Disrupting the Repeat Domain of Premelanosome Protein (PMEL) Produces Dysamyloidosis and Dystrophic Ocular Pigment Reflective of Pigmentary Glaucoma

Pigmentary glaucoma has recently been associated with missense mutations in PMEL that are dominantly inherited and enriched in the protein’s fascinating repeat domain. PMEL pathobiology is intriguing because PMEL forms functional amyloid in healthy eyes, and this PMEL amyloid acts to scaffold melanin deposition. This is an informative contradistinction to prominent neurodegenerative diseases where amyloid formation is neurotoxic and mutations cause a toxic gain of function called “amyloidosis”. Preclinical animal models have failed to model this PMEL “dysamyloidosis” pathomechanism and instead cause recessively inherited ocular pigment defects via PMEL loss of function; they have not addressed the consequences of disrupting PMEL’s repetitive region. Here, we use CRISPR to engineer a small in-frame mutation in the zebrafish homolog of PMEL that is predicted to subtly disrupt the protein’s repetitive region. Homozygous mutant larvae displayed pigmentation phenotypes and altered eye morphogenesis similar to presumptive null larvae. Heterozygous mutants had disrupted eye morphogenesis and disrupted pigment deposition in their retinal melanosomes. The deficits in the pigment deposition of these young adult fish were not accompanied by any detectable glaucomatous changes in intraocular pressure or retinal morphology. Overall, the data provide important in vivo validation that subtle PMEL mutations can cause a dominantly inherited pigment pathology that aligns with the inheritance of pigmentary glaucoma patient pedigrees. These in vivo observations help to resolve controversy regarding the necessity of PMEL’s repeat domain in pigmentation. The data foster an ongoing interest in an antithetical dysamyloidosis mechanism that, akin to the amyloidosis of devastating dementias, manifests as a slow progressive neurodegenerative disease

The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general

The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general.</p

The genetic characterization of mendelian ocular disorders in the population of Newfoundland and Labrador

Background -- Recently, ocular genetics have shown the first successes in genetic therapies, and treatment of genetic diseases making identification of disease genes of great importance. Gene discovery is most successful through the study of genetic founder populations, such as that of Newfoundland and Labrador. -- Objective -- The objective of this thesis was to identify disease genes in three forms of Mendelian ocular disease: anterior segment dysgenesis (ASD), achromatopsia (ACHM), and microphthalmia-dwarfism (MDW). This was undertaken to find novel genes and mutations to further our understanding genetic pathways involved in each condition. -- Results -- Of the 11 families recruited for this study, 8 were solved through identification of pathogenic variants. The ASD phenotype was discovered to be caused by a novel mutation in FOXE3, seven ACHM families through mutations in CNGA3 and CNGB3, one ACHM family was found to actually have a rare disease called Jalili Syndrome through a novel mutation in CNNM4, and two MDW families helped determine a putative disease locus on 16q21. -- Conclusions -- The identification of seven mutations (two novel, five previously described) have solved the genetic etiology in eight of eleven families providing insight into the disease pathways for these families. This allows for genetic counseling and the possibility for genetically based therapies in the future

FOXC1 modulates MYOC secretion through regulation of the exocytic proteins RAB3GAP1, RAB3GAP2 and SNAP25

<div><p>The neurodegenerative disease glaucoma is one of the leading causes of blindness in the world. Glaucoma is characterized by progressive visual field loss caused by retinal ganglion cell (RGC) death. Both surgical glaucoma treatments and medications are available, however, they only halt glaucoma progression and are unable to reverse damage. Furthermore, many patients do not respond well to treatments. It is therefore important to better understand the mechanisms involved in glaucoma pathogenesis. Patients with Axenfeld-Rieger syndrome (ARS) offer important insight into glaucoma progression. ARS patients are at 50% risk of developing early onset glaucoma and respond poorly to treatments, even when surgical treatments are combined with medications. Mutations in the transcription factor FOXC1 cause ARS. Alterations in FOXC1 levels cause ocular malformations and disrupt stress response in ocular tissues, thereby contributing to glaucoma progression. In this study, using biochemical and molecular techniques, we show that FOXC1 regulates the expression of RAB3GAP1, RAB3GAP2 and SNAP25, three genes with central roles in both exocytosis and endocytosis, responsible for extracellular trafficking. FOXC1 positively regulates RAB3GAP1 and RAB3GAP2, while either increase or decrease in FOXC1 levels beyond its normal range results in decreased SNAP25. In addition, we found that FOXC1 regulation of RAB3GAP1, RAB3GAP2 and SNAP25 affects secretion of Myocilin (MYOC), a protein associated with juvenile onset glaucoma and steroid-induced glaucoma. The present work reveals that FOXC1 is an important regulator of exocytosis and establishes a new link between FOXC1 and MYOC-associated glaucoma.</p></div

FOXC1 over-expression changes protein levels of RAB3GAP1, RAN3GAP2 and SNAP25 in HeLa cells.

