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.

<ol>
	<li>Stucki, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+impairments+contribute+to+Spinocerebellar+ataxia+type+1+progression+and+can+be+ameliorated+by+the+mitochondria-targeted+antioxidant+MitoQ.">Mitochondrial impairments contribute to Spinocerebellar ataxia type 1 progression and can be ameliorated by the mitochondria-targeted antioxidant MitoQ.</a> Free Radic Biol Med. 97, 427-440 (2016).</li><li>Breuer, M. E., Willems, P. H., Russel, F. G., Koopman, W. J., Smeitink, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+mitochondrial+dysfunctions+in+the+brain,+from+mice+to+men.">Modeling mitochondrial dysfunctions in the brain, from mice to men.</a> J Inherit Metab Dis. 35 (2), 193-210 (2012).</li><li>Breuer, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mitochondrial+OXPHOS+dysfunction+in+the+development+of+neurologic+diseases.">The role of mitochondrial OXPHOS dysfunction in the development of neurologic diseases.</a> Neurobiol Dis. , (2012).</li><li>Hroudov&#225;, J., Singh, N., Fizar, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunctions+in+neurodegenerative+diseases,+relevance+to+Alzheimer's+disease.">Mitochondrial dysfunctions in neurodegenerative diseases, relevance to Alzheimer's disease.</a> Biomed Res Int. 2014, 175062 (2014).</li><li>Burright, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCA1+transgenic+mice,+a+model+for+neurodegeneration+caused+by+an+expanded+CAG+trinucleotide+repeat.">SCA1 transgenic mice, a model for neurodegeneration caused by an expanded CAG trinucleotide repeat.</a> Cell. 82 (6), 937-948 (1995).</li><li>Clark, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purkinje+cell+expression+of+a+mutant+allele+of+SCA1+in+transgenic+mice+leads+to+disparate+effects+on+motor+behaviors,+followed+by+a+progressive+cerebellar+dysfunction+and+histological+alterations.">Purkinje cell expression of a mutant allele of SCA1 in transgenic mice leads to disparate effects on motor behaviors, followed by a progressive cerebellar dysfunction and histological alterations.</a> J Neurosci. 17 (19), 7385-7395 (1997).</li><li>Kuznetsov, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+function+in+situ+in+permeabilized+muscle+fibers,+tissues+and+cells.">Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells.</a> Nat Protoc. 3 (6), 965-976 (2008).</li><li>Deng-Bryant, Y., Singh, I. N., Carrico, K. M., Hall, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effects+of+tempol,+a+catalytic+scavenger+of+peroxynitrite-derived+free+radicals,+in+a+mouse+traumatic+brain+injury+model.">Neuroprotective effects of tempol, a catalytic scavenger of peroxynitrite-derived free radicals, in a mouse traumatic brain injury model.</a> J Cereb Blood Flow Metab. 28 (6), 1114-1126 (2008).</li><li>Vaishnav, R. A., Singh, I. N., Miller, D. M., Hall, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+peroxidation-derived+reactive+aldehydes+directly+and+differentially+impair+spinal+cord+and+brain+mitochondrial+function.">Lipid peroxidation-derived reactive aldehydes directly and differentially impair spinal cord and brain mitochondrial function.</a> J Neurotrauma. 27 (7), 1311-1320 (2010).</li><li>Matthews, R. T., Yang, L., Browne, S., Baik, M., Beal, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coenzyme+Q10+administration+increases+brain+mitochondrial+concentrations+and+exerts+neuroprotective+effects.">Coenzyme Q10 administration increases brain mitochondrial concentrations and exerts neuroprotective effects.</a> Proc Natl Acad Sci U S A. 95 (15), 8892-8897 (1998).</li><li>Ferrante, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effects+of+creatine+in+a+transgenic+mouse+model+of+Huntington's+disease.">Neuroprotective effects of creatine in a transgenic mouse model of Huntington's disease.</a> J Neurosci. 20 (12), 4389-4397 (2000).</li><li>Ferrante, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+effects+of+coenzyme+Q10+and+remacemide+in+transgenic+mouse+models+of+Huntington's+disease.">Therapeutic effects of coenzyme Q10 and remacemide in transgenic mouse models of Huntington's disease.