miRNA Changes in retinal ganglion cells after optic nerve crush and glaucomatous damage

The purpose of this study was to characterize the miRNA profile of purified retinal ganglion cells (RGC) from healthy and diseased rat retina. Diseased retina includes those after a traumatic optic nerve crush (ONC), and after ocular hypertension/glaucoma. Rats were separated into four groups: healthy/intact, 7 days after laser-induced ocular hypertension, 2 days after traumatic ONC, and 7 days after ONC. RGC were purified from rat retina using microbeads conjugated to CD90.1/Thy1. RNA were sequenced using Next Generation Sequencing. Over 100 miRNA were identified that were significantly different in diseased retina compared to healthy retina. Considerable differences were seen in the miRNA expression of RGC 7 days after ONC, whereas after 2 days, few changes were seen. The miRNA profiles of RGC 7 days after ONC and 7 days after ocular hypertension were similar, but discrete miRNA differences were still seen. Candidate mRNA showing different levels of expression after retinal injury were manipulated in RGC cultures using mimics/AntagomiRs. Of the five candidate miRNA identified and subsequently tested for therapeutic efficacy, miR-194 inhibitor and miR-664-2 inhibitor elicited significant RGC neuroprotection, whereas miR-181a mimic and miR-181d-5p mimic elicited significant RGC neuritogenesis

Transgenic mice expressing the Tyr437His mutant of human myocilin protein develop glaucoma

PURPOSE. To developed a genetic mouse model of primary open-angle glaucoma induced by expression of mutated human myocilin in transgenic mice and to test whether expression of mutated human myocilin in the eye angle structures produces more significant damage to the eye than does mutated mouse myocilin. METHODS. Recombineering in Escherichia coli was used to introduce the Tyr437His point mutation into a BAC carrying the full-length human MYOCILIN (MYOC) gene and long flanking regions. This BAC was used to produce transgenic mice. The expression of myocilin in the iridocorneal angle tissues and aqueous humor was studied by immunohistochemistry and Western blot analysis. Intraocular pressure was measured noninvasively with a fiber optic transducer. Retinal ganglion cells were retrograde labeled with fluorescent gold, and counted 5 days after labeling. RESULTS. BAC transgenic mice expressed elevated levels of myocilin in tissues of the iridocorneal angle. Expression of mutated myocilin induced its intracellular accumulation and prevented secretion of both mutated and wild-type myocilin into the aqueous humor. Transgenic mice demonstrated a moderate elevation of intraocular pressure, which was more pronounced at night than in daytime. In the peripheral retina, transgenic mice lost 20% of the retinal ganglion cells and 55% of large retinal ganglion cells. Axonal degeneration was observed at the periphery of the optic nerve. CONCLUSIONS. Expression of equivalent levels of mutated human or mouse myocilin in the eyes of transgenic mice produce comparable pathologic changes that are similar to those observed in patients with glaucoma. (Invest Ophthalmol Vis Sci. 2008;49:1932-1939) DOI:10.1167/iovs.07-1339 G laucoma, a group of neurodegenerative disorders, is one of the leading causes of blindness in the world. It is characterized by the death of retinal ganglion cells, the degeneration of axons in the optic nerve, and a specific deformation of the optic nerve head. In glaucoma, peripheral vision declines first, whereas the loss of central vision occurs much later. Elevated intraocular pressure (IOP) is one of the main risk factors in glaucoma, but it is still not known how elevated IOP kills retinal ganglion cells. Primary open-angle glaucoma is the most common form of glaucoma and will affect more than 60 million people and blind approximately 4.5 million worldwide by 2010. 1 Despite the high frequency and severity of glaucoma, little is known about the molecular mechanisms underlying the pathologic effects of glaucoma. It is now well established that a genetic component may contribute to glaucoma, and several glaucoma-associated genes have been identified. The first identified and the most studied gene is myocilin (MYOC), which is heavily expressed in and secreted by the trabecular meshwork, 2-4 one of the key components of the eye's aqueous humor outflow system. The myocilin protein belongs to a family of glycosylated proteins containing a C-terminal olfactomedin domain. 5 Dominant mutations in MYOC are found in 3% to 4% of patients with primary open-angle glaucoma, 7-9 Mutant myocilin protein is not secreted and actually blocks the secretion of nonmutated myocilin. 10 -12 It has been suggested that the intracellular accumulation of myocilin aggregates is deleterious to the trabecular meshwork cells and leads to the deterioration of their function and subsequent elevation of IOP. 13,14 Among the identified mutations, the Tyr437His mutation in the olfactomedin domain of myocilin is associated with a severe form of glaucoma, for which the average age at diagnosis is 20 years. 15 Animal models of glaucoma represent a useful tool for studying the molecular mechanisms of glaucoma, and several genetic mouse models of glaucoma have been developed. Mice deficient in the glutamate transporters Glast or Eaac1 demonstrate retinal ganglion cell and optic nerve degeneration without elevated IOP. 16 DBA/2J mice develop a progressive form of secondary angle-closure glaucoma resembling pigmentary dispersion glaucoma in humans. 17 A transgenic mouse strain with a targeted mutation in the gene for the ␣1 subunit of collagen type I demonstrates a gradual elevation of IOP and progressive optic nerve axon loss. 19 Expression of mutated myocilin in the iridocorneal angle and sclera induced its accumulation in cell cytoplasm and prevented its secretion into the extracellular space. Transgenic mice demonstrated a moderate elevation of intraocular pressure, the loss of ϳ20% of the retinal ganglion cells in the peripheral retina, and axonal degeneration in the optic nerve. Although mouse and human myocilin proteins have many similarities, 22 It has been suggested that expression of mutated human myocilin in the eyes of transgenic mice produces more dramatic effects than expression of mutated mouse myocilin and From th

