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

Summary

We present a biochemical and behavioral protocol to evaluate the efficacy of mitochondria-targeted water-soluble compounds for the treatment of Spinocerebellar ataxia type 1 (SCA1) and other cerebellar neurodegenerative diseases.

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 With genetic evidence (unpublished data) and proteomic evidence of mitochondrial dysfunction in the SCA1 mouse model, we evaluate the efficacy of treatment with the water-soluble metabolic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.

Introduction

Mitochondria are the key producers of adenosine triphosphate (ATP), an essential coenzyme for cellular energy, with the majority of mitochondrial ATP produced through oxidative phosphorylation (OXPHOS) using the electron transport chain. The brain, given its high metabolic demands and its reliance on oxidative phosphorylation for powering neural activity, is highly susceptible to mitochondrial dysfunction. As a result, mitochondrial dysfunction is triggered during the aging process ¹ and is implicated in the pathogenesis of multiple neurodegenerative diseases ^2,3,<....

Protocol

This protocol follows the IACUC guidelines at Skidmore College for working with mice.

1. Treatment with Water-soluble Compounds

1. Dissolve succinic acid to a concentration of 0.75 mg/mL in cage drinking water. Note that any water-soluble compound of interest at the desired concentration could be substituted at this stage. Stir solution to make sure that the compound is fully dissolved.
2. After mice reach the desired age of treatment, replace the home cage drinking water of the treatment condition group with the solution in step 1.1.
3. Measure the weight of both treatment and control group water bottles daily to ensure that both treated....

Results

Through pharmacological targeting of cerebellar mitochondria with succinic acid we are able to prevent mitochondrial dysfunction in a mouse model of the cerebellar neurodegenerative disease SCA1. The canonical electron donor of succinate dehydrogenase, succinic acid, was dissolved in home cage drinking water of SCA1 mice for one month, with behavioral assessment beginning during the second week of treatment and neuropathological assessment following treatment (Figure .......

Discussion

If these methods are used as described, they are capable of detecting and alleviating oxidative phosphorylation-mediated mitochondrial dysfunction in cerebellar neurodegenerative disease mice models. The combined biochemical and behavioral assays are multifaceted methods for determining the extent of mitochondrial contribution to cerebellar neurodegenerative disease pathology. By treating mice with succinic acid to stimulate metabolism and boost mitochondrial function, we are able to show a rescue of cerebellar behaviora.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adenosine diphosphate	Sigma Aldrich	A2754	ADP
Ascorbate	Sigma Aldrich	A7631
Bovine serum albumin	Sigma Aldrich	A2153	BSA
4',6-Diamidino-2-phenylindole	Sigma Aldrich	D9542	DAPI
Digitonin	Sigma Aldrich	D141
Dithiothreitol	Sigma Aldrich	D0632	DTT
Donkey serum	Sigma Aldrich	D9663
Glutamate	Sigma Aldrich	1446600
Malate	Sigma Aldrich	6994
Mannitol	Sigma Aldrich	M4125
Paraformaldehyde	Sigma Aldrich	P6148
Potassium-lactobionate	Bio-Sugars	69313-67-3
Rotenone	Sigma Aldrich	R8875
Saponin	Sigma Aldrich	47036
Succinic Acid	Sigma Aldrich	S3674
N,N,N′,N′-Tetramethyl-p-phenylenediamine	Sigma Aldrich	T7394	TMPD
Triton X-100	Sigma Aldrich	T9284
Urea	Sigma Aldrich	U0631
Vectashield mounting medium	Vector Labs	H-1000
Antibodies
11NQ antibody (anti-ataxin-1 )			Servadio, et al. 1995, PMID: 7647801
Alexa Fluor 488 anti-mouse secondary antibody	Life Technologies	A-11015
Alexa Fluor 594 anti-rabbit secondary antibody	Life Technologies	A-11012
Calbindin antibody (goat)	Santa Cruz	C-20
Animals
Control transgenic mice	Harry Orr, Ph.D.	A02	Burright, et al. 1997, PMID: 9217978
SCA1 mice	Harry Orr, Ph.D.	B05	Burright, et al. 1997, PMID: 9217978
Wildtype mice	The Jackson Laboratory	001800
Equipment
ESM-100L microtome	ERMA		Sledge microtome
Fluoview FV1200 Confocal Microscope	Olympus
Glycerol-gelatin slides	FD Neuro Technologies	PO101
Hamilton syringe	Sigma Aldrich	VCAT 80465
OXYT1 Oxytherm Electrode Control Unit	Hansatech Instruments
P.T.F.E. paper	Cole-Parmer	UX-08277-15
Rotallion Rotarod	PPP&G		contact corresponding author for information
Ultimate 3000 HPLC	Dionex
Software
ImageJ	National Institute of Health		http://imagej.nih.gov/ij/
Cell counter plugin (for ImageJ)	National Institute of Health		http://rsb.info.nih.gov/ij/plugins/cell-counter.html
3P&G Rota-Rod v3.3.3 (rotarod software)	PPP&G		contact corresponding author for information
Phidget21.dll (required for rotarod software)	DLL-Files.com		https://www.dll-files.com/phidget21.dll.html