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.
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 With genetic evidence (unpublished data) and proteomic evidence of mitochondrial dysfunction in the SCA1 mouse model, we evaluate the efficacy of treatment with the water-soluble metabolic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.
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 2,3,4. Therefore, it follows that mitochondria are attractive therapeutic targets for neurodegeneration.
In this protocol, we have adopted the use of Spinocerebellar ataxia type 1 (SCA1) as a model neurodegenerative disease for the study of mitochondrial dysfunction and the development of mitochondrial-targeted therapies. SCA1 is caused by a polyglutamine (polyQ) repeat expansion mutation in the ataxin-1 gene product which triggers progressive degeneration of the Purkinje neurons of the cerebellum and neurons of other brain regions. The transgenic mouse line used here (designated as the SCA1 mouse), which expresses a polyQ-mutant ataxin-1 transgene under the control of a Purkinje-cell specific promoter, allows for the targeted analysis of the Purkinje-cell component of SCA1 5. SCA1 mice undergo gradual Purkinje cell degeneration and develop ataxic gait 6.
Mitochondrial complex dysfunction and mitochondrial-targeted treatment efficacy can be evaluated with a battery of molecular and behavioral assays. Mitochondrial complex dysfunction is measured ex vivo by respiration assays that detect altered oxygen consumption within cerebellar tissue in the presence of electron transport chain substrates and inhibitors 7. Respiration assays have previously been used with permeabilized tissue, mitochondrial isolates, and whole tissue 7,8,9. They allow for direct assessment of mitochondrial function unlike morphological data collection methods such as transmission electron microscopy or immunofluorescence staining. The use of whole tissue rather than isolated mitochondria prevents biased selection of healthy mitochondria that may occur during the isolation process 7. When adapted to the protocol as shown, the respiration assay is a valuable method for detecting mitochondrial dysfunction in cerebellar neurodegenerative disease states.
Non-specific activators of metabolism can be used to infer mitochondrial dysfunction in transgenic mice models of neurodegenerative disease and aid in the development of new therapies. Quercetin, coenzyme q10 and creatine have all been shown to ameliorate neurodegenerative disease pathology in patients and in animal models of neurodegenerative disease 10,11,12,13,14,15,16. Here we present a novel metabolic activator, succinic acid, to stimulate metabolism and boost mitochondrial function in neurodegenerative disease. To ensure that the activator is crossing the blood brain barrier, HPLC was employed to detect delivery to neural tissue in treated mice 17.
To evaluate the therapeutic effects of metabolically targeted water soluble compounds such as succinic acid, a battery of behavioral paradigms and immunopathological studies can be used. Due to the motor coordination deficits found in cerebellar neurodegenerative disease, the footprint runway assay, beam assay and accelerating rotating rod assay are used to detect rescue of behavioral pathology 6, 18, 19. These measures are supplemented with immunopathological assessment of cerebellar cytoarchitecture by assessing molecular layer thickness (defined as Purkinje cell dendritic arbor length) and Purkinje cell soma counts within a defined lobule of cerebellar tissue 6, 20, 21. Here we present multiple neuropathological and behavioral methods for detection and treatment of mitochondrial dysfunction with metabolically targeted water soluble compounds.
We use ex vivo analysis of mitochondrial respiration to analyze mitochondrial dysfunction in the SCA1 transgenic mouse. Furthermore, we show that disease symptoms and pathology are improved by the water-soluble mitochondrial booster succinic acid, further implicating mitochondrial dysfunction in SCA1 disease progression.
This protocol follows the IACUC guidelines at Skidmore College for working with mice.
1. Treatment with Water-soluble Compounds
2. Footprint Analysis
3. Beam Analysis
4. Accelerating Rotarod
5. Cerebellar Extraction and Fixation
6. Immunopathology Assay
7. Quantifying Molecular Layer Thickness and Purkinje Cell Numbers
8. HPLC Analysis of Cerebellar Succinic Acid Concentrations Post-treatment
9. Ex Vivo Analysis of Cerebellar Mitochondrial Functionality Using an Oxygen Electrode Control Unit
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 1A). Succinic acid treatment 22 improves performance of SCA1 mice as measured by the footprint assay, beam assay and accelerating rotarod assay. The behavioral improvements detected indicates enhanced motor coordination and motor learning (Figure 1B-D).
