Co-Injection of Sulfotyrosine Facilitates Retinal Uptake of Hyaluronic Acid Nanospheres Following Intravitreal Injection.

Gene and drug delivery to the retina is a critical therapeutic goal. While the majority of inherited forms of retinal degeneration affect the outer retina, specifically the photoreceptors and retinal pigment epithelium, effective targeted delivery to this region requires invasive subretinal delivery. Our goal in this work was to evaluate two innovative approaches for increasing both the persistence of delivered nanospheres and their penetration into the outer retina while using the much less invasive intravitreal delivery method. We formulated novel hyaluronic acid nanospheres (HA-NS, 250 nm and 500 nm in diameter) conjugated to fluorescent reporters and delivered them intravitreally to the adult Balb/C mouse retina. They exhibited persistence in the vitreous and along the inner limiting membrane (ILM) for up to 30 days (longest timepoint examined) but little retinal penetration. We thus evaluated the ability of the small molecule, sulfotyrosine, to disrupt the ILM, and found that 3.2 µg/µL sulfotyrosine led to significant improvement in delivery to the outer retina following intravitreal injections without causing retinal inflammation, degeneration, or loss of function. Co-delivery of sulfotyrosine and HA-NS led to robust improvements in penetration of HA-NS into the retina and accumulation along the interface between the photoreceptors and the retinal pigment epithelium. These exciting findings suggest that sulfotyrosine and HA-NS may be an effective strategy for outer retinal targeting after intravitreal injection.

A CLN6-CLN8 complex recruits lysosomal enzymes at the ER for Golgi transfer.

Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and transferred to the Golgi complex by interaction with the Batten disease protein CLN8 (ceroid lipofuscinosis, neuronal, 8). Here we investigated the relationship of this pathway with CLN6, an ER-associated protein of unknown function that is defective in a different Batten disease subtype. Experiments focused on protein interaction and trafficking identified CLN6 as an obligate component of a CLN6-CLN8 complex (herein referred to as EGRESS: ER-to-Golgi relaying of enzymes of the lysosomal system), which recruits lysosomal enzymes at the ER to promote their Golgi transfer. Mutagenesis experiments showed that the second luminal loop of CLN6 is required for the interaction of CLN6 with the enzymes but dispensable for interaction with CLN8. In vitro and in vivo studies showed that CLN6 deficiency results in inefficient ER export of lysosomal enzymes and diminished levels of the enzymes at the lysosome. Mice lacking both CLN6 and CLN8 did not display aggravated pathology compared with the single deficiencies, indicating that the EGRESS complex works as a functional unit. These results identify CLN6 and the EGRESS complex as key players in lysosome biogenesis and shed light on the molecular etiology of Batten disease caused by defects in CLN6.

Gene Therapy Rescues Retinal Degeneration in Receptor Expression-Enhancing Protein 6 Mutant Mice.

Hereditary retinal dystrophy is clinically defined as a broad group of chronic and progressive disorders that affect visual function by causing photoreceptor degeneration. Previously, we identified mutations in the gene encoding receptor expression-enhancing protein 6 (REEP6), in individuals with autosomal recessive retinitis pigmentosa (RP), the most common form of inherited retinal dystrophy. One individual was molecularly diagnosed with biallelic REEP6 mutations, a missense mutation over a frameshift mutation. In this study, we generated Reep6 compound heterozygous mice, Reep6L135P/-, which mimic the patient genotype and recapitulate the early-onset retinal degeneration phenotypes observed in the individual with RP. To determine the feasibility of rescuing the Reep6 mutant phenotype via gene replacement therapy, we delivered Reep6.1, the mouse retina-specific isoform of REEP6, to photoreceptors of Reep6 mutant mice on postnatal day 20. Evaluation of the therapeutic effects 2 months posttreatment showed improvements in the photoresponse as well as preservation of photoreceptor cells. Importantly, guanylyl cyclase 1 (GC1) expression was also restored to the outer segment after treatment. Furthermore, rAAV8-Reep6.1 single treatment in Reep6 mutant mice 1 year postinjection showed significant improvements in retinal function and morphology, suggesting that the treatment is effective even after a prolonged period. Findings from this study show that gene replacement therapy in the retina with rAAV overexpressing Reep6 is effective, preserving photoreceptor function in Reep6 mutant mice. These findings provide evidence that rAAV8-based gene therapy can prolong survival of photoreceptors in vivo and can be potentially used as a therapeutic modality for treatment of patients with RP.