<p>Western blot analysis of HeLa protein lysates after transfection with either pcDNA4-Xpress-Empty or pcDNA4-Xpress-FOXC1(WT). Blots were probed with antibodies for FOXC1, RAB3GAP1, RAB3GAP2, SNAP25 and α-Tubulin (loading control). Normalized scaled values were calculated for <b>A)</b> RAB3GAP1 (example blot shown in <b>B</b>). Normalized and scaled values compared to level of FOXC1 knockdown are presented in <b>C)</b> RAB3GAP2 (example blot shown in <b>D</b>) and <b>E)</b> SNAP25 (example blot shown in <b>F</b>) *<i>P</i>˂0.05. Experiments were repeated at least three times.</p

Luciferase transactivation by FOXC1 through upstream regions of <i>RAB3GAP1</i>, <i>RAB3GAP2</i> and <i>SNAP25</i>.

<p>Transactivation experiments with plasmid expressing FOXC1 (WT) or mutant FOXC1(p.S131L), and reporter construct (pGL3 or pGL3.TK) containing <b>(A)</b> The 225bp <i>RAB3GAP1</i> upstream (pGL3.R3G1) and pGL3.FOXO1 (positive control) <b>(B)</b> Upstream region of RAB3GAP1 (pGL3.R3G1), and constructs with each possible combination of putative FOXC1 binding sites deleted. <b>(C)</b> The 211bp <i>RAB3GAP2</i> upstream region, the region with the BS deleted, pGL3.TK.R3G2.del and pGL3.TK.6xFBS (positive control) <b>(D)</b> The 151bp <i>SNAP25</i> upstream region (pGL3.TK. SNAP25), and the region with the BS deleted (SNAP25.del). All Experiments were repeated at least three times in triplicate. Error bars represent standard error. N.S Not significant, *<i>P</i>˂0.05, **<i>P</i>˂0.01 versus pGL3.TK or pGL3.</p

FOXC1 knockdown decreases protein levels of RAB3GAP1, RAB3GAP2 and SNAP25 in HeLa cells.

<p><b>A)</b> Western blot analysis of HeLa protein lysates after siRNA transfection with siRNA Scrambled control, or siRNA FOXC1. As FOXC1 has a number of phosphorylation sites, a number of bands are observed at ~70kDa, indicated by a curly brace. Blots were probed for FOXC1, RAB3GAP1, RAB3GAP2, SNAP25 and α-Tubulin (loading control). Normalized and scaled values for <b>B)</b> RAB3GAP1 <b>C)</b> RAB3GAP2 and <b>D)</b> SNAP25. All Western blots were repeated at least three times. *<i>P</i>˂0.05, **<i>P</i>˂0.01, ***<i>P</i><0.001.</p

<i>FOXC1</i> knockdown decreases RNA levels of <i>RAB3GAP1</i>, <i>RAB3GAP2</i> and <i>SNAP25</i>.

<p>qRT-PCR experiments using RNA isolated from HeLa cells transfected with scrambled siRNA. qPCR was used to evaluate changes in RNA levels of <b>A)</b><i>FOXC1</i> <b>B)</b><i>RAB3GAP1</i> <b>C)</b><i>RAB3GAP2</i>, <b>D)</b> Total <i>SNAP25</i> (<i>SNAP25ab</i>) <b>E)</b> <i>SNAP25</i> isoform a (<i>SNAP25a</i>), <b>F)</b> <i>SNAP25</i> isoform b (<i>SNAP25b</i>) and <i>HPRT1</i> (housekeeping gene control). Fold change in RNA levels was calculated using the ΔΔCt method normalized to HPRT1 and scaled to siRNA scrambled control. Experiments were performed three times in triplicate. Error bars represent standard error. *<i>P</i>˂0.05.</p

RAB3GAP2 knockdown decreases intracellular and extracellular levels of exogenous MYOC in HeLa cells.

<p>Western blot analysis using HeLa cell lysate, and cell media collected after transfection with pRc-MYOC (WT) and either siRNA Control or siRNA RAB3GAP1. Blots of lysates were probed and bands were quantified, normalized, and scaled for A) RAB3GAP2, and MYOC using TFIID as a loading control and blots of media were probed for MYOC using ponceau stain intensity as a loading control (right). <b>B)</b> Example blots of lysates (left) and media (right). Error bars represent standard error. *<i>P</i>˂0.05.</p