</a> J Neurosci. 22 (5), 1592-1599 (2002).</li><li>Hersch, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creatine+in+Huntington+disease+is+safe,+tolerable,+bioavailable+in+brain+and+reduces+serum+8OH2'dG.">Creatine in Huntington disease is safe, tolerable, bioavailable in brain and reduces serum 8OH2'dG.</a> Neurology. 66 (2), 250-252 (2006).</li><li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy+with+coenzyme+Q10+and+creatine+produces+additive+neuroprotective+effects+in+models+of+Parkinson's+and+Huntington's+diseases.">Combination therapy with coenzyme Q10 and creatine produces additive neuroprotective effects in models of Parkinson's and Huntington's diseases.</a> J Neurochem. 109 (5), 1427-1439 (2009).</li><li>Yang, X., Dai, G., Li, G., Yang, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coenzyme+Q10+reduces+beta-amyloid+plaque+in+an+APP/PS1+transgenic+mouse+model+of+Alzheimer's+disease.">Coenzyme Q10 reduces beta-amyloid plaque in an APP/PS1 transgenic mouse model of Alzheimer's disease.</a> J Mol Neurosci. 41 (1), 110-113 (2010).</li><li>Sandhir, R., Mehrotra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quercetin+supplementation+is+effective+in+improving+mitochondrial+dysfunctions+induced+by+3-nitropropionic+acid,+implications+in+Huntington's+disease.">Quercetin supplementation is effective in improving mitochondrial dysfunctions induced by 3-nitropropionic acid, implications in Huntington's disease.</a> Biochim Biophys Acta. 1832 (3), 421-430 (2013).</li><li>Ergonul, P. G., Nergiz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+organic+acids+in+olive+fruit+by+HPLC.">Determination of organic acids in olive fruit by HPLC.</a> 'Czech Food Sci. 28 (3), 202-205 (2010).</li><li>Jones, B. J., Roberts, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quantiative+measurement+of+motor+inco-ordination+in+naive+mice+using+an+acelerating+rotarod.">The quantiative measurement of motor inco-ordination in naive mice using an acelerating rotarod.</a> J Pharm Pharmacol. 20 (4), 302-304 (1968).</li><li>Luong, T. N., Carlisle, H. J., Southwell, A., Patterson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+motor+balance+and+coordination+in+mice+using+the+balance+beam.">Assessment of motor balance and coordination in mice using the balance beam.</a> J Vis Exp. (49), (2011).</li><li>Servadio, A., Koshy, B., Armstrong, D., Antalffy, B., Orr, H. T., Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+analysis+of+the+ataxin-1+protein+in+tissues+from+normal+and+spinocerebellar+ataxia+type+1+individuals.">Expression analysis of the ataxin-1 protein in tissues from normal and spinocerebellar ataxia type 1 individuals.</a> Nat Genet. 10 (1), 94-98 (1995).</li><li>Klement, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ataxin-1+nuclear+localization+and+aggregation,+role+in+polyglutamine-induced+disease+in+SCA1+transgenic+mice.">Ataxin-1 nuclear localization and aggregation, role in polyglutamine-induced disease in SCA1 transgenic mice.</a> Cell. 95 (1), 41-53 (1998).</li><li>Serra, H. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+profiling+links+SCA1+pathophysiology+to+glutamate+signaling+in+Purkinje+cells+of+transgenic+mice.">Gene profiling links SCA1 pathophysiology to glutamate signaling in Purkinje cells of transgenic mice.</a> Hum Mol Genet. 13 (20), 2535-2543 (2004).</li><li>Carter, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+progressive+motor+deficits+in+mice+transgenic+for+the+human+Huntington's+disease+mutation.">Characterization of progressive motor deficits in mice transgenic for the human Huntington's disease mutation.</a> J Neurosci. 19 (8), 3248-3257 (1999).</li><li>Anjomani Virmouni, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+GAA-repeat-expansion-based+mouse+model+of+Friedreich's+ataxia.">A novel GAA-repeat-expansion-based mouse model of Friedreich's ataxia.</a> Dis Model Mech. 8 (3), 225-235 (2015).</li></ol>

