Comparative morphology of mitochondria in optic nerve and cell body of retinal ganglion cells in a mouse model of spastic paraplegia type 2

Abstract text (incl. figure legends and references)

Oligodendrocytes facilitate rapid action potential propagation by ensheathing central nervous system axons, thereby existing in a close metabolic relationship with the myelinated axon. This "axo-glial" unit critically depends on proper energy supply by axonal mitochondria to meet their high energy demands for saltatory action potential propagation. In neurodegenerative diseases such as PLP1-related disorders, axonal swellings together with alterations in morphology and function of axonal mitochondria serve as hallmarks for dys- and demyelinating disease states. So far it remained elusive how lack of proteolipid protein (PLP) in oligodendrocytes and myelin impairs axonal function, but a deficit in axonal energy supply due to mitochondrial dysfunction is one plausible explanation. Here we used retinal ganglion cells (RGCs) with their myelinated axons in the optic nerve as model tissue for studying axonal mitochondria in a Plp-deficient mouse model of spastic paraplegia type 2.

To investigate the influence of myelin on the local mitochondrial heterogeneity in RGC neurons we compared mitochondrial morphology in myelinated and non-myelinated parts of the axon. For this purpose, we developed a method to reliably target RGC mitochondria in distinct regions: the optic nerve, the optic nerve head (ONH), and the retinal ganglion cell body for electron microscopy and 3D reconstruction.

A hybrid method was established in combining chemical fixation with high-pressure freezing (HPF) and freeze substitution to obtain a well-preserved mitochondrial ultrastructure. HPF is a well-established method to study the ultrastructure of cells and tissue in a close-to-native state, but complex tissue requires dissection before freezing. To minimize handling artefacts and disturbance of tissue organization, prefixed tissue was sliced with a vibratome producing sections of required thickness, from which the most suitable sections were selected for HPF. Careful vibratome sectioning of the eye was indispensable for targeting the optic nerve head. For the structural investigation, we applied transmission electron microscopy (TEM) and electron tomography for 3D analysis of individual mitochondria.

Mitochondria in the RG cell body and optic nerve head region revealed very little morphologic difference between wild-type mice and Plp^−/y mice. However, we observed inclusions in RGC mitochondria of Plp^−/y mice, a finding that requires further investigation. At the ONH, mitochondrial clusters containing mitochondria with a round shape and mostly tubular cristae were prominent in both genotypes. The optic nerve revealed clear genotype-dependent changes in mitochondrial morphology. This is consistent with a recent study by Steyer et al. 2020, J Struc Biol 210(2):107492), in which 3D reconstruction of data sets obtained by focused ion beam scanning electron microscopy (FIB-SEM) showed that axons in Plp^−/y mice contain twice as many mitochondria per 10 µm axon, with the mitochondria additionally appearing shorter and rounder.

Overall, we were able to develop a method to reliably target the optic nerve head with attached retina and optic nerve to study mitochondrial heterogeneity in RGC axons in a mouse model of spastic paraplegia type 2. This will help us to better understand the consequences for axonal mitochondria architecture and function in Plp-deficiency.