Name: treating sca1 mice with water-soluble compounds to non-specifically boost mitochondrial function
Uploaded: 2017-01-22T13:00:00.000+00:00
Duration: 11 min 47 s
Description: Watch this Scientific Journal Video about Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function at JoVE.com

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>

著者らは、開示することは何もありません。

説明したように、これらの方法を使用する場合、それらは、小脳の神経変性疾患のマウスモデルにおける酸化的リン酸化によって媒介されるミトコンドリア機能不全を検出し、緩和することが可能です。組み合わせた生化学的および行動アッセイは、小脳の神経変性疾患の病態にミトコンドリアの寄与の程度を決定するための多面的な方法です。代謝を促進し、ミトコンドリア機能を高める?...

ミトコンドリア電子輸送鎖を使用して、酸化的リン酸化（OXPHOS）を経て製造ミトコンドリアのATPの大部分で、アデノシン三リン酸（ATP）、細胞エネルギーのために必須の補酵素の主要な生産者です。脳は、その高い代謝要求と神経活動に電力を供給するための酸化的リン酸化への依存を与え、ミトコンドリア機能障害の影響を非常に受けやすいです。その結果、ミトコンドリア機能障害は、老化プロセス中にトリガされます 1及び複数の神経変性疾患2、3、4の病因に関与しています。したがって、ミトコンドリアは、神経変性のための魅力的な治療標的であることになります。 このプロトコルでは、我々はミトコンドリアの研究のためのモデルの神経変性疾患などの脊髄小脳失調症1型（SCA1）の使用を採用していますリットル機能障害およびミトコンドリア標的療法の開発。 SCA1は、他の脳領域のプルキンエ小脳のニューロンとニューロンの進行性変性をトリガアタキシン1遺伝子産物におけるポリグルタミン（ポリQ）リピート伸長変異によって引き起こされます。プルキンエ細胞特異的プロモーターの制御下でポリQ突然変異体アタキシン1導入遺伝子を発現する（SCA1マウスと命名）は、ここで使用されるトランスジェニックマウス系統は、SCA1 5のプルキンエ細胞成分の標的分析を可能にします。 SCA1マウスは緩やかなプルキンエ細胞の変性を受け、失調性歩行6を開発します。 ミトコンドリア複合体の機能不全とミトコンドリア標的治療の有効性は、分子や行動アッセイの電池を用いて評価することができます。ミトコンドリア複合体機能障害は小脳組織内の変更された酸素消費量を検出する呼吸アッセイによりex vivoで測定され、電子伝達鎖基質および阻害剤7が存在します。呼吸アッセイは、以前に透過性の組織、ミトコンドリアの単離物、および全組織7、8、9で使用されてきました。これらは、透過型電子顕微鏡または免疫蛍光染色などの形態学的データの収集方法とは異なり、ミトコンドリア機能を直接評価することを可能にします。組織全体ではなく、単離ミトコンドリアの使用は、単離工程7の間に発生する可能性があり、健康なミトコンドリアのバイアスされた選択を防ぐことができます。示されるように、プロトコルに適合したときに、呼吸アッセイは、小脳の神経変性疾患状態においてミトコンドリア機能障害を検出するための有益な方法です。 代謝の非特異的活性化は、神経変性diseasのトランスジェニックマウスモデルにおいてミトコンドリア機能障害を推定するために使用することができeと新しい治療法の開発に役立ちます。ケルセチン、コエンザイムQ10およびクレアチンは、全ての患者において神経変性病10、11、12、13、14、15、16の動物モデルにおいて神経変性疾患の病状を改善することが示されています。ここでは、代謝を促進し、神経変性疾患でミトコンドリアの機能を高めるための新規な代謝賦活剤、コハク酸を、提示します。活性化剤は、血液脳関門を通過していることを確実にするために、HPLCを処置したマウス17の神経組織への送達を検出するために使用しました。 コハク酸のような代謝的に標的と水溶性化合物の治療効果を評価するために、行動パラダイム及び免疫病理学的研究の電池を使用することができます。デュ小脳の神経変性疾患、フットプリントの滑走路アッセイ、ビームアッセイ加速回転棒アッセイに見られる運動協調の障害に電子が行動病理6、18、19の救助を検出するために使用されます。これらの措置は、小脳組織6、20、21の定義された小葉内の分子（プルキンエ細胞の樹状突起樹の長さとして定義された）層の厚さとプルキンエ細胞体数を評価することによって小脳細胞構築の免疫病理学的評価を補っています。ここでは、代謝的に標的と水溶性化合物を用いたミトコンドリア機能障害の検出および治療のための複数の神経病理学的および行動の方法を提示します。 私たちは、SCA1のTRANにミトコンドリア機能障害を分析するために、ミトコンドリア呼吸のex vivo分析を使用しますsgenicマウス。さらに、我々は、疾患の症状および病状がさらにSCA1疾患の進行におけるミトコンドリア機能障害が関与し、水溶性のミトコンドリアブースタコハク酸によって改善されることを示しています。