The authors have nothing to disclose.

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...

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.

<table><tbody><tr><td>Adenosine diphosphate</td><td>Sigma Aldrich</td><td>A2754</td><td>ADP</td></tr><tr><td>Ascorbate</td><td>Sigma Aldrich</td><td>A7631</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A2153</td><td>BSA</td></tr><tr><td>4&#039;,6-Diamidino-2-phenylindole</td><td>Sigma Aldrich</td><td>D9542</td><td>DAPI</td></tr><tr><td>Digitonin</td><td>Sigma Aldrich</td><td>D141</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma Aldrich</td><td>D0632</td><td>DTT</td></tr><tr><td>Donkey serum</td><td>Sigma Aldrich</td><td>D9663</td><td></td></tr><tr><td>Glutamate</td><td>Sigma Aldrich</td><td>1446600</td><td></td></tr><tr><td>Malate</td><td>Sigma Aldrich</td><td>6994</td><td></td></tr><tr><td>Mannitol</td><td>Sigma Aldrich</td><td>M4125</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td></td></tr><tr><td>Potassium-lactobionate</td><td>Bio-Sugars</td><td>69313-67-3</td><td></td></tr><tr><td>Rotenone</td><td>Sigma Aldrich</td><td>R8875</td><td></td></tr><tr><td>Saponin</td><td>Sigma Aldrich</td><td>47036</td><td></td></tr><tr><td>Succinic Acid</td><td>Sigma Aldrich</td><td>S3674</td><td></td></tr><tr><td>&lt;em&gt;N,N,N&amp;prime;,N&amp;prime;&lt;/em&gt;-Tetramethyl-&lt;em&gt;p&lt;/em&gt;-phenylenediamine</td><td>Sigma Aldrich</td><td>T7394</td><td>TMPD</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T9284</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich</td><td>U0631</td><td></td></tr><tr><td>Vectashield mounting medium</td><td>Vector Labs</td><td>H-1000</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>11NQ antibody (anti-ataxin-1 )</td><td></td><td></td><td>Servadio, &lt;i&gt;et al.&lt;/i&gt; 1995, PMID: 7647801</td></tr><tr><td>Alexa Fluor 488 anti-mouse secondary antibody</td><td>Life Technologies</td><td>A-11015</td><td></td></tr><tr><td>Alexa Fluor 594 anti-rabbit secondary antibody</td><td>Life Technologies</td><td>A-11012</td><td></td></tr><tr><td>Calbindin antibody (goat)</td><td>Santa Cruz</td><td>C-20</td><td></td></tr><tr><td>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Control transgenic mice</td><td>Harry Orr, Ph.D.</td><td>A02</td><td>Burright, &lt;i&gt;et al.&lt;/i&gt; 1997, PMID: 9217978</td></tr><tr><td>SCA1 mice</td><td>Harry Orr, Ph.D.</td><td>B05</td><td>Burright, &lt;i&gt;et al.&lt;/i&gt; 1997, PMID: 9217978</td></tr><tr><td>Wildtype mice</td><td>The Jackson Laboratory</td><td>001800</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ESM-100L microtome</td><td>ERMA</td><td></td><td>Sledge microtome</td></tr><tr><td>Fluoview FV1200 Confocal Microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Glycerol-gelatin slides</td><td>FD Neuro Technologies</td><td>PO101</td><td></td></tr><tr><td>Hamilton syringe</td><td>Sigma Aldrich</td><td>VCAT 80465</td><td></td></tr><tr><td>OXYT1 Oxytherm Electrode Control Unit</td><td>Hansatech Instruments</td><td></td><td></td></tr><tr><td>P.T.F.E. paper</td><td>Cole-Parmer</td><td>UX-08277-15</td><td></td></tr><tr><td>Rotallion Rotarod</td><td>PPP&amp;amp;G</td><td></td><td>contact corresponding author for information</td></tr><tr><td>Ultimate 3000 HPLC</td><td>Dionex</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td>National Institute of Health</td><td></td><td>&lt;a href=&quot;http://imagej.nih.gov/ij/&quot;&gt;http://imagej.nih.gov/ij/&lt;/a&gt;</td></tr><tr><td>Cell counter plugin (for ImageJ)</td><td>National Institute of Health</td><td></td><td>&lt;a href=&quot;http://rsb.info.nih.gov/ij/plugins/cell-counter.html&quot;&gt;http://rsb.info.nih.gov/ij/plugins/cell-counter.html&lt;/a&gt;</td></tr><tr><td>3P&amp;amp;G Rota-Rod v3.3.3 (rotarod software)</td><td>PPP&amp;amp;G</td><td></td><td>contact corresponding author for information</td></tr><tr><td>Phidget21.dll (required for rotarod software)</td><td>DLL-Files.com</td><td></td><td>&lt;a href=&quot;https://www.dll-files.com/phidget21.dll.html&quot;&gt;https://www.dll-files.com/phidget21.dll.html&lt;/a&gt;</td></tr></tbody></table>

