Loss of Optineurin <i>In Vivo</i> Results in Elevated Cell Death and Alters Axonal Trafficking Dynamics

<div><p>Mutations in Optineurin have been associated with ALS, glaucoma, and Paget’s disease of bone in humans, but little is known about how these mutations contribute to disease. Most of the cellular consequences of Optineurin loss have come from <i>in vitro</i> studies, and it remains unclear whether these same defects would be seen <i>in vivo</i>. To answer this question, we assessed the cellular consequences of Optineurin loss in zebrafish embryos to determine if they showed the same defects as have been described in the <i>in vitro</i> studies. We found that loss of Optineurin resulted in increased cell death, as well as subtle cell morphology, cell migration and vesicle trafficking defects. However, unlike experiments on cells in culture, we found no indication that the Golgi apparatus was disrupted or that NF-κB target genes were upregulated. Therefore, we conclude that <i>in vivo</i> loss of Optineurin shows some, but not all, of the defects seen in <i>in vitro</i> work.</p></div

<i>optn</i> embryos have increased cell death.

<p><b>A–D.</b> Compressed z-stacks of 25 hpf WT (A, C) or <i>MZoptn</i> (B, D) embryos labeling dying cells with Acridine Orange (A, B) or α-activated caspase-3 (C, D). Anterior is to the left and dorsal is up. <b>A, B.</b><i>MZoptn</i> embryos have more Acridine Orange labeled cells (arrows). <b>C, D.</b><i>MZoptn</i> embryos have more caspase-3 labeled cells (arrows). <b>E–G.</b> TEM images from cross sections of WT (E) or <i>MZoptn</i> embryos (F, G). E, F are normal looking cells, G is apoptotic cell. In G, the nucleus is condensed (N), the nuclear envelope is missing (arrowhead) and the mitochondria are enlarged (arrows) compared to normal cell sin E, F. <b>H–J.</b> Quantification of cell death by acridine orange staining in 25 hpf heads (H) or 2 dpf head and trunks (I) or caspase staining in 25 hpf heads and trunks (J). ** indicates p≤0.01.</p

<i>MZoptn</i> embryos have defects in the dynamics of vesicle trafficking.

<p><b>A–J, M–P.</b> Frames from time-lapse imaging of Texas Red-dextran trafficked through longitudinal spinal axons in WT (A–H) or <i>MZoptn</i> (M–P) embryos. Moving puncta are indicated by arrows and time codes are indicated. WT punctum shown in A–K traveled a little slower than average. <i>MZoptn</i> punctum shown in L–P traveled faster than average. <b>K, L.</b> Quantification of the speed of indicated puncta of WT panels (K) or <i>MZoptn</i> panels (L). Each time-point corresponds to the average speed of the puncta over the preceding 15 seconds. <b>Q–T.</b> Quantification of all puncta from WT and <i>MZoptn</i> embryos showing overall speed of puncta (Q), speed of puncta when they are not pausing (R), % of time puncta spend pausing (S) and time that each puncta spends on individual pauses (T). * indicates p≤0.05.</p

<i>sa0143</i> is a null mutation of <i>optn</i> with reduced protein and transcript levels.

<p><b>A.</b> Trace files from sequencing reactions of adult fish showing region of <i>sa0143</i> mutation and corresponding amino acids. Arrow indicates mutated base. <b>B, C.</b> Live pictures of adult WT (B) and <i>optn</i> (C) fish showing no difference in morphology. <b>D.</b> Western blot of protein extracts from 3 dpf embryos. Green = anti-Optn, Red = anti-γ-tubulin as a loading control. Asterisks indicate likely non-specific bands. The Optn band is absent in the <i>MZoptn</i> lane. <b>E.</b> Representative gel of semi-quantitative RT-PCR run with cDNA from retinas of 6 mpf fish. Top blot was used with primers against exons 1–3 of <i>optn</i>, bottom blot was used with primers against <i>ef1a</i> as a loading control. PCR was stopped at cycle times indicated below the blots. <b>F.</b> Quantification of 3 semi-quantitative RT-PCR experiments. Levels were analyzed for 28-cycle bands and normalized to WT bands for each experiment. ** indicates p≤0.01.</p

<i>MZoptn</i> embryos have defects in the migration of neural crest cells.

<p><b>A–J.</b> Compressed z-stacks of WT (A, C, E, G, I) or <i>MZoptn</i> (B, D, F, H, J) embryos. Embryos are mounted laterally (A–H) or ventrally (I, J) with anterior to the left. <b>A, B.</b> α-acetylated alpha-tubulin staining of axon tracts in 25 hpf embryos. The <i>MZoptn</i> embryos have normal axon pathfinding (arrows). <b>C, D.</b> α-SV2 staining of spinal motor axons in 2 dpf embryos. These axons are guided normally in <i>MZoptn</i> embryos (arrows). <b>E, F.</b> GFP labeled endothelial cells in 2 dpf embryos show that intersegmental vessels are patterned correctly in <i>MZoptn</i> embryos (arrows). <b>G, H.</b> GFP labeling of neural crest cells in 23 hpf embryos. The ventrally directed streams of cells (arrows) in the trunk are in the right location, but the width of the streams is wider in <i>MZoptn</i> embryos along the anterior-posterior axis (horizontal bars correspond to width of above stream). <b>I, J.</b> GFP labeling of neural crest cells that are contributing to the jaw cartilage in 4 dpf embryos. The elements of the jaw cartilage in <i>MZoptn</i> embryos appear normal (arrows), including Meckel’s cartilage (m), palatoquadrate (pq), ceratohyal (ch), hyosympathetic (hs), ethmoid plate (ep) and ceratobranchials (cb). <b>K.</b> Quantification of the width of the neural crest streams in G, H. * indicates p≤0.05.</p

Optineurin protein sequences are conserved across species.

<p>Alignment of optineurin protein sequences from human, mouse and zebrafish. Black shaded amino acids are conserved across species, gray shaded amino acids are similar. Identified protein binding sites are indicated above the corresponding parts of the protein. Asterisks indicated amino acids associated with disease in human patients when mutated. Number sign indicates location of <i>sa0143</i> zebrafish mutation.</p

<i>MZoptn</i> embryos have subtle morphological defects at early developmental ages.

<p><b>A-F.</b> Representative images of live WT (A, C, E) or <i>MZoptn</i> (B, D, F) embryos at 25 hpf (A, B) or 5 dpf (C-F). Anterior is to the left. <b>A, B.</b> The area of the eyes (arrows) and length of the yolk tube extension (arrowheads) are smaller in <i>MZoptn</i> embryos. <i>MZoptn</i> embryos occasionally have a curve to the tail (open arrowhead). <b>C-F.</b> Pigment cells in <i>MZoptn</i> embryos have more filopodia. E and F are higher magnification views of boxed areas in C and D. Insets in E and F are higher magnifications of pigment cells indicated by arrows. <b>G-I.</b> Quantification of eye size at 25 hpf (I), length of yolk tube extension at 25 hpf (J) and isoperimetric quotient of pigment cells at 5 dpf (K). * indicates p≤0.05, ** indicates p≤0.01.</p