Mutation in the intracellular chloride channel CLCC1 associated with autosomal recessive retinitis pigmentosa.

We identified a homozygous missense alteration (c.75C>A, p.D25E) in CLCC1, encoding a presumptive intracellular chloride channel highly expressed in the retina, associated with autosomal recessive retinitis pigmentosa (arRP) in eight consanguineous families of Pakistani descent. The p.D25E alteration decreased CLCC1 channel function accompanied by accumulation of mutant protein in granules within the ER lumen, while siRNA knockdown of CLCC1 mRNA induced apoptosis in cultured ARPE-19 cells. TALEN KO in zebrafish was lethal 11 days post fertilization. The depressed electroretinogram (ERG) cone response and cone spectral sensitivity of 5 dpf KO zebrafish and reduced eye size, retinal thickness, and expression of rod and cone opsins could be rescued by injection of wild type CLCC1 mRNA. Clcc1+/- KO mice showed decreased ERGs and photoreceptor number. Together these results strongly suggest that intracellular chloride transport by CLCC1 is a critical process in maintaining retinal integrity, and CLCC1 is crucial for survival and function of retinal cells.

STIM1 Is Required for Remodeling of the Endoplasmic Reticulum and Microtubule Cytoskeleton in Steering Growth Cones.

The spatial and temporal regulation of calcium signaling in neuronal growth cones is essential for axon guidance. In growth cones, the endoplasmic reticulum (ER) is a significant source of calcium signals. However, it is not clear whether the ER is remodeled during motile events to localize calcium signals in steering growth cones. The expression of the ER-calcium sensor, stromal interacting molecule 1 (STIM1) is necessary for growth cone steering toward the calcium-dependent guidance cue BDNF, with STIM1 functioning to sustain calcium signals through store-operated calcium entry. However, STIM1 is also required for growth cone steering away from semaphorin-3a, a guidance cue that does not activate ER-calcium release, suggesting multiple functions of STIM1 within growth cones (Mitchell et al., 2012). STIM1 also interacts with microtubule plus-end binding proteins EB1/EB3 (Grigoriev et al., 2008). Here, we show that STIM1 associates with EB1/EB3 in growth cones and that STIM1 expression is critical for microtubule recruitment and subsequent ER remodeling to the motile side of steering growth cones. Furthermore, we extend our data in vivo, demonstrating that zSTIM1 is required for axon guidance in actively navigating zebrafish motor neurons, regulating calcium signaling and filopodial formation. These data demonstrate that, in response to multiple guidance cues, STIM1 couples microtubule organization and ER-derived calcium signals, thereby providing a mechanism where STIM1-mediated ER remodeling, particularly in filopodia, regulates spatiotemporal calcium signals during axon guidance.SIGNIFICANCE STATEMENT Defects in both axon guidance and endoplasmic reticulum (ER) function are implicated in a range of developmental disorders. During neuronal circuit development, the spatial localization of calcium signals controls the growth cone cytoskeleton to direct motility. We demonstrate a novel role for stromal interacting molecule 1 (STIM1) in regulating microtubule and subsequent ER remodeling in navigating growth cones. We show that STIM1, an activator of store-operated calcium entry, regulates the dynamics of microtubule-binding proteins EB1/EB3, coupling ER to microtubules, within filopodia, thereby steering growth cones. The STIM1-microtubule-ER interaction provides a new model for spatial localization of calcium signals in navigating growth cones in the nascent nervous system.

Interference with the Cannabinoid Receptor CB1R Results in Miswiring of GnRH3 and AgRP1 Axons in Zebrafish Embryos.

The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron's axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake.

The Dlx5 and Foxg1 transcription factors, linked via miRNA-9 and -200, are required for the development of the olfactory and GnRH system.

During neuronal development and maturation, microRNAs (miRs) play diverse functions ranging from early patterning, proliferation and commitment to differentiation, survival, homeostasis, activity and plasticity of more mature and adult neurons. The role of miRs in the differentiation of olfactory receptor neurons (ORNs) is emerging from the conditional inactivation of Dicer in immature ORN, and the depletion of all mature miRs in this system. Here, we identify specific miRs involved in olfactory development, by focusing on mice null for Dlx5, a homeogene essential for both ORN differentiation and axon guidance and connectivity. Analysis of miR expression in Dlx5(-/-) olfactory epithelium pointed to reduced levels of miR-9, miR-376a and four miRs of the -200 class in the absence of Dlx5. To functionally examine the role of these miRs, we depleted miR-9 and miR-200 class in reporter zebrafish embryos and observed delayed ORN differentiation, altered axonal trajectory/targeting, and altered genesis and position of olfactory-associated GnRH neurons, i.e. a phenotype known as Kallmann syndrome in humans. miR-9 and miR-200-class negatively control Foxg1 mRNA, a fork-head transcription factor essential for development of the olfactory epithelium and of the forebrain, known to maintain progenitors in a stem state. Increased levels of z-foxg1 mRNA resulted in delayed ORN differentiation and altered axon trajectory, in zebrafish embryos. This work describes for the first time the role of specific miR (-9 and -200) in olfactory/GnRH development, and uncovers a Dlx5-Foxg1 regulation whose alteration affects receptor neuron differentiation, axonal targeting, GnRH neuron development, the hallmarks of the Kallmann syndrome.

