Abstract
Purpose: :
Glaucoma is a neurodegenerative disease of the eye and optic nerve, characterized by retinal ganglion cell (RGC) axonal degeneration and eventual soma loss in the retina. As glaucoma progresses, axonal transport is disrupted, interfering with mitochondrial transport. An inability for mitochondria to freely move and remodel may contribute to degeneration and progression of glaucoma. We propose that disease-induced metabolic challenges create imbalance of mitochondrial fusion proteins in specific cellular compartments, which, combined with compromised axonal transport, contributes to increased mitochondrial dysfunction.
Methods: :
Measures of protein levels, RNA levels, and cell-specificity of mitofusin (Mfn) are evaluated. Protein is from pooled samples for Western blot analysis. NanoPro (NP) quantifies protein from one-animal sample pools. Our new development of NP assays allows for probing of Mfn1, Mfn2 and ubiquitin expression on an animal-by-animal basis. RNA transcript levels are assessed from pooled samples. Laser-capture microdissection (LCM) will isolate in vivo RGCs and be used with the NP for determining cell-specific Mfn and ubiquitin expression. In vitro cultures of RGCs exposed to high pressure will be compared with LCM-isolated RGCs and assessed for damage done to the fusion proteins and resultant mitochondrial behavioral changes.
Results: :
Western blot shows higher levels of the fusion proteins Mfn1 and Mfn2 in retina, optic nerve (ON) and superior colliculus (SC) between control and age-matched diseased tissues.Mfn1 RNA expression is steady in both retina and SC, with expression in ON trending to increased expression with age in control, while decreasing with age and disease. In Mfn2, the same trend was apparent in the ON, while expression in control retinal tissue was steady, but significantly lower in disease.NP shows differences in Mfn2. In diseased ON, a dramatic decrease in the amount of Mfn2 is seen, while retinal levels dramatically increase in disease.
Conclusions: :
Analyses of RNA expression shows transcript changes in ON and retinal tissue in conjunction with both age and disease. Analysis of protein expression changes show mitofusin protein levels are directly affected by the progression of glaucoma in the ON and retina. There is adequate Mfn2 present in the RGCs, but it is not being transported down the axon to where it is needed.
Keywords: mitochondria • ganglion cells • protein purification and characterization