This study was carried out in the Center for the Study of Mitochondrial Pediatric Diseases (http://www.mitopedia.org), funded by the Mariani Foundation. VT is a member of the European Reference Network for Rare Neuromuscular Diseases (ERN EURO-NMD).

NOTE: The use of human fibroblasts may require ethical approval. Fibroblasts used in this study were derived from MD patients and stored in the Institutional biobank in compliance with ethical requirements. Informed consent was provided for the use of the cells. Perform all cell culture procedures under a sterile laminar flow cabinet at room temperature (RT, 22-25 &#176;C). Use sterile-filtered solutions suitable for cell culture and sterile equipment. Grow all cell lines in a humidified incubator at 37 &#176;C with 5% CO2. Mycoplasma tests should be conducted weekly to ensure mycoplasma-free cultures. 143BTK- rho0 cells can be generated as previously described5.
1. Culture of cells
<ol>
	<li>Before starting any procedure, verify the presence of the mutation in fibroblasts derived from MD patient(s) and quantify the percentage of heteroplasmy or homoplasmy by Restriction Fragment Length Polymorphism (RFLP) and/or whole mtDNA sequencing analyses14.</li>
	<li>Seed fibroblasts in four 35 mm Petri dishes, each containing 2 mL of Complete Culture Medium (Table 1). Let the cells grow until 80% confluent (48 h).</li>
	<li>Grow 143BTK- rho0 cells in 8 mL of Supplemented&#160;Culture Medium (Table 1) in a 100 mm Petri dish.</li>
	<li>Maintain the cells in an incubator at 37 &#176;C with 5% CO2.</li>
	<li>Check the absence of mtDNA in rho0 cells by sequencing techniques14.</li>
</ol>
2. Enucleation of fibroblasts
<ol>
	<li>Sterilize four 250 mL centrifuge-suitable bottles by autoclave sterilization at 121 &#176;C for a 20 min cycle. Dry them in a laboratory oven or at RT.</li>
	<li>Prewarm the centrifuge at 37 &#176;C.</li>
	<li>Wash the 35 mm dishes containing the fibroblasts twice, using 2 mL of 1x phosphate-buffered saline (PBS) without (w/o) calcium and magnesium.</li>
	<li>Clean the outer surface of the dishes with 70% ethanol and wait until the alcohol evaporates.</li>
	<li>Remove the lids from the dishes and the screw caps from the bottles. Place each dish, without the lid, upside down on the bottom of each 250 mL centrifuge bottle.</li>
	<li>Slowly add 32 mL of Enucleation Medium to each bottle (Table 1), allowing the medium to enter the dish and come into contact with the cells. Remove any bubbles from the dishes using a long glass Pasteur pipette, curving the tip in a Bunsen flame. 
	NOTE: It is important to remove the bubbles to allow the medium to enter the dish and come in contact with the cells.</li>
	<li>Close each bottle with the screw cap and transfer them to the centrifuge.</li>
	<li>Centrifuge for 20 min at 37 &#176;C and 8,000 &#215; g, acceleration max, deceleration slow. Pay attention to balance the centrifuge: counterweight each bottle. If necessary, adjust the weight by adding a suitable volume of Enucleation Medium.</li>
	<li>During centrifugation, use vacuum or a 10 mL serological pipette to aspirate and discard the medium from the 143BTK- rho0 culture plates and wash them twice using 4 mL of 1x PBS w/o calcium and magnesium.</li>
	<li>Add 2 mL of trypsin to cover the cell monolayer completely.</li>
	<li>Place the dishes in a 37 &#176;C incubator for ~2 min.</li>
	<li>Remove the dishes from the incubator, observe cell detachment using an inverted microscope for live cells (objectives 4x or 10x), and inhibit the enzyme activity by adding 2 mL of Supplemented Culture Medium.</li>
	<li>Aspirate the 4 mL of the cell suspension in the dish with a 10 mL pipette and transfer it to a 15 mL conical tube.</li>
