CRISPR-Cas9–based treatment of myocilin-associated glaucoma

Primary open-angle glaucoma (POAG) is a leading cause of irreversible vision loss worldwide, with elevated intraocular pressure (IOP) a major risk factor. Myocilin (MYOC) dominant gain-of-function mutations have been reported in ∼4% of POAG cases. MYOC mutations result in protein misfolding, leading to endoplasmic reticulum (ER) stress in the trabecular meshwork (TM), the tissue that regulates IOP. We use CRISPR-Cas9–med iated genome editing in cultured human TM cells and in a MYOC mouse model of POAG to knock down expression of mutant MYOC, resulting in relief of ER stress. In vivo genome editing results in lower IOP and prevents further glaucomatous damage. Importantly, using an ex vivo human organ culture system, we demonstrate the feasibility of human genome editing in the eye for this important disease. Keywords: myocilin; CRISPR; glaucoma; trabecular meshwork; genome editingNational Institutes of Health (U.S.) (Grant R01 EY024259)National Institutes of Health (U.S.) (Grant R01 EY026177)National Institutes of Health (U.S.) (Grant R00 EY022077

A Novel Protein LZTFL1 Regulates Ciliary Trafficking of the BBSome and Smoothened

Many signaling proteins including G protein-coupled receptors localize to primary cilia, regulating cellular processes including differentiation, proliferation, organogenesis, and tumorigenesis. Bardet-Biedl Syndrome (BBS) proteins are involved in maintaining ciliary function by mediating protein trafficking to the cilia. However, the mechanisms governing ciliary trafficking by BBS proteins are not well understood. Here, we show that a novel protein, Leucine-zipper transcription factor-like 1 (LZTFL1), interacts with a BBS protein complex known as the BBSome and regulates ciliary trafficking of this complex. We also show that all BBSome subunits and BBS3 (also known as ARL6) are required for BBSome ciliary entry and that reduction of LZTFL1 restores BBSome trafficking to cilia in BBS3 and BBS5 depleted cells. Finally, we found that BBS proteins and LZTFL1 regulate ciliary trafficking of hedgehog signal transducer, Smoothened. Our findings suggest that LZTFL1 is an important regulator of BBSome ciliary trafficking and hedgehog signaling

The centriolar satellite protein AZI1 interacts with BBS4 and regulates ciliary trafficking of the BBSome.

Bardet-Biedl syndrome (BBS) is a well-known ciliopathy with mutations reported in 18 different genes. Most of the protein products of the BBS genes localize at or near the primary cilium and the centrosome. Near the centrosome, BBS proteins interact with centriolar satellite proteins, and the BBSome (a complex of seven BBS proteins) is believed to play a role in transporting ciliary membrane proteins. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. Here, we show that a centriolar satellite protein, AZI1 (also known as CEP131), interacts with the BBSome and regulates BBSome ciliary trafficking activity. Furthermore, we show that AZI1 interacts with the BBSome through BBS4. AZI1 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon AZI1 depletion. Under conditions in which the BBSome does not normally enter cilia, such as in BBS3 or BBS5 depleted cells, knock down of AZI1 with siRNA restores BBSome trafficking to cilia. Finally, we show that azi1 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer's vesicle abnormalities and melanosome transport delay. These findings associate AZI1 with the BBS pathway. Our findings provide further insight into the regulation of BBSome ciliary trafficking and identify AZI1 as a novel BBS candidate gene

Autophagy stimulation reduces ocular hypertension in a murine glaucoma model via autophagic degradation of mutant myocilin

Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) damage is associated with primary open-angle glaucoma (POAG). Myocilin mutations resulting in elevated IOP are the most common genetic causes of POAG. We have previously shown that mutant myocilin accumulates in the ER and induces chronic ER stress, leading to TM damage and IOP elevation. However, it is not understood how chronic ER stress leads to TM dysfunction and loss. Here, we report that mutant myocilin activated autophagy but was functionally impaired in cultured human TM cells and in a mouse model of myocilin-associated POAG (Tg-MYOCY437H). Genetic and pharmacological inhibition of autophagy worsened mutant myocilin accumulation and exacerbated IOP elevation in Tg-MYOCY437H mice. Remarkably, impaired autophagy was associated with chronic ER stress–induced transcriptional factor CHOP. Deletion of CHOP corrected impaired autophagy, enhanced recognition and degradation of mutant myocilin by autophagy, and reduced glaucoma in Tg-MYOCY437H mice. Stimulating autophagic flux via tat-beclin 1 peptide or torin 2 promoted autophagic degradation of mutant myocilin and reduced elevated IOP in Tg-MYOCY437H mice. Our study provides an alternate treatment strategy for myocilin-associated POAG by correcting impaired autophagy in the TM

AZI1 knockdown increases ciliary localization of BBS4.

<p><b>A</b>) RPE-1 cells expressing GFP-BBS4 were depleted of AZI1, and the number of cells with ciliary GFP (BBS4) was counted. BBS4 is stained with GFP (green), Acetylated α-tubulin was used to detect cilia (red). <b>B</b>) Graph showing significant increases in ciliary localization of BBS4 upon AZI1 knockdown. <b>C</b>) Depletion of AZI1 in BBS3 and BBS5 depleted cells restores ciliary BBSome localization. RPE-1 cells were transfected with siRNA as indicated, and BBS9 (red) localization was analyzed. Cilia (green) in the insets of figures <b>A</b> and <b>C</b> are slightly shifted to show ciliary localization of BBS proteins. <b>D</b>) Cilia containing BBS9 at different conditions were counted and presented graphically. All data are presented in mean +/− SEM. Significance is calculated using the Student's t-test for C and E, and one way ANOVA for A. P<0.05 is considered significant for each analysis.</p

AZI1 co-localizes with BBS4 to the centriolar satellite.