SPATA7 maintains a novel photoreceptor-specific zone in the distal connecting cilium.

Photoreceptor-specific ciliopathies often affect a structure that is considered functionally homologous to the ciliary transition zone (TZ) called the connecting cilium (CC). However, it is unclear how mutations in certain ciliary genes disrupt the photoreceptor CC without impacting the primary cilia systemically. By applying stochastic optical reconstruction microscopy technology in different genetic models, we show that the CC can be partitioned into two regions: the proximal CC (PCC), which is homologous to the TZ of primary cilia, and the distal CC (DCC), a photoreceptor-specific extension of the ciliary TZ. This specialized distal zone of the CC in photoreceptors is maintained by SPATA7, which interacts with other photoreceptor-specific ciliary proteins such as RPGR and RPGRIP1. The absence of Spata7 results in the mislocalization of DCC proteins without affecting the PCC protein complexes. This collapse results in destabilization of the axonemal microtubules, which consequently results in photoreceptor degeneration. These data provide a novel mechanism to explain how genetic disruption of ubiquitously present ciliary proteins exerts tissue-specific ciliopathy phenotypes.

NMNAT1 E257K variant, associated with Leber Congenital Amaurosis (LCA9), causes a mild retinal degeneration phenotype.

NMNAT1 (nicotinamide mononucleotide adenylyltransferase 1) encodes a rate-limiting enzyme that catalyzes the biosynthesis of NAD+ and plays a role in neuroprotection. Mutations in NMNAT1 have been identified to cause a recessive, non-syndromic early form of blindness genetically defined as Leber Congenital Amaurosis 9 (LCA9). One of the most common alleles reported so far in NMNAT1 is the c.769G > A (E257K) missense mutation, which occurs in 70% of all LCA9 cases. However, given its relatively high population frequency and the observation of individuals with homozygous E257K variant without phenotype, the pathogenicity of this allele has been questioned. To address this issue, we have studied the pathogenic effects of this allele by generating a knock-in mouse model. Interestingly, no obvious morphological or functional defects are observed in Nmnat1 E257K homozygous mice up to one year old, even after light-damage. Together with the previous clinical reports, we propose that the E257K allele is a weak hypomorphic allele that has significantly reduced penetrance in the homozygous state. In contrast, compound heterozygous Nmnat1E257K/- mice exhibit photoreceptor defects which are exacerbated upon exposure to light. Furthermore, retina tissue- specific Nmnat1 conditional knockout mice exhibit photoreceptor degeneration before the retina has terminally differentiated. These findings suggest that NMNAT1 plays an important role in photoreceptors and is likely involved in both retinal development and maintenance of photoreceptor integrity.

POU6f1 Mediates Neuropeptide-Dependent Plasticity in the Adult Brain.

The mouse olfactory bulb (OB) features continued, activity-dependent integration of adult-born neurons, providing a robust model with which to examine mechanisms of plasticity in the adult brain. We previously reported that local OB interneurons secrete the neuropeptide corticotropin-releasing hormone (CRH) in an activity-dependent manner onto adult-born granule neurons and that local CRH signaling promotes expression of synaptic machinery in the bulb. This effect is mediated via activation of the CRH receptor 1 (CRHR1), which is developmentally regulated during adult-born neuron maturation. CRHR1 is a GS-protein-coupled receptor that activates CREB-dependent transcription in the presence of CRH. Therefore, we hypothesized that locally secreted CRH activates CRHR1 to initiate circuit plasticity programs. To identify such programs, we profiled gene expression changes associated with CRHR1 activity in adult-born neurons of the OB. Here, we show that CRHR1 activity influences expression of the brain-specific Homeobox-containing transcription factor POU Class 6 Homeobox 1 (POU6f1). To elucidate the contributions of POU6f1 toward activity-dependent circuit remodeling, we targeted CRHR1+ neurons in male and female mice for cell-type-specific manipulation of POU6f1 expression. Whereas loss of POU6f1 in CRHR1+ neurons resulted in reduced dendritic complexity and decreased synaptic connectivity, overexpression of POU6f1 in CRHR1+ neurons promoted dendritic outgrowth and branching and influenced synaptic function. Together, these findings suggest that the transcriptional program directed by POU6f1 downstream of local CRH signaling in adult-born neurons influences circuit dynamics in response to activity-dependent peptide signaling in the adult brain.SIGNIFICANCE STATEMENT Elucidating mechanisms of plasticity in the adult brain is helpful for devising strategies to understand and treat neurodegeneration. Circuit plasticity in the adult mouse olfactory bulb is exemplified by both continued cell integration and synaptogenesis. We previously reported that these processes are influenced by local neuropeptide signaling in an activity-dependent manner. Here, we show that local corticotropin-releasing hormone (CRH) signaling induces dynamic gene expression changes in CRH receptor expressing adult-born neurons, including altered expression of the transcription factor POU6f1 We further show that POU6f1 is necessary for proper dendrite specification and patterning, as well as synapse development and function in adult-born neurons. Together, these findings reveal a novel mechanism by which peptide signaling modulates adult brain circuit plasticity.