Rescue of behavioral performance was found in concert with a preservation of cerebellar tissue (Figure 2). Succinic acid treatment significantly preserved Purkinje cell bodies and increased molecular layer thickness (indicative of Purkinje cell dendritic arbor extension) in the cerebellar primary fissure in treated SCA1 mice compared to untreated SCA1 mice. Purkinje cell dendritic length and Purkinje cell body counts provide strong measures of overall cerebellar cytoarchitecture 5.
HPLC was performed on the cerebellar tissue from treated mice to verify that succinic acid dissolved in the cage drinking water successfully crossed the blood brain barrier and penetrated cerebellar tissue. A dose response curve varying the length of treatment further supports ad libitum treatment of succinic acid as a viable way of reaching cerebellar tissue (Figures 3A-B). Our respiration experiments are on-going and our results will be published in a research manuscript. Here we show that we can successfully record the rate of respiration through cerebellar oxidative phosphorylation complexes upon addition of varying electron transport chain substrates and inhibitors (Figure 3C). Ultimately, we will use the respiration assay to show oxidative phosphorylation deficits in SCA1 mouse cerebellum and the reversal of those deficits by succinic acid treatment.
Figure 1: Treatment Scheme and Representative Data from the Footprint Assay, Beam Assay and Accelerating Rotarod.(A) Succinic acid was dissolved in home cage drinking water of SCA1 mice for four weeks beginning at 17 weeks of age. SCA1 mice begin to display the ataxia phenotype at 5 weeks of age. Not depicted in the schematic are three control cohorts: succinic acid-treated wildtype mice, vehicle-treated SCA1 mice and vehicle-treated wildtype mice. Behavioral assessments were conducted during the treatment period as follows: footprint assay during week 17, beam assay during week 18 and accelerating rotarod during week 19. At 20 weeks of age, mice were sacrificed and cerebellar tissue was harvested for immunopathology assays, HPLC assays and respiration assays. (B) Representative data from the footprint assay showing a vehicle-treated wildtype gait (top), vehicle-treated SCA1 gait (middle) and a succinic acid-treated SCA1 gait (bottom). (C) Beam analysis data showing improvement in beam performance (measured as number of trials for a successful run) of SCA1 mice with succinic acid treatment. (* p < 0.05). (D) Succinic acid treatment significantly improves accelerating rotarod performance of SCA1 22 mice as shown by increased latency to fall (* p < 0.05). Error bars in C-D reflect the standard error of the mean. Significance was determined by one-way ANOVA and multiple comparisons post-hoc analysis. Please click here to view a larger version of this figure.
Figure 2: Representative Data from the Purkinje Cell Counts and Molecular Layer Thickness Immunopathology Assays. (A) Representative results from the Purkinje cell counts assay showing a vehicle-treated control transgenic cerebellar primary fissure (top), vehicle-treated SCA1 cerebellar primary fissure (middle) and a succinic acid-treated SCA1 cerebellar primary fissure (bottom) stained for ATXN1-positive nuclei (red) and counterstained with DAPI (blue). White lines depict the 200 μm regions of tissue designated for cell counts. For this particular analysis, a control transgenic mouse over-expressing a non-pathogenic ATXN1 gene under control of a Purkinje cell-specific promoter was used instead of a wildtype mouse because the transgenic control features easily detectable levels of nuclear Purkinje ATXN1 and the wildtype mouse does not. The control transgenic mouse does not display an ataxic phenotype or cerebellar neurodegeneration. (B) Representative results from the molecular layer thickness assay showing a vehicle-treated wildtype cerebellar primary fissure (top), vehicle-treated SCA1 cerebellar primary fissure (middle) and a succinic acid-treated SCA1 cerebellar primary fissure (bottom) stained for calbindin, a marker of Purkinje cells (white). Two of the three cerebellar cell layers can be visualized with calbindin. The middle Purkinje cell layer (consisting of Purkinje cell bodies) is visible along with the outermost molecular layer (consisting of Purkinje cell dendrites). The molecular layer appears in the middle of the primary fissure due to the natural folds of the tissue. Please click here to view a larger version of this figure.