<p><b>A</b>) AZI1 and PCM1 co-localize at the centrosome. IMCD3 cells were transfected with HA-AZI1 construct and stained with antibody against HA (green) and PCM1 (red). AZI1 and PCM1 co-localize at the centrosome, in the presence (row 1) as well as absence of cilia (row 2). γ-tubulin and acetylated α-tubulin staining were used to identify the basal body and cilia. <b>B</b>) BBS4 co-localizes with AZI1, which resembles its co-localization with PCM1. RPE-1 cells stably expressing GFP-BBS4 were used; anti-GFP antibody was used to detect BBS4 (green); anti-AZI1 (row 1) or anti-PCM1 (row 2) antibody is shown in red. <b>C</b>) Western blotting shows expression levels of BBS and other satellite proteins upon AZI1 knockdown. Three different siRNAs were used, and efficient and specific knockdown of AZI1 by all three siRNAs are shown. Antibody against various BBS and satellite proteins were used to show that loss of AZI1 does not cause significant differences in the expression of those proteins. GAPDH is used as a loading control. <b>D</b>) Centriolar satellite localization of AZI1 (red) is confirmed in RPE-1 cells. siRNA based depletion diminished AZI1 from centriolar satellites. Depletion of PCM1 also depletes AZI from satellites but AZI1 at core centriolar areas remain intact. Insets show enlarged view of the centrosomal region. Green is γ-tubulin and acetylated α-tubulin staining for basal body and cilia, respectively. Scale bar, 10 µm.</p

Azi1 (Cep131) knockdown causes defects in KV formation, melanosome trafficking and vision defects in zebrafish.

<p>Embryos were untreated or injected with 125 µM Bbs4 MO or 150 µM cep131 MO as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004083#pgen.1004083-Yen1" target="_blank">[28]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004083#pgen.1004083-Tayeh1" target="_blank">[29]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004083#pgen.1004083-Pretorius1" target="_blank">[31]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004083#pgen.1004083-Baye1" target="_blank">[33]</a>. <b>A</b>) <i>azi1</i>, as well as <i>bbs4</i> morpholino injection causes a mild to severe body curvature phenotype in the 48 hpf larva. Images indicate examples of normal, mild or severe phenotypes. <b>B</b>) Quantification of body curvature. More of the <i>azi1</i> morphants have severe body curvature than <i>bbs4</i> morphants. <b>C</b>) Micrographs show KVs from MO injected and uninjected 8–10 somite stage embryos. <b>D</b>) <i>bbs4</i> MO caused KV abnormalities in 30% of embryos, and <i>azi1</i> MO caused 35% abnormal KV compared with 12% in control embryos (P<0.001 Fisher's exact test). <b>E</b>) <i>bbs4</i> MO and <i>azi1</i> MO delayed melanosome retrograde transport to 4.04+/−0.43 min, and 3.03+/−0.12 min, respectively compared to the control MO, in which the complete transport occurs within 1.92+/−0.04 minutes. <b>F</b>) Vision assays were performed on the morphants by observing their response to dark/light cues as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004083#pgen.1004083-Baye1" target="_blank">[33]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004083#pgen.1004083-Easter1" target="_blank">[35]</a>. For each fish the vision response tested 5 times. For C and D data are presented mean +/− SEM, P<0.01 by one-way ANOVA with Tukey post-test. The number of embryos used is presented in the figure.</p

A model showing centrosomal complex of BBS4, BBSome complex formation, and its ciliary localization.

<p><b>A</b>) 1. Partial BBSome complex (without BBS4) arrives near the centrosome, where BBS4 is part of a satellite complex. At the centrosome BBS4 is incorporated into the BBSome and a stable holo-BBSome complex is formed, which is trafficked to cilia while the rest of the satellite complex remain at the centrosome. A potential rate-limiting factor of BBSome entry into cilia is availability of the BBSome complex that can enter cilia. <b>B</b>) Loss of AZI1 weakens the interaction of BBS4 with the satellite complex, and more BBSome complex is available for entry into cilia.</p

AZI1 knockdown reduces ciliogenesis but increases ciliary localization of the BBSome.

<p><b>A</b>) RPE-1 cells were transfected with siRNA against <i>BBS4</i>, <i>PCM1</i>, and <i>AZI1</i>. Approximately 500 cells per sample were counted. <b>B</b>) AZI1 depletion increases the ciliary localization of BBS9 compared to the control knockdown. Red staining represents BBS9, and cilia are stained with acetylated α-tubulin. Nuclei are stained blue with DAPI. <b>C</b>) Graph showing a significant increase in cells with ciliary BBS9 upon AZI1 knockdown by different siRNAs. <b>D</b>) BBS8 (red) is used as a BBSome marker to confirm increased ciliary localization of the BBSome upon AZI1 knockdown. Cilia (green) in the insets of figures <b>B</b> and <b>D</b> are slightly shifted to show ciliary localization of BBS proteins. <b>E</b>) The graph shows a significant increase in the number of ciliated cells with BBS8 or BBS9 upon AZI1 knockdown, and decrease in ciliary BBS8 or BBS9 localization upon AZI1 overexpression. Approximately 250 ciliated cells were counted in control as well as AZI1 knockdown culture of RPE-1 cells. All data are presented in mean +/− SEM. Significance is calculated using Student's t-test for C and E, and one way ANOVA for A. P<0.05 is considered significant for each analysis.</p