Conditional loss of Spata7 in photoreceptors causes progressive retinal degeneration in mice.

The mammalian retina consists of multiple cell layers including photoreceptor cells, which are light sensing neurons that play essential functions in the visual process. Previously, we identified mutations in SPATA7, encoding spermatogenesis associated protein 7, in families with Leber Congenital Amaurosis (LCA) and juvenile Retinitis Pigmentosa (RP), and showed that Spata7 null mice recapitulate the human disease phenotype of retinal degeneration. SPATA7 is expressed in the connecting cilium of photoreceptor (PR) cells in the mouse retina, as well as in retinal pigment epithelium (RPE) cells, but the functional role of Spata7 in the RPE remains unknown. To investigate whether Spata7 is required in PRs, the RPE, or both, we conditionally knocked out Spata7 in photoreceptors and RPE cells using Crx-Cre and Best1-Cre transgenic mouse lines, respectively. In Spata7 photoreceptor-specific conditional (cKO) mice, both rod and cone photoreceptor dysfunction and degeneration is observed, characterized by progressive thinning of the outer nuclear layer and reduced response to light; however, RPE-specific deletion of Spata7 does not impair retinal function or cell survival. Furthermore, our findings show that both Rhodopsin and RPGRIP1 are mislocalized in the Spata7Flox/-; Crx-Cre cKO mice, suggesting that loss of Spata7 in photoreceptors alone can result in altered trafficking of these proteins in the connecting cilium. Together, our findings suggest that loss of Spata7 in photoreceptors alone is sufficient to cause photoreceptor degeneration, but its function in the RPE is not required for photoreceptor survival; therefore, loss of Spata7 in photoreceptors alters both rod and cone function and survival, consistent with the clinical phenotypes observed in LCA and RP patients with mutations in SPATA7.

Isochlorogenic acid A promotes melanin synthesis in B16 cell through the ß-catenin signal pathway.

Isochlorogenic acid A, also called 3,5-dicaffeoylquinic acid (3,5-diCQA), is a widespread phenolic compound in the plant. Recent studies have shown that it has antioxidant and anti-inflammatory activity. In addition, oxidative stress and inflammation induced by solar ultraviolet radiation is a very significant reason for skin depigmentation. Therefore, in this study, we evaluated the effect of 3,5-diCQA on B16 cells and explored its molecular mechanism. Results showed that 3,5-diCQA upregulated intracellular melanin production in a time- and dose-dependent manner. Tyrosinase (TYR) activity was also increased after treatment with 3,5-diCQA in a dose-dependent manner. Expressions of TYR, TYR-related protein1, TYR-related protein2, and microphthalmia-associated transcription factor were upregulated in a dose-dependent manner after 48 h of treatment with 3,5-diCQA. Results also showed that 3,5-diCQA promoted the phosphorylation of Akt at Thr308 and glycogen synthase kinase-3ß at Ser 9. Moreover, 3,5-diCQA increased the content of ß-catenin in cell cytoplasm and nucleus by reducing the content of phosphorylated ß-catenin (p-ß-catenin). All these results suggest that 3,5-diCQA may mediate the acceleration of melanin synthesis by the ß-catenin signal pathway.

REEP6 deficiency leads to retinal degeneration through disruption of ER homeostasis and protein trafficking.