Olfactomedin 2: Expression in the Eye and Interaction with Other Olfactomedin Domain–Containing Proteins

This study reports the expression of olfactomedin 2 during the course of mouse and rat eye development and its interaction with other olfactomedin family members

Changes in mRNA levels of the Myoc/Tigr gene in the rat eye after experimental elevation of intraocular pressure or optic nerve transection. Invest Ophthalmol Vis Sci.

PURPOSE. To isolate the rat Myoc/Tigr gene and investigate changes in its expression pattern in normal eyes and in eyes with either pressure-induced optic nerve damage or optic nerve transection. METHODS. Expression pattern of the rat Myoc/Tigr gene was investigated by Northern blot hybridization. Optic nerve damage and death of ganglion cells in the retina were induced unilaterally, by injection of hypertonic saline solution, episcleral vein cauterization, or optic nerve transection. The levels of mRNA for Myoc/Tigr were compared between several tissues of the control and surgically altered eyes, by using semiquantitative RT-PCR, real-time PCR, and Northern blot analysis. RESULTS. The rat Myoc/Tigr gene is 10 kb long and contains three exons. Among the eye tissues analyzed, Myoc/Tigr mRNA was detected in the combined tissues of the eye angle, sclera, cornea, retina, and optic nerve head. With pressure-induced optic nerve degeneration, the level of Myoc/Tigr mRNA decreased in the retina and the combined tissues of the eye angle, but increased in the optic nerve head. After optic nerve transection, the level of Myoc/Tigr mRNA increased in the retina, but did not change in the combined tissues of the eye angle. CONCLUSIONS. The decreased level of Myoc/Tigr mRNA in the retina after induction of elevated intraocular pressure compared with that in the control retina cannot be explained by ganglion cell death alone. Differences in Myoc/Tigr mRNA levels in eye tissues after elevation of intraocular pressure or optic nerve transection may reflect the activation of different signaling pathways involved in regulation of this gene. (Invest Ophthalmol Vis Sci. 2001;42:3165-3172

Myocilin Is a Modulator of Wnt Signaling▿

It is well documented that mutations in the MYOCILIN gene may lead to juvenile- and adult-onset primary open-angle glaucoma. However, the functions of wild-type myocilin are still not well understood. To study the functions of human myocilin and its two proteolytic fragments, these proteins were expressed in HEK293 cells. Conditioned medium from myocilin-expressing cells, as well as purified myocilin, induced the formation of stress fibers in primary cultures of human trabecular meshwork or NIH 3T3 cells. Stress fiber-inducing activity of myocilin was blocked by antibodies against myocilin, as well as secreted inhibitors of Wnt signaling, secreted Frizzled-related protein 1 (sFRP1) or sFRP3, and β-catenin small interfering RNA. Interaction of myocilin with sFRP1, sFRP3, and several Frizzled receptors was confirmed by immunoprecipitation experiments and by binding of myocilin to the surface of cells expressing cysteine-rich domains of different Frizzled and sFRPs. Treatment of NIH 3T3 cells with myocilin and its fragments induced intracellular redistribution of β-catenin and its accumulation on the cellular membrane but did not induce nuclear accumulation of β-catenin. Overexpression of myocilin in the eye angle tissues of transgenic mice stimulated accumulation of β-catenin in these tissues. Myocilin and Wnt proteins may perform redundant functions in the mammalian eye, since myocilin modulates Wnt signaling by interacting with components of this signaling pathway