Expression Omnibus for the advantage of the scientific community. We show that these cells express the majority of markers of cone cell origin. Using immunostaining and confocal microscopy, alongside scanning electron microscopy, we show that these cells grow long primary cilia, reminiscent of photoreceptor outer segments, and localize many cilium proteins to the axoneme, membrane and transition zone. We show that siRNA knockdown of cilia genes Ift88 results in loss of cilia, and that this can be assayed by high-throughput screening. We present evidence that the 661W cell line is a useful cell model for studying retinal ciliopathies

Topological Analysis of Small Leucine-Rich Repeat Proteoglycan Nyctalopin

Nyctalopin is a small leucine rich repeat proteoglycan (SLRP) whose function is critical for normal vision. The absence of nyctalopin results in the complete form of congenital stationary night blindness. Normally, glutamate released by photoreceptors binds to the metabotropic glutamate receptor type 6 (GRM6), which through a G-protein cascade closes the non-specific cation channel, TRPM1, on the dendritic tips of depolarizing bipolar cells (DBCs) in the retina. Nyctalopin has been shown to interact with TRPM1 and expression of TRPM1 on the dendritic tips of the DBCs is dependent on nyctalopin expression. In the current study, we used yeast two hybrid and biochemical approaches to investigate whether murine nyctalopin was membrane bound, and if so by what mechanism, and also whether the functional form was as a homodimer. Our results show that murine nyctalopin is anchored to the plasma membrane by a single transmembrane domain, such that the LRR domain is located in the extracellular space

Evidence for a retroviral insertion in TRPM1 as the cause of congenital stationary night blindness and leopard complex spotting in the horse

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ²=1022.00, p<<0.0005), and CSNB, testing 43 horses (χ2=43, p<<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder.Rebecca R. Bellone … David L. Adelson, Sim Lin Lim … et al

A study of basic mineralogical, physical-chemical, and engineering index properties of laterite soils

Increasing construction activity in tropical environments has greatly expanded the requirement for engineering knowledge related to the basic properties of tropical soils. One of the most frequently encountered yet least understood variety of tropical soils are those residual surface deposits commonly identifies as laterite soils. Because of a general lack of factual data regarding index properties of these soils, generalizations attempting to characterize the engineering behavior of laterite soils have frequently been devoid of factual constraints. A detailed study has been made of selected soils, reported to be laterite soils, to establish data related to the basic chemical, mineralogical and engineering index properties of such material. All of the soils were developed in a tropical environment between the 11th and 18th North parallels of latitude. The findings have been studied to establish a correlation between the chemical, mineralogical and engineering index properties in order to point the way to more intelligent understanding and engineering usage of these soils. The findings of the laboratory study show that, from the civil engineering point of view, no distinguishing chemical, mineralogical or engineering property could be found which is sufficiently definitive to set these soils apart, as a group, from other soils. Based upon the findings, it is concluded that the persistent usage of the term "laterite" by civil engineers as descriptive of a broad group of tropical soils with similar properties, is at best misleading. The results of the correlation study show a strong relation to exist between the cation exchange activity and the activity ratio for the soils tested. This relation may be approximated by the expression CEAc=0.784(Ac)¹². A strong relation was also found to exist between cation exchange activity, activity ratio and the type of clay mineral composition for each soil tested. The relation may be clearly depicted by the use of a correlation graph which was developed for this purpose as a part of this study

A study of basic mineralogical, physical-chemical, and engineering index properties of laterite soils

Increasing construction activity in tropical environments has greatly expanded the requirement for engineering knowledge related to the basic properties of tropical soils. One of the most frequently encountered yet least understood variety of tropical soils are those residual surface deposits commonly identifies as laterite soils. Because of a general lack of factual data regarding index properties of these soils, generalizations attempting to characterize the engineering behavior of laterite soils have frequently been devoid of factual constraints. A detailed study has been made of selected soils, reported to be laterite soils, to establish data related to the basic chemical, mineralogical and engineering index properties of such material. All of the soils were developed in a tropical environment between the 11th and 18th North parallels of latitude. The findings have been studied to establish a correlation between the chemical, mineralogical and engineering index properties in order to point the way to more intelligent understanding and engineering usage of these soils. The findings of the laboratory study show that, from the civil engineering point of view, no distinguishing chemical, mineralogical or engineering property could be found which is sufficiently definitive to set these soils apart, as a group, from other soils. Based upon the findings, it is concluded that the persistent usage of the term "laterite" by civil engineers as descriptive of a broad group of tropical soils with similar properties, is at best misleading. The results of the correlation study show a strong relation to exist between the cation exchange activity and the activity ratio for the soils tested. This relation may be approximated by the expression CEAc=0.784(Ac)¹². A strong relation was also found to exist between cation exchange activity, activity ratio and the type of clay mineral composition for each soil tested. The relation may be clearly depicted by the use of a correlation graph which was developed for this purpose as a part of this study