Unraveling the CLCC1 interactome: Impact of the Asp25Glu variant and its interaction with SigmaR1 at the Mitochondrial-Associated ER Membrane (MAM).

The endoplasmic reticulum (ER) plays an indispensable role in cellular processes, including maintenance of calcium homeostasis, and protein folding, synthesized and processing. Disruptions in these processes leading to ER stress and the accumulation of misfolded proteins can instigate the unfolded protein response (UPR), culminating in either restoration of balanced proteostasis or apoptosis. A key player in this intricate balance is CLCC1, an ER-resident chloride channel, whose essential role extends to retinal development, regulation of ER stress, and UPR. The importance of CLCC1 is further underscored by its interaction with proteins localized to mitochondria-associated endoplasmic reticulum membranes (MAMs), where it participates in UPR induction by MAM proteins. In previous research, we identified a p.(Asp25Glu) pathogenic CLCC1 variant associated with retinitis pigmentosa (RP) (CLCC1 hg38 NC_000001.11; NM_001048210.3, c.75C > A; UniprotKB Q96S66). In attempt to decipher the impact of this variant function, we leveraged liquid chromatography-mass spectrometry (LC-MS) to identify likely CLCC1-interacting proteins. We discovered that the CLCC1 interactome is substantially composed of proteins that localize to ER compartments and that the Asp25Glu variant results in noticeable loss and gain of specific protein interactors. Intriguingly, the analysis suggests that the CLCC1Asp25Glu mutant protein exhibits a propensity for increased interactions with cytoplasmic proteins compared to its wild-type counterpart. To corroborate our LC-MS data, we further scrutinized two novel CLCC1 interactors, Calnexin and SigmaR1, chaperone proteins that localize to the ER and MAMs. Through microscopy, we demonstrate that CLCC1 co-localizes with both proteins, thereby validating our initial findings. Moreover, our results reveal that CLCC1 co-localizes with SigmaR1 not merely at the ER, but also at MAMs. These findings reinforce the notion of CLCC1 interacting with MAM proteins at the ER-mitochondria interface, setting the stage for further exploration into how these interactions impact ER or mitochondria function and lead to retinal degenerative disease when impaired.

Tyrosinase (TYR) gene sequencing and literature review reveals recurrent mutations and multiple population founder gene mutations as causative of oculocutaneous albinism (OCA) in Pakistani families.

PURPOSE: To investigate eight previously unreported Pakistani families with genetically undefined OCA for mutations in TYR. METHODS: Sanger sequencing of TYR has been performed in eight families with OCA phenotype. Mutation analysis was performed to establish the pathogenic role of novel mutation. Bioinformatics analysis was performed to predict the structural and functional impacts on protein due to the mutation. RESULTS: In this study, we identified six likely pathogenic variants of TYR (c.272 G>A, c.308 G>A, c.346C>T, c.715 C>T, c.832 C>T and c.1255 G>A), including one novel variant (c.308 G>A; p.Cys103Tyr), segregating as appropriate in each family. Cys103 lies in the highly conserved region of the tyrosinase enzyme, and p.Cys103Tyr is predicted to disturb enzymatic function via alteration of the configurational orientation of TYR leading to a more rigid polypeptide structure. We have also reviewed the mutation spectrum of TYR in Pakistani ethnicity. Published data on OCA families proposed that ~40% have been associated with genetic variations in the TYR gene. The mutations reported in this study have now been described with varying frequencies in Pakistani families, including very rare/unique mutations. CONCLUSION: A literature review of TYR gene mutations in Pakistani populations, combined with our genetic data, identified a number of gene mutations likely to represent regional ancestral founder mutations of relevance to Pakistani populations, in addition to sporadic and recurrent 'hotspot' mutations present repeatedly in other regions worldwide.