Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function

Abstract

Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ataxias and other movement disorders marked by progressive degeneration of the cerebellum. The goal of this protocol is to assess mitochondrial dysfunction in Spinocerebellar ataxia type 1 (SCA1) and assess the efficacy of pharmacological targeting of metabolic respiration via the water-soluble compound succinic acid to slow disease progression. This approach is applicable to other cerebellar diseases and can be adapted to a host of water-soluble therapies. Ex vivo analysis of mitochondrial respiration is used to detect and quantify disease-related changes in mitochondrial function. With genetic evidence (unpublished data) and proteomic evidence of mitochondrial dysfunction in the SCA1 mouse model, we evaluate the efficacy of treatment with the water-soluble metabolic booster succinic acid by dissolving this compound directly into the home cage drinking water. The ability of the drug to pass the blood brain barrier can be deduced using high performance liquid chromatography (HPLC). The efficacy of these compounds can then be tested using multiple behavioral paradigms including the accelerating rotarod, balance beam test and footprint analysis. Cytoarchitectural integrity of the cerebellum can be assessed using immunofluorescence assays that detect Purkinje cell nuclei and Purkinje cell dendrites and soma. These methods are robust techniques for determining mitochondrial dysfunction and the efficacy of treatment with water-soluble compounds in cerebellar neurodegenerative disease.

Short-term succinic acid treatment mitigates cerebellar mitochondrial OXPHOS dysfunction, neurodegeneration and ataxia in a Purkinje-specific spinocerebellar ataxia type 1 (SCA1) mouse model

“…Mitochondrial respiration experiments were adapted from Kuznetsov, et al [ 34 ]; our methodology for this study is described elsewhere [ 35 ]. Briefly, whole cerebella from treated and untreated FVB and B05 mice were quickly extracted from CO 2 -asphyxiated wild type and B05 mice (treated and untreated) under IACUC guidelines and prepared in homogenate buffer (0.25 M sucrose, 0.5 mM EDTA, 50 mM Tris-HCl, pH 7.4), and added to the calibrated respirometer set to 30°C (Oxytherm System, Hansatech Instruments, UK).…”

“…HPLC respiration experiments were adapted from Ergönül and Nergiz [ 36 ]; our adapted methodology for this study is described in detail elsewhere [ 35 ]. In brief, PBS-washed cerebellar, cortical, liver and kidney tissue from treated and untreated FVB and B05 mice were finely minced in a chilled dounce homogenizer in 75:25 water: methanol, by volume.…”

“…Molecular layer thickness was measured in Image J by scorers that were blinded to the genotype and treatment conditions. Two designated 200 μm stretches of primary fissure along the Purkinje layer were identified in each image, and three random measurements were made within each 200 μm stretch from the proximal end of the Purkinje dendritic arbor at the base of the soma to the distal end of the dendritic arbor [ 35 , 37 ]. Six measurements from three cerebellar sections per animal were averaged and expressed as the mean ± SEM.…”

“…To analyze Purkinje cell counts, cerebellar sections from A02 transgenic mice and B05 transgenic mice (treated and untreated) were prepared as described above, incubated in 11NQ anti-ATXN1 antibody (1:2000) [ 9 ], and detected with 1:500 Alexa 594-goat anti-rabbit secondary antibody (ThermoFisher Scientific). Following confocal imaging of primary fissures, two designated 200 μm stretches along the Purkinje layer were identified, and the number of ATXN1-positive nuclei within each stretch were counted manually [ 35 ] by blinded scorers. Six measurements from three cerebellar sections per animal were averaged and expressed as the mean number of soma ± SEM.…”

“…Gait assessment was measured by labeling hind feet of mice with blue or red non-toxic water-based paint, placing subjects on a paper-lined runway and allowing them to walk towards a goal box, as described in detail previously [ 33 , 35 ]. Successful trials were defined as a minimum of five sequential footprints in which the subject walked towards the goal box without turning or stopping [ 35 ]. Step length, gait width, alternation coefficient, and linear movement were assessed from the footprints as described previously [ 33 ] by blinded scorers.…”