Increase in mitochondrial DNA mutations impairs retinal function and renders the retina vulnerable to injury Yu X. G. Kong, 1 Nicole Van Bergen, 1 Ian A. Trounce, 1,2 Bang V. Bui, 3 Vicki Chrysostomou, 1 Hayley Waugh, 1 Algis Vingrys 3 and Jonathan G. Crowston 1,2 1 Centre for Eye Research Australia, University of Melbourne, Victoria, Australia 2 Royal Victorian Eye and Ear Hospital, Victoria, Australia 3 Department of Optometry & Vision Sciences, University of Melbourne, Victoria, Australia Summary Mouse models that accumulate high levels of mitochon- drial DNA (mtDNA) mutations owing to impairments in mitochondrial polymerase c (PolG) proofreading function have been shown to develop phenotypes consistent with accelerated aging. As increase in mtDNA mutations and aging are risk factors for neurodegenerative diseases, we sought to determine whether increase in mtDNA muta- tions renders neurons more vulnerable to injury. We therefore examined the in vivo functional activity of reti- nal neurons and their ability to cope with stress in trans- genic mice harboring a neural-targeted mutant PolG gene with an impaired proofreading capability (Kasahara, et al. (2006) Mol Psychiatry 11(6):577–93, 523). We confirmed that the retina of these transgenic mice have increased mtDNA deletions and point mutations and decreased the expression of mitochondrial oxidative phosphorylation enzymes. Associated with these changes, the PolG trans- genic mice demonstrated accelerated age-related loss in retinal function as measured by dark-adapted electroreti- nogram, particularly in the inner and middle retina. Fur- thermore, the retinal ganglion cell–dominant inner retinal function in PolG transgenic mice showed greater vulnera- bility to injury induced by raised intraocular pressure, an insult known to produce mechanical, metabolic, and oxi- dative stress in the retina. These findings indicate that an accumulation of mtDNA mutations is associated with impairment in neural function and reduced capacity of neurons to resist external stress in vivo, suggesting a potential mechanism whereby aging central nervous sys- tem can become more vulnerable to neurodegeneration. Key words : aging; electroretinogram; glaucoma; mito- chondria; polymerase gamma retina. Introduction Aging is associated with an increase in mitochondrial DNA (mtDNA) mutations in the form of both deletions (Cortopassi & Arnheim, 1990; Arnheim & Cortopassi, 1992; Corral-Debrinski et al., 1992; Simonetti et al., 1992; Khrapko & Vijg, 2007) and point mutations (Simon & Lin, 2004; Cantuti-Castelvetri et al., 2005). This is particularly pronounced in postmitotic tissues with high levels of energy requirement such as the brain and heart (Cortopassi et al., 1992). Impaired mitochondrial function con- sequent to mtDNA abnormalities have been determined in age- related neurodegenerative disorders, such as Parkinsons’s disease (Schnopp et al., 1996), Alzheimer’s disease (Coskun et al., 2004), and optic neuropathies (Carelli et al., 2004) includ- ing glaucoma (Abu-Amero et al., 2006). However, the in vivo effects of age-related increases in mitochondrial DNA mutations on neuronal activity and the ability for neurons to cope with injury have not been studied in detail. Mitochondrial DNA mutations can arise from errors in DNA replication. One enzyme central to the process of mtDNA repli- cation is polymerase c (PolG; Kaguni, 2004; Graziewicz et al., 2006), which consists of a catalytic subunit with polymerase and exonuclease activity and a small accessory subunit that enhances binding (Lim et al., 1999). The exonuclease activity proofreads nascent mtDNA and ensures faithful replication of the mitochondrial genome (Longley et al., 2001). In humans, mutations in PolG have been shown to result in mtDNA insta- bility causing chronic progressive external ophthalmoplegia and other neurodegenerative diseases (Graziewicz et al., 2006; Horvath et al., 2006). Mice carrying systemic mutations in the exonucleolytic proofreading domain of PolG (Kujoth et al., 2005; Trifunovic et al., 2005) exhibit early senescence, includ- ing signs of osteopenia, alopecia, infertility, cardiomyopathy, and early death. These mice also demonstrate auditory system dysfunctions consistent with age-related hearing loss (Niu et al., 2007). The phenotype is associated with defects in the mitochondrial oxidative phosphorylation (OXPHOS) pathway (Edgar et al., 2009) and increased apoptosis (Kujoth et al., 2005; Dai et al., 2010). These models have increased levels of both mtDNA deletions and point mutations, and controversy continues as to which class of mutations may be most respon- sible for the observed pathology (Khrapko et al., 2006; Ver- mulst et al., 2007, 2009; Kraytsberg et al., 2009; Edgar et al., 2010). A C E L 6 9 0 B Dispatch: 1.3.11 Journal: ACEL CE: Sindhuja Journal Name Manuscript No. Author Received: No. of pages: 12 PE: Ramya Correspondence Professor Jonathan G. Crowston, Department of Ophthalmology, University of Melbourne, Parkville, 3010, Victoria, Australia. Tel.: +613 9929 8429; fax: +613 9662 3859; e-mail: crowston@unimelb.edu.au Accepted for publication 8 February 2011 ª 2011 The Authors Aging Cell ª 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 1 Aging Cell (2011) pp1–12 Doi: 10.1111/j.1474-9726.2011.00690.x 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55