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy. We recently identified mutations in REEP6, which encodes the receptor expression enhancing protein 6, in several families with autosomal recessive RP. REEP6 is related to the REEP and Yop1p family of ER shaping proteins and potential receptor accessory proteins, but the role of REEP6 in the retina is unknown. Here we characterize the disease mechanisms associated with loss of REEP6 function using a Reep6 knockout mouse generated by CRISPR/Cas9 gene editing. In control mice REEP6 was localized to the inner segment and outer plexiform layer of rod photoreceptors. The Reep6-/- mice exhibited progressive photoreceptor degeneration from P20 onwards. Ultrastructural analyses at P20 by transmission electron microscopy and 3View serial block face scanning EM revealed an expansion of the distal ER in the Reep6-/- rods and an increase in their number of mitochondria. Electroretinograms revealed photoreceptor dysfunction preceded degeneration, suggesting potential defects in phototransduction. There was no effect on the traffic of rhodopsin, Rom1 or peripherin/rds; however, the retinal guanylate cyclases GC1 and GC2 were severely affected in the Reep6 knockout animals, with almost undetectable expression. These changes correlated with an increase in C/EBP homologous protein (CHOP) expression and the activation of caspase 12, suggesting that ER stress contributes to cell death. Collectively, these data suggest that REEP6 plays an essential role in maintaining cGMP homeostasis though facilitating the stability and/or trafficking of guanylate cyclases and maintaining ER and mitochondrial homeostasis.

Mutations in the Spliceosome Component CWC27 Cause Retinal Degeneration with or without Additional Developmental Anomalies.

Pre-mRNA splicing factors play a fundamental role in regulating transcript diversity both temporally and spatially. Genetic defects in several spliceosome components have been linked to a set of non-overlapping spliceosomopathy phenotypes in humans, among which skeletal developmental defects and non-syndromic retinitis pigmentosa (RP) are frequent findings. Here we report that defects in spliceosome-associated protein CWC27 are associated with a spectrum of disease phenotypes ranging from isolated RP to severe syndromic forms. By whole-exome sequencing, recessive protein-truncating mutations in CWC27 were found in seven unrelated families that show a range of clinical phenotypes, including retinal degeneration, brachydactyly, craniofacial abnormalities, short stature, and neurological defects. Remarkably, variable expressivity of the human phenotype can be recapitulated in Cwc27 mutant mouse models, with significant embryonic lethality and severe phenotypes in the complete knockout mice while mice with a partial loss-of-function allele mimic the isolated retinal degeneration phenotype. Our study describes a retinal dystrophy-related phenotype spectrum as well as its genetic etiology and highlights the complexity of the spliceosomal gene network.

Mutations in REEP6 Cause Autosomal-Recessive Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy.

Conditional knockout of retinal determination genes in differentiating cells in Drosophila.

Conditional gene knockout in postmitotic cells is a valuable technique which allows the study of gene function with spatiotemporal control. Surprisingly, in contrast to its long-term and extensive use in mouse studies, this technology is lacking in Drosophila. Here, we use a novel method for generating complete loss of eyes absent (eya) or sine oculis (so) function in postmitotic cells posterior to the morphogenetic furrow (MF). Specifically, genomic rescue constructs with flippase recognition target (FRT) sequences flanking essential exons are used to generate conditional null alleles. By removing gene function in differentiating cells, we show that eya and so are dispensable for larval photoreceptor differentiation, but are required for differentiation during pupal development. Both eya and so are necessary for photoreceptor survival and the apoptosis caused by loss of eya or so function is likely a secondary consequence of inappropriate differentiation. We also confirm their requirement for cone cell development and reveal a novel role in interommatidial bristle (IOB) formation. In addition, so is required for normal eye disc morphology. This is the first report of a knockout method to study eya and so function in postmitotic cells. This technology will open the door to a large array of new functional studies in virtually any tissue and at any stage of development or in adults.

Integrative subcellular proteomic analysis allows accurate prediction of human disease-causing genes.

Proteomic profiling on subcellular fractions provides invaluable information regarding both protein abundance and subcellular localization. When integrated with other data sets, it can greatly enhance our ability to predict gene function genome-wide. In this study, we performed a comprehensive proteomic analysis on the light-sensing compartment of photoreceptors called the outer segment (OS). By comparing with the protein profile obtained from the retina tissue depleted of OS, an enrichment score for each protein is calculated to quantify protein subcellular localization, and 84% accuracy is achieved compared with experimental data. By integrating the protein OS enrichment score, the protein abundance, and the retina transcriptome, the probability of a gene playing an essential function in photoreceptor cells is derived with high specificity and sensitivity. As a result, a list of genes that will likely result in human retinal disease when mutated was identified and validated by previous literature and/or animal model studies. Therefore, this new methodology demonstrates the synergy of combining subcellular fractionation proteomics with other omics data sets and is generally applicable to other tissues and diseases.

