The investigation of axonal pathology in the cerebellum of patients with mitochondrial disease

Abstract

Cerebellar ataxia affects 68% of adult patients with mitochondrial disease and is associated with progressive loss of co-ordination, impaired balance, and speech difficulties. In these patients, the cerebellum shows numerous neuropathological changes, and a prominent feature is the appearance of axonal torpedoes which represent swollen axons from Purkinje cells. Axonal torpedoes occur in the proximal portion of the Purkinje cell axon projecting into the granular cell layer and are mainly comprised of hyper phosphorylated neurofilament H. Although they have been reported in mitochondrial disease, their significance and contribution to disease is not known. Immunohistochemistry and immunofluorescence was used to characterise and quantify axonal torpedoes in the cerebellums of ten patients with mitochondrial disease and fourteen controls. A triple immunofluorescent assay was developed to reliably quantify the level of respiratory chain protein expression in axonal torpedoes compared to Purkinje cell bodies and their axons. A major limitation of current immunofluorescent techniques is the ability to use very thin brain sections (5μm) due to the intrinsic properties of the tissue causing scattering of light emitted from the fluorophores reducing the resolution of the image. To overcome this limitation, I have optimised the novel clearing technique known as CLARITY (Clear, Lipid-exchanged, Acrylamide-hybridized Rigid, Imaging/immunostaining compatible, Tissue hYdrogel) to use on both mouse and human cerebellar sections from both control individuals and patients. The optimisation of CLARITY has allowed for the use of 250μm thick cerebellar sections to further characterise the morphology of axonal torpedoes in 3D volume as well as determining the degree of axonal changes between patients and controls on a global scale. This study provides a detailed characterisation of axonal pathology that occurs in the cerebellum of patients with mitochondrial disease. The success in optimising the clearing method that produces quality staining of neuronal structures in thick (250μm) cerebellar tissue from both mouse and human tissue will allow for further investigation of changes in the vascular, dendritic or axonal networks in 3D volume on a global scale.