Axonal mtDNA Copy Number Correlates With the Neurodegenerative Pattern in Optic Nerve Cross-Sections of Leber’s Hereditary Optic Neuropathy Patients

Abstract

Purpose: : To describe the correlation between axonal mtDNA amount and pattern of neurodegeneration in optic nerve cross-sections from control and Leber’s hereditary optic neuropathy (LHON) individuals. LHON, a blinding disorder due to mtDNA point mutations affecting complex I, is characterized by selective neurodegeneration of retinal ganglion cells (RGCs). Increased mitochondrial biogenesis is described as a compensatory mechanism in LHON and mtDNA copy number may be relevant for RGCs loss.

Methods: : We investigated 12 eyes from six controls and 4 eyes from two LHON/11778 patients, collected at autopsy. Retrobulbar optic nerve 5 µm thick cross-sections were stained with Luxol Fast Blue and nerve bundles from temporal, superior, nasal, inferior, and central regions were microdissected by laser capturing. Microdissected areas were measured (µm²) and subjected to histomorphometric analyses (Metamorph 6.0) to count glial cells. Total DNA was extracted and mtDNA amount was quantified by Real-Time-PCR. The number of mtDNA molecules were normalized as mtDNA copy/µm³ (area x 5 µm) or as mtDNA copy/nucleus (glial cells). Pattern of degeneration was evaluated in the LHON optic nerves by immunostaining with antibodies against myelin basic protein and by paraphenylenediamine staining.

Results: : Content of mtDNA per µ³ in temporal and central regions was significantly lower (p<0.05) than the superior, nasal and inferior regions. Similar results were observed for mtDNA/nucleus ratio. These results fit the profile of neurodegeneration seen in optic nerve cross-section from the LHON patients, where a "butterfly-like" pattern of axonal loss affects first the temporal quadrant, eventually expanding into the superior/inferior quadrants and leaving the nasal the most spared.

Conclusions: : This study demonstrates that the papillomacular bundle axons of the temporal quadrant are the most vulnerable having the lowest amount of mtDNA. These observations support the proposal that mtDNA copy number is relevant to neurodegeneration of RGCs and increase of mitochondrial biogenesis may be a successful compensatory mechanism.

Keywords: neuro-ophthalmology: optic nerve • mitochondria • genetics