	<li>Count the cells using a Burker hemocytometer chamber or an automated counter.</li>
	<li>At the end of centrifugation (step 2.8), aspirate the medium from the bottles and discard it.</li>
	<li>Remove the dishes by inverting the bottles on a sterile gauze previously sprayed with 70% ethanol. Clean the outer surface of the dishes and their lids with 70% ethanol. Wait until the alcohol evaporates, and then close the dishes.</li>
	<li>Before proceeding, check for cytoplast (ghost) formation using an inverted microscope for live cells (objective 4x or 10x). Look for extremely elongated fibroblasts due to the extrusion of their nuclei induced by cytochalasin B.</li>
	<li>To each 35 mm dish, add 1 &#215; 106 of 143BTK- rho0 cells resuspended in 2 mL of 143BTK- rho0 culture medium supplemented with 5% fetal bovine serum (FBS).</li>
	<li>Leave the dishes for 3 h in a humidified incubator at 37 &#176;C and 5% CO2 and let the 143BTK- rho0 cells settle on the ghosts. Do not disturb the dishes.</li>
</ol>
3. Fusion of the enucleated fibroblasts with rho&#160;0 cells
<ol>
	<li>After 3 h of incubation, aspirate and discard the medium from the dishes.</li>
	<li>Wash the adherent cells twice with 2 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) high glucose w/o serum or with Minimum Essential Medium (MEM).</li>
	<li>Aspirate and discard the medium.</li>
	<li>Add 500 &#181;L of PEG solution (see the Table of Materials) to the cells and incubate for exactly 1 min.</li>
	<li>Aspirate and discard the PEG solution.</li>
	<li>Wash the cells three times using 2 mL of DMEM high glucose w/o serum or with MEM.</li>
	<li>Add 2 mL of Fusion Medium (Table 1) and incubate overnight in the incubator at 37 &#176;C with 5% CO2.</li>
</ol>
4. Cybrid selection and expansion
<ol>
	<li>After overnight incubation, remove the plates from the incubator, trypsinize the cells as described above (steps 2.9-2.13), and transfer the content of each 35 mm dish into a 100 mm dish.</li>
	<li>Add 8 mL of Selection Medium (Table 1) and place the plates in the incubator at 37 &#176;C with 5% CO2.</li>
	<li>Change the medium every 2-3 days.</li>
	<li>Wait for ~10-15 days of selection until colonies of cells appear.</li>
	<li>Freeze one of the four Petri dishes by collecting all the clones and generating a &#34;massive&#34; culture as a backup of the cybrids, which can be eventually recloned and used for further investigations.</li>
	<li>Trypsinize the cells in the remaining culture dishes, count, and seed into one or more Petri dishes at 50-100 cells/dish in the Supplemented Culture Medium (Table 1) until clones appear. Let them grow for some days.</li>
	<li>Pellet the remaining cells by centrifugation at 1,200 &#215; g for 3 min at RT and discard the supernatant.</li>
	<li>Extract DNA from the pellet (see the Table of Materials).</li>
	<li>Perform genotyping by variable number of tandemly repeated (VNTR) analysis as previously reported15.</li>
	<li>Pick up clones from the Petri dish with cloning cylinders or a pipette tip, using a stereomicroscope to avoid pooling of different clones, and transfer them to a 96-well plate, each well containing 200 &#181;L of Supplemented Culture Medium (Table 1).</li>
	<li>Expand every clone until there are enough cells for freezing and extracting DNA.</li>
	<li>Verify the mutation percentage of each clone by RFLP or other sequencing methods. Ideally, try to obtain both clones with wild-type mtDNA (0% mutation) and clones with different mutation percentages, both adding up to homoplasmic mutant mtDNA (100% mutation). See Figure 1 for a schematic diagram of the cybrid generation protocol.</li>
</ol>

