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>
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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 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&#39;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.

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 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.
<img alt="Figure 1" src="/files/ftp_upload/53758/53758fig1.jpg" class="xfigimg" /> 
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 &#60; 0.05). (D) Succinic acid treatment significantly improves accelerating rotarod performance of SCA1 22 mice as shown by increased latency to fall (* p &#60; 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. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53758/53758fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53758/53758fig2.jpg" class="xfigimg" /> 
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 &#956;m 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. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53758/53758fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53758/53758fig3.jpg" class="xfigimg" /> 
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). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53758/53758fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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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

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 ...

Biochemistry

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

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 ...

Assessment of Open Probability of the Mitochondrial Permeability Transition Pore in the Setting of Coenzyme Q Excess

The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in health and disease. The mPTP mediates leakage of protons across the IMM during low-conductance opening and is specifically inhibited by cyclosporine A (CsA). Coenzyme Q (CoQ) is a regulator of the mPTP, and tissue-specific differences have been found in CoQ content and open probability of the mPTP in forebrain and heart mitochondria in a newborn mouse model of fragile X syndrome (FXS, Fmr1 knockout). We developed a technique to determine the voltage threshold for mPTP opening in this mutant strain, exploiting the role of the mPTP as a proton leak channel.
To do so, oxygen consumption and membrane potential (&#916;&#936;) were simultaneously measured in isolated mitochondria using polarography and a tetraphenylphosphonium (TPP+) ion-selective electrode during leak respiration. The threshold for mPTP opening was determined by the onset of CsA-mediated inhibition of proton leak at specific membrane potentials. Using this approach, differences in voltage gating of the mPTP were precisely defined in the context of CoQ excess. This novel technique will permit future investigation for enhancing the understanding of physiological and pathological regulation of low-conductance opening of the mPTP.

This method exploits the contribution of the mitochondrial permeability transition pore to low-conductance proton leak to determine the voltage threshold for pore opening in neonatal fragile X syndrome mice with increased cardiomyocyte mitochondrial coenzyme Q content compared to wildtype control.

The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in ...

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

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