Mutations in POMGNT1 cause non-syndromic retinitis pigmentosa.

A growing number of human diseases have been linked to defects in protein glycosylation that affects a wide range of organs. Among them, O-mannosylation is an unusual type of protein glycosylation that is largely restricted to the muscular and nerve system. Consistently, mutations in genes involved in the O-mannosylation pathway result in infantile-onset, severe developmental defects involving skeleton muscle, brain and eye, such as the muscle-eye-brain disease (MIM no. 253280). However, the functional importance of O-mannosylation in these tissues at later stages remains largely unknown. In our study, we have identified recessive mutations in POMGNT1, which encodes an essential component in O-mannosylation pathway, in three unrelated families with autosomal recessive retinitis pigmentosa (RP), but without extraocular involvement. Enzymatic assay of these mutant alleles demonstrate that they greatly reduce the POMGNT1 enzymatic activity and are likely to be hypomorphic. Immunohistochemistry shows that POMGNT1 is specifically expressed in photoreceptor basal body. Taken together, our work identifies a novel disease-causing gene for RP and indicates that proper protein O-mannosylation is not only essential for early organ development, but also important for maintaining survival and function of the highly specialized retinal cells at later stages.

Next-generation sequencing-based molecular diagnosis of 12 inherited retinal disease probands of Uyghur ethnicity.

Inherited retinal disease (IRD) is a category of genetic disorders affecting retina. Understanding the molecular basis of IRD is vital for clinical and genetic classification of patients. Uyghur people is an isolated ethnic group mainly residing in northwestern China with genetic admixture from Europeans and East Asians. The genetic etiology of IRD in this specific population still remains unknown. Here, by next-generation sequencing (NGS), we screened mutations in over 200 known retinal disease genes in a cohort of 12 unrelated Uyghur IRD probands. Out of the 12 probands, six are solved with high confidence, two with low confidence, while the remaining four are unsolved. We identified known disease-causing alleles in this cohort that suggest ancient Uyghur migration and also discovered eight novel disease-associated variants. Our results showed NGS-based mutation screening as a reliable approach for molecular diagnosis. In addition, this approach can also be applied to reveal the genetic history of a specific ethnic group.

Hypomorphic mutations identified in the candidate Leber congenital amaurosis gene CLUAP1.

PURPOSE: Leber congenital amaurosis (LCA) is an early-onset form of retinal degeneration. Six of the 22 known LCA genes encode photoreceptor ciliary proteins. Despite the identification of 22 LCA genes, the genetic basis of ~30% of LCA patients remains unknown. We sought to investigate the cause of disease in the remaining 30% by examining cilia-associated genes. METHODS: Whole-exome sequencing was performed on an LCA cohort of 212 unsolved probands previously screened for mutations in known retinal-disease genes. Immunohistochemistry using mouse retinas was used to confirm protein localization and zebrafish were used to perform rescue experiments. RESULTS: A homozygous nonsynonymous mutation was found in a single proband in CLUAP1, a gene required for ciliogenesis and cilia maintenance. Cluap1 knockout zebrafish exhibit photoreceptor cell death as early as 5 days after fertilization, and rescue experiments revealed that our proband's mutation is significantly hypomorphic. CONCLUSION: Consistent with the knowledge that CLUAP1 plays an important role in cilia function and that cilia are critical to photoreceptor function, our results indicate that hypomorphic mutations in CLUAP1 can result in dysfunctional photoreceptors without systemic abnormalities. This is the first report linking mutations in CLUAP1 to human disease and establishes CLUAP1 as a candidate LCA gene.Genet Med 18 10, 1044-1051.

ADIPOR1 Is Mutated in Syndromic Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a genetically heterogeneous retinal disorder. Despite the numerous genes associated with RP already identified, the genetic basis remains unknown in a substantial number of patients and families. In this study, we performed whole-exome sequencing to investigate the molecular basis of a syndromic RP case that cannot be solved by mutations in known disease-causing genes. After applying a series of variant filtering strategies, we identified an apparently homozygous frameshift mutation, c.31delC (p.Q11Rfs*24) in the ADIPOR1 gene. The reported phenotypes of Adipor1-null mice contain retinal dystrophy, obesity, and behavioral abnormalities, which highly mimic those in the syndromic RP patient. We further confirmed ADIPOR1 retina expression by immunohistochemistry. Our results established ADIPOR1 as a novel disease-causing gene for syndromic RP and highlight the importance of fatty acid transport in the retina.