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The authors have no conflicts of interest to disclose.

The mtDNA has a very high mutation rate compared to nDNA because of the lack of protective histones and its location close to the respiratory chain, which exposes the molecule to damaging oxidative effects not efficiently counteracted by the repair systems16. The first pathogenic mtDNA mutations were identified in 198817,18, and since then, a large number of mutations have been described. NGS technology is a relevant approach to screen the...

Mitochondrial disorders (MDs) are a group of multisystem syndromes caused by an impairment in mitochondrial functions due to mutations in either nuclear (nDNA) or mitochondrial (mtDNA) DNA1. They are among the most common inherited metabolic diseases, with a prevalence of 1:5,000. mtDNA-associated diseases follow the rules of mitochondrial genetics: maternal inheritance, heteroplasmy and threshold effect, and mitotic segregation2. Human mtDNA is a double-stranded DNA circle of 16.6 kb, which contains a short control region with sequences needed for replication and transcription, 13 protein-coding genes (all subunits of the respiratory chain), 22 tRNA, and 2 rRNA genes3.
In healthy individuals, there is one single mtDNA genotype (homoplasmy), whereas more than one genotype coexists (heteroplasmy) in pathological conditions. Deleterious heteroplasmic mutations must overcome a critical threshold to disrupt OXPHOS and cause diseases that can affect any organ at any age4. The dual genetics of OXPHOS dictates inheritance: autosomal recessive or dominant and X-linked for nDNA mutations, maternal for mtDNA mutations, plus sporadic cases both for nDNA and mtDNA.
At the beginning of the mitochondrial medicine era, a landmark experiment by King and Attardi5 established the basis to understand the origin of a mutation responsible for an MD by creating hybrid cells containing nuclei from tumor cell lines in which mtDNA was entirely depleted (rho0 cells) and mitochondria from patients with MDs. Next-generation sequencing (NGS) techniques were not available at that time, and it was not easy to determine whether a mutation was present in the nuclear or mitochondrial genome. The method, described in 1989, was then used by several researchers working in the field of mitochondrial medicine6,7,8,9; a detailed protocol has been recently published10, but no video has been made yet. Why should such a protocol be relevant nowadays when NGS could precisely and rapidly identify where a mutation is located? The answer is that cybrid generation is still the state-of-the-art protocol to understand the pathogenic role of any novel mtDNA mutation, correlate the percentage of heteroplasmy with the severity of the disease, and perform a biochemical investigation in a homogeneous nuclear system in which the contribution of the autochthonous nuclear background of the patient is absent11,12,13.
This protocol describes how to obtain cytoplasts from confluent, patient-derived fibroblasts grown in 35 mm Petri dishes. Centrifugation of the dishes in the presence of cytochalasin B allows the isolation of enucleated cytoplasts, which are then fused with rho0 cells in the presence of polyethylene glycol (PEG). The resulting cybrids are then cultivated in selective medium until clones arise. The representative results section shows an example of molecular characterization of the resulting cybrids to prove that the mtDNA is identical to that of the donor patients&#39; fibroblasts and that the nDNA is identical to the nuclear DNA of the tumoral rho0 cell line.

<table><tbody><tr><td>5-Bromo-2&#039;-Deoxyuridine</td><td>Sigma-Aldrich (Merck)</td><td>B5002-500MG</td><td></td></tr><tr><td>6 well Plates</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>96 well Plates</td><td>Corning</td><td>3596</td><td></td></tr><tr><td>Blood and Cell Culture DNA extraction kit</td><td>QIAGEN</td><td>13323</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>Avanti J-25</td><td>7,200 rcf, 37 &amp;deg;C</td></tr><tr><td>Centrifuge bottles, 250 mL</td><td>Beckman Coulter</td><td>356011</td><td></td></tr><tr><td>Cytochalasin B from Drechslera dematioidea</td><td>Sigma-Aldrich (Merck)</td><td>C2743-200UL</td><td></td></tr><tr><td>Dialyzed FBS</td><td>Gibco</td><td>26400-036 100mL</td><td></td></tr><tr><td>DMEM High Glucose (w/o L-Glutamine W/Sodium Pyruvate)</td><td>EuroClone</td><td>ECB7501L</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline - PBS (w/o Calcium w/o Magnesium)</td><td>EuroClone</td><td>ECB4004L</td><td></td></tr><tr><td>Ethanol Absolute Anhydrous</td><td>Carlo Erba</td><td>414601</td><td></td></tr><tr><td>FetalClone III (Bovine Serum Product)</td><td>Cytiva - HyClone Laboratories</td><td>SH30109.03</td><td></td></tr><tr><td>Glass pasteur pipettes</td><td>VWR</td><td>M4150NO250SP4</td><td></td></tr><tr><td>Inverted Research Microscope For Live Cell Microscopy</td><td>Nikon</td><td>ECLIPSE TE200</td><td></td></tr><tr><td>JA-14 Fixed-Angle Aluminum Rotor</td><td>Beckman Coulter</td><td>339247</td><td></td></tr><tr><td>Laboratory autoclave Vapormatic 770</td><td>Labotech</td><td>29960014</td><td></td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>EuroClone</td><td>ECB 3000D</td><td></td></tr><tr><td>Minimum Essential Medium MEM</td><td>Euroclone</td><td>ECB2071L</td><td></td></tr><tr><td>MycoAlert Mycoplasma Detection Kit</td><td>Lonza</td><td>LT07-318</td><td></td></tr><tr><td>PEG (Polyethylene glicol solution)</td><td>Sigma-Aldrich (Merck)</td><td>P7181-5X5ML</td><td></td></tr><tr><td>Penicillin-Streptomycin (solution 100x)</td><td>EuroClone</td><td>ECB3001D</td><td></td></tr><tr><td>Primo TC Dishes 100 mm</td><td>EuroClone</td><td>ET2100</td><td></td></tr><tr><td>Primo TC Dishes 35 mm</td><td>EuroClone</td><td>ET2035</td><td></td></tr><tr><td>Sodium Pyruvate 100 mM</td><td>EuroClone</td><td>ECM0542D</td><td></td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ1000</td><td></td></tr><tr><td>Trypsin 2.5% in HBSS</td><td>EuroClone</td><td>ECB3051D</td><td></td></tr><tr><td>Uridine</td><td>Sigma-Aldrich (Merck)</td><td>U3003-5G</td><td></td></tr></tbody></table>