<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

ミトコンドリア機能障害は老化のプロセスで、いくつかの遺伝性脊髄小脳失調症と小脳の進行性変性によってマークされ、他の運動障害などの神経変性疾患において重要な役割を果たしています。このプロトコルの目的は、脊髄小脳失調症1型（SCA1）におけるミトコンドリア機能障害を評価し、疾患の進行を遅らせるために、水溶性の化合物のコハク酸を介して代謝呼吸の薬理学的標的化の有効性を評価することです。このアプローチは、他の小脳疾患に適用可能であり、水溶性の治療のホストに適合させることができます。 ミトコンドリア呼吸のex vivo分析は、ミトコンドリア機能の疾患に関連する変化を検出し、定量化するために使用されます。遺伝的証拠（未発表データ）とSCA1マウスモデルにおけるミトコンドリア機能障害のプロテオミクス証拠により、我々は、水溶性代謝ブースターSを用いた治療の有効性を評価しますホームケージの飲料水に直接この化合物を溶解することによりuccinic酸。血液脳関門を通過する薬剤の能力は、高速液体クロマトグラフィー（HPLC）を用いて推定することができます。これらの化合物の有効性は、その後、加速ロータロッド、バランスビームテスト及びフットプリント分析を含む、複数の行動パラダイムを用いて試験することができます。小脳の細胞構築完全性は、プルキンエ細胞核とプルキンエ細胞の樹状突起と細胞体を検出する免疫​​アッセイを用いて評価することができます。これらの方法は、ミトコンドリア機能障害および小脳の神経変性疾患における水溶性化合物を用いた治療の有効性を決定するための強固な技術です。

コハク酸と小脳ミトコンドリアの薬理学的標的化を通して、私たちは、小脳の神経変性疾患のSCA1のマウスモデルにおけるミトコンドリア機能障害を防止することができます。コハク酸デヒドロゲナーゼ、コハク酸、の正規の電子供与体は、治療（ 図 1A）は、次の治療および神経病理学的評価の第2週の間、行動評価の始まりと、1ヶ月間SCA1マ?...

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

ミトコンドリア機能を非特異的に高めるための水溶性化合物による SCA1 マウスの治療 |医学 |ビデオ

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.

非具体的に水溶性化合物でSCA1マウスの治療は、ミトコンドリア機能を高めます

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

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10.5K ビュー.  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. 

ビデオ: 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.

有機塩素系農薬に曝露した肝細胞におけるミトコンドリア機能の検出実験プロトコル

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

環境学

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.

ミトコンドリアの呼吸鎖 Supercomplexes の分離と分析のためのハイブリッドクリア/ブルーネイティブ電気泳動

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

生化学

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.

慢性的な刺激の収縮活動に伴うラット骨格筋の表現型適応を研究への応用

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

発生生物学

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.

細胞外フラックス解析を用いた単一3D微細組織スフェロイドのミトコンドリアエネルギー代謝の探索

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

がん研究

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.

高解像度呼吸法による坐骨神経のミトコンドリア機能の評価

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 ...

超複合体分析のための小さな組織サンプル中のミトコンドリア分画

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.

著者スポットライト:健康と疾患における酸化的リン酸化システムのダイナミクスとミトコンドリアの役割の解明

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

ミトコンドリアスーパーコンプレックス解析のためのミトコンドリアの単離 (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 ...

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.

非具体的に水溶性化合物でSCA1マウスの治療は、ミトコンドリア機能を高めます

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有機塩素系農薬に曝露した肝細胞におけるミトコンドリア機能の検出実験プロトコル

ミトコンドリアの呼吸鎖 Supercomplexes の分離と分析のためのハイブリッドクリア/ブルーネイティブ電気泳動

慢性的な刺激の収縮活動に伴うラット骨格筋の表現型適応を研究への応用

細胞外フラックス解析を用いた単一3D微細組織スフェロイドのミトコンドリアエネルギー代謝の探索

高解像度呼吸法による坐骨神経のミトコンドリア機能の評価

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

ミトコンドリアスーパーコンプレックス解析のためのミトコンドリアの単離 (Small Tissue and Cell Culture Samples)

ミトコンドリアスーパーコンプレックス解析のためのミトコンドリアの単離 (Small Tissue and Cell Culture Samples)

ミトコンドリアスーパーコンプレックス解析のためのミトコンドリアの単離 (Small Tissue and Cell Culture Samples)

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