ATF6 Is Mutated in Early Onset Photoreceptor Degeneration With Macular Involvement.

PURPOSE: Photoreceptor degeneration (PRD) is a genetically heterogeneous retinal disorder. Although a number of genes involved in PRD have been identified, their genetic basis remains unknown in a significant number of patients. In this study, we aimed to identify novel disease-causing genes of PRD. METHODS: Comprehensive ocular examinations were performed in a 2-year-old patient diagnosed with early onset PRD. Retinal capture sequencing was performed to screen causative mutations in known retinal disease-causing loci. Whole-exome sequencing (WES) and a series of variant-filtering strategies were applied for identifying novel disease-causing genes. Retina ATF6 expression was confirmed by immunohistochemistry. RT-PCR was performed to identify ATF6 mRNA in the patient. RESULTS: The patient showed typical PRD features, with macular involvement and ellipsoid zone irregularities. Results of retinal capture sequencing were negative. WES data led to identification of biallelic loss-of-function mutations in the ATF6 gene. The first variant generates a premature stop codon (NCBI accession no. NM_007348: c.1126C>T, p.R376*) and the second variant affects a splicing donor site (NM_007348: c.1533+1G>C). Sanger sequencing confirmed the 2 alleles are from 1 parent each. Both of the variants are extremely rare in the population. The splicing variant causes either intron inclusion or exon skipping in the patient, thus severely disrupting ATF6 functional domains. ATF6 is expressed in three neuronal cell layers of mouse retina. CONCLUSIONS: Our results support ATF6 as a novel disease-causing gene for PRD and suggest that disrupted protein quality control mechanisms may be a novel pathological mechanism underlying human retinal degeneration.

Spata7 is a retinal ciliopathy gene critical for correct RPGRIP1 localization and protein trafficking in the retina.

Leber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) are severe hereditary diseases that causes visual impairment in infants and children. SPATA7 has recently been identified as the LCA3 and juvenile RP gene in humans, whose function in the retina remains elusive. Here, we show that SPATA7 localizes at the primary cilium of cells and at the connecting cilium (CC) of photoreceptor cells, indicating that SPATA7 is a ciliary protein. In addition, SPATA7 directly interacts with the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1), a key connecting cilium protein that has also been linked to LCA. In the retina of Spata7 null mutant mice, a substantial reduction of RPGRIP1 levels at the CC of photoreceptor cells is observed, suggesting that SPATA7 is required for the stable assembly and localization of the ciliary RPGRIP1 protein complex. Furthermore, our results pinpoint a role of this complex in protein trafficking across the CC to the outer segments, as we identified that rhodopsin accumulates in the inner segments and around the nucleus of photoreceptors. This accumulation then likely triggers the apoptosis of rod photoreceptors that was observed. Loss of Spata7 function in mice indeed results in a juvenile RP-like phenotype, characterized by progressive degeneration of photoreceptor cells and a strongly decreased light response. Together, these results indicate that SPATA7 functions as a key member of a retinal ciliopathy-associated protein complex, and that apoptosis of rod photoreceptor cells triggered by protein mislocalization is likely the mechanism of disease progression in LCA3/ juvenile RP patients.

Kaliziri extract upregulates tyrosinase, TRP-1, TRP-2 and MITF expression in murine B16 melanoma cells.

BACKGROUND: Kaliziri extract (KZE) is a traditional Uyghur medicine (TUM), used by traditional hospitals in China as an injection for treatment of vitiligo for more than 30 years. Clinical application has shown that this medicine has obvious therapeutic effects. However, its phytochemical analysis and mechanism have not been examined. METHODS: KZE was extracted from seeds of Kaliziri [Vernonia anthelmintica (L.) Willd.] in ethanol-water (80:20, v/v), its components were identified by LC-MS/MS, and the signaling pathway of melanin synthesis in KZE treated murine B16 melanoma cells was examined by western blotting. RESULTS: Liquid chromatography-mass spectrometry analysis confirmed that the main components of KZE are flavonoids. KZE increased the tyrosinase activity and melanin content in a dose-dependent manner at concentrations of 5-40 µg/ml, and treatment with 20 µg/ml of KZE enhanced the expression of tyrosinase in B16 cells in a time-dependent manner. CONCLUSIONS: KZE induced melanogenesis by increasing the expression of TYR, TRP-1, TRP-2 and MITF in B16 cells.