青光眼是全球引發雙眼失明的疾病主因之一，其特點是導致視網膜神經節細胞和神經纖維層逐漸病變且薄化，初期伴隨周邊的?野缺損，最終導致全盲。依病因可分為原發性和續發性，原發性意謂自發性。先前有研究指出原發性開放青光眼 (primary open-angle glaucoma) 與 optineurin 的突變有關，另外肌萎縮性脊髓?索硬化症還有佩吉特氏病也與 OPTN 相關。Optineurin 是一種多功能蛋白，有一些捲曲螺旋和 C 端與泛素結的結構，其參與在許多訊息傳遞的路徑上，像是 NF-κB 的活化、自噬作用或者是自體免疫反應。青光眼發病的主要症狀為視野缺損、視力喪失，主要是由於神經節細胞中的視神經死亡。Optineurin 作為其中的關鍵蛋白，包括參與囊泡分泌和回收的運輸路徑、NF-κB 訊號、有絲分裂、高基氏體機轉和抗病毒信號，影響甚鉅，但此相關研究仍缺乏，機轉不明，因此釐清此基因相關的功能及分析，為本篇研究的主要目的。
本研究針對斑馬魚 OPTN 基因進行研究。如以半定量 RT-PCR觀察斑馬魚早期胚胎各個發育時期的基因表現量，發現表現量隨著發育時期，呈正相關，接著利用全胚胎原位雜交的方式觀察基因在發育後期表現情形。為瞭解 OPTN 在斑馬魚體內的生理功能，我們利用反股寡核酸 (Antisense Morpholino oligonucleotides，MO) 顯微注射抑制 OPTN 基因的功能，為了確認 MO 對 OPTN 基因的抑制是專性的，我們建構了帶有紅色螢光蛋白的 OPTN 表現質體 (OPTN- TaqRFP），藉由統計螢光表現率來探討 MO 的專一性，結果發現OPTN 的 MO 可以有效的抑制紅色螢光表現，因此我們確認其具專一性 knockdown OPTN 的基因表現。取 3dpf 注射 MO 的斑馬魚，進行組織切片，量測其眼視網膜厚度變化情形，此數據可供往後研究原發性開放青光眼之早期病程。
Glaucoma is a leading cause of irreversible visual impairment and blindness resulting from progressive degeneration of retinal ganglion cells (RGCs) and optic neuropathy. Optineurin was originally identified as a gene responsible for primary open-angle glaucoma. The gene, OPTN, codes for the protein optineurin, which is involved in a variety of functions including regulation of endocytic trafficking, autophagy, immune response, mitosis and NF-κB signal transduction. Many missense mutations in optineurin have been reported and the association of the mutation with the disease varies in different populations. Optineurin is localized to pathological structures also seen in several neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, etc. However, functional alterations caused by mutations in optineurin are poorly understood.
In this study, we investigated the physiological functions of OPTN in the zebrafish embryonic development. RT-PCR analysis of zebrafish OPTN transcripts were revealed significantly in the 120hpf embryos. Meanwhile, whole-mount in situ hybridization (WISH) signals were also observed in the 120hpf embryos. In addition, we established the fusion protein construct OPTN-TagRFP that was injected into embryos in the presence or absence of OPTN-targeting morpholinos. We have found that OPTN- targeting MO was able to specifically knockdown the expression of TagRFP-tagged OPTN fusion proteins in a dose-dependent manner. These
data may be useful for studying early POAG pathophysiology.