an in vitro approach to study mitochondrial dysfunction: a cybrid model

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human mitochondrial disorders. From a genetic point of view, the OXPHOS represents a unique example because it results from the complementation of two distinct genetic systems: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, OXPHOS defects can be due to mutations affecting nuclear and mitochondrial encoded genes.
The groundbreaking work by King and Attardi, published in 1989, showed that human cell lines depleted of mtDNA (named rho0) could be repopulated by exogenous mitochondria to obtain the so-called &#34;transmitochondrial cybrids.&#34; Thanks to these cybrids containing mitochondria derived from patients with mitochondrial disorders (MDs) and nuclei from rho0 cells, it is possible to verify whether a defect is mtDNA- or nDNA-related. These cybrids are also a powerful tool to validate the pathogenicity of a mutation and study its impact at a biochemical level. This paper presents a detailed protocol describing cybrid generation, selection, and characterization.

Generating cybrids requires 3 days of laboratory work plus a selection period (~2 weeks) and additional 1-2 weeks for the growth of clones. The critical steps are the quality of cytoplasts and the selection period. The morphology of cybrids resembles that of the rho0 donor cells. Assignment of the correct mtDNA and nDNA in the cybrids is mandatory to confirm the identity of the cells. An example is given in Figure 2. In this case, we generated cybrids starting from fibroblasts der...

Watch this Scientific Journal Video about An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model at JoVE.com

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Transmitochondrial cybrids are hybrid cells obtained by fusing mitochondrial DNA (mtDNA)-depleted cells (rho0 cells) with cytoplasts (enucleated cells) derived from patients affected by mitochondrial disorders. They allow the determination of the nuclear or mitochondrial origin of the disease, evaluation of biochemical activity, and confirmation of the pathogenetic role of mtDNA-related variants.

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human ...

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JoVE Journal

Genetics

3.6K Views.  Fondazione IRCCS Istituto Neurologico Carlo Besta. Transmitochondrial cybrids are hybrid cells obtained by fusing mitochondrial DNA (mtDNA)-depleted cells (rho0 cells) with cytoplasts (enucleated cells) derived from patients affected by mitochondrial disorders. They allow the determination of the nuclear or mitochondrial origin of the disease, evaluation of biochemical activity, and confirmation of the pathogenetic role of mtDNA-related variants.

Video: An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

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

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Biochemistry

Transmitochondrial Cybrid Generation From Suspension-Growing Cancer Cells

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (&#961;0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

Author Spotlight: Transmitochondrial Cybrid Generation Using Cancer Cell Lines  

This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process.

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

Analyzing Mitochondrial Physiology and Bioenergetics in Fruit Flies

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

Author Spotlight: Exploring Mitochondrial Function and Chemical Toxicity Using Drosophila melanogaster 

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

Neuroscience

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Biology

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

An In Situ Immunostaining Protocol for Parkinson's Disease Study

Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model

Mitochondria play a central role in the energy metabolism of cells, and their function is especially important for neurons due to their high energy demand. Therefore, mitochondrial dysfunction is a pathological hallmark of various neurological disorders, including Parkinson's disease. The shape and organization of the mitochondrial network is highly plastic, which allows the cell to respond to environmental cues and needs, and the structure of mitochondria is also tightly linked to their health. Here, we present a protocol to study mitochondrial morphology in situ based on immunostaining of the mitochondrial protein VDAC1 and subsequent image analysis. This tool could be particularly useful for the study of neurodegenerative disorders because it can detect subtle differences in mitochondrial counts and shape induced by aggregates of &#945;-synuclein, an aggregation-prone protein heavily involved in the pathology of Parkinson's disease. This method allows one to report that substantia nigra pars compacta dopaminergic neurons harboring pS129 lesions show mitochondrial fragmentation (as suggested by their reduced Aspect Ratio, AR) compared to their healthy neighboring neurons in a pre-formed fibril intracranial injection Parkinson model.

Author Spotlight: Establishing a New Fluorescence-Based Protocol for In Vivo Mitochondrial Morphology Analysis in Parkinson's Disease

This study presents a method to analyze the morphology of mitochondria based on immunostaining and image analysis in mouse brain tissue in situ. It also describes how this allows one to detect changes in mitochondrial morphology induced by protein aggregation in Parkinson's disease models.

Mitochondria play a central role in the energy metabolism of cells, and their function is especially important for neurons due to their high energy ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

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

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1^B9-Null Mutant Using High-resolution Respirometry

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model

Studying Mitochondrial Structure and Function in Drosophila Ovaries