treating sca1 mice with water-soluble compounds to non-specifically boost mitochondrial function

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.

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...

Watch this Scientific Journal Video about Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function at JoVE.com

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

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

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 ...

treating-sca1-mice-with-water-soluble-compounds-to-non-specifically

Research

Education

JoVE Core

JoVE Journal

Biology

Medicine

Cellular Processes

Cellular Respiration

SCIENTISTS IN ACTION

10.5K Views.  Skidmore College. 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. 

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

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

Environment

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

Biochemistry

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Developmental Biology

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

Cancer Research

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular mechanisms that maintain oocyte quality remain unclear, thus it is difficult to increase the birth rate of women over 35 years old at present. Oocytes contain more mitochondria than any type of cell in the body, and any mitochondrial dysfunction can lead to reduced oocyte quality. In the 1990s, oocyte cytoplasmic transfer resulted in great success in human reproduction but was accompanied by ethical controversies. Autologous mitochondrial transplantation is expected to be a useful technique to increase the quality of oocytes that have decreased due to age. In the present study, we used adipose-derived stem cells from aged mice as a mitochondria donor to increase the quality of oocytes of aged mice. Further development of autologous mitochondrial transfer technology will provide a new and effective treatment for infertility in aged women.

Here, we describe the isolation of mitochondria from mouse adipose-derived mesenchymal stem cells, and then transfer the mitochondria into aged mouse oocytes to improve the quality of the oocytes.

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular ...

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

Author Spotlight: Unveiling Oxidative Phosphorylation System Dynamics and Mitochondrial Roles in Health and Disease

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

Mechanistic Insights into Salidroside's Mitochondrial Protection via AMPK/Sirt1/HIF-1&#945; Pathway in Hypoxic HT22 Cells

Salidroside (Sal), an active ingredient of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba has been found to exert mitochondrial protective effects by improving metabolism and enhancing the energy supply of brain cells under hypoxic conditions. However, its mechanism of action has not been fully clarified. In the present study, high-performance liquid chromatography was first employed to analyze the effects of Sal on nucleotide (ATP, ADP, and AMP) levels. The cellular thermal shift assay (CETSA), a widely used molecular interaction method for validating and quantifying drug target engagement in cells and tissues across different species, was then chosen to confirm the affinity of Sal for AMPK/Sirt1/HIF-1&#945; pathway-related proteins. The results revealed that Sal increased ATP and ADP levels in hypoxic HT22 cells while reducing AMP levels. Moreover, Sal exhibited stable binding to AMPK&#945;, p-AMPK&#945;, Sirt1, and HIF-1&#945; proteins. In conclusion, Sal may exert mitochondrial protective effects by modulating the AMPK/Sirt1/HIF-1&#945; pathway to regulate nucleotide content. This study provides a methodological reference for nucleotide content analysis in cell samples and contributes to the identification and discovery of targets for compounds derived from traditional Chinese medicine.

The present study identified a unique mechanism by which salidroside exerts mitochondrial protective effects on hypoxic HT22 cells, partly through the AMPK/Sirt1/HIF-1&#945; pathway.

Salidroside (Sal), an active ingredient of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba has been found to exert mitochondrial protective effects by ...

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

Summary

Explore More Videos

Chapters in this video

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

Mechanistic Insights into Salidroside's Mitochondrial Protection via AMPK/Sirt1/HIF-1α Pathway in Hypoxic HT22 Cells