Figure 3: Representative Results from the HPLC and Respiration Assays. (A) Standard curve of known concentrations up to 250 ppm succinic acid diluted in acetate buffer and plotted against the area beneath the measured HPLC peak. The equation of the line was used to calculate unknown succinic acid concentrations from treated tissue samples. (B) Representative dose response succinic acid peaks from mouse cerebellar tissue following different times of treatment (either 0, 1 or 5 weeks). Under the conditions described, succinic acid typically eluted between 10 and 12 min. (C) Representative respiration run from vehicle-treated wildtype mouse cerebellar tissue. The blue line depicts the change in oxygen concentration, the dashed red line depicts the change in respiration rate and the solid red line depicts a smoothed version of the dashed line. D = addition of digitonin, G/M = addition of glutamate/malate, A = addition of ADP, R = addition of rotenone, S = addition of succinic acid, AA = addition of antimycin A and A/T = addition of Asc/TMPD. All substrates were added at the times depicted in the figure and at the concentrations described in the protocol (step 9.5). Please click here to view a larger version of this figure.
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 behavioral deficits and cerebellar degeneration.
The methodology presented here can be used to test a wide variety of water-soluble metabolically targeted compounds for potential treatment of cerebellar neurodegenerative disease. Succinic acid is readily soluble in water at 0.75 mg/mL which allows for simple delivery via cage drinking water and does not necessitate the creation of supplemented food pellets. Other water-soluble compounds can be easily substituted into the protocol. If using metabolically targeted compounds that are not water-soluble, our methodology can be readily adapted for treatment with supplemented food pellets 11, 12. When testing a new compound, it is critical to verify cerebellar penetration of the drug before commencing treatment via HPLC analysis or other suitable detection method.
The battery of pathological assessments was chosen due to their reported efficacy at detecting cerebellar neurodegenerative pathologies. The footprint assay, beam assay and rotarod assay are widely used assessments of cerebellar neurodegenerative disease including various SCAs, and Friedreich's ataxia 6, 23, 24. These particular behavioral assessments strongly correlate with cerebellar degeneration. However, other behavioral paradigms can be substituted or added as needed.
The labeling of Purkinje cells in SCA1 mice with ataxin-1 and calbindin is widely used to detect cerebellar degeneration in SCA1 models 5, 20, 21. Other neuronal-selective antibodies and tissue regions can be used to visualize the mitigation of neurodegeneration as needed.
Respiration analysis is a powerful tool for the specific detection of dysfunction or repair within individual mitochondrial complexes from cerebellar tissue. Care should be taken to minimize the time between harvesting the tissue and running the assay to achieve reproducible results. Also, it is important to note the homogeneity of the tissue being analyzed. Specifically, cerebellar tissue consists overwhelmingly of granule neurons which do not express the SCA1 transgene in our model. Therefore, a critical step of this protocol is to determine if whole cerebellar respiration is a viable method for detection of oxidative complex function. Respiration changes in the transgenic SCA1 mouse cerebellum may be subtle and may require increased numbers of subjects to achieve statistically significant results.
The combination of behavioral and neuropathological methods detailed in this protocol provide distinct quantification of SCA1 disease and can robustly detect improvement upon treatment with water-soluble compounds. The significance of this protocol is in its wide adaptability to various treatments and a diverse array of mouse models. Furthermore, many of the techniques used in this protocol do not require expensive equipment or complicated techniques and are therefore suitable for an undergraduate research setting. Future applications of this protocol will be used to assess the degree of treatment efficacy upon age-dependent delivery of the compound.
The authors have nothing to disclose.
We would like to thank Dr. Harry Orr at the University of Minnesota for his generous gift of transgenic mice. We would also like to thank the following Skidmore College alum for their work performing the preceding experiments: Monica Villegas, Porter Hall, Mitchell Spring, Nicholas Toker, Jenny Zhang, Chloe Larson and Cheyanne Slocum. Furthermore, we would like to thank Skidmore College for funding the development of these methods.
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 |