私たちは貴重な入力については、図3と教授ジョナス伝のためにデータを提供するためハンナワーナーに感謝します。スウェーデンの研究評議会（グラント522-2006-6416（MA）、521-2007-5407（MA）、オーケウイバーグ財団（MA、JH）、スウェーデン王立科学アカ​​デミー（MA、JH）と、スウェーデンの化学社会（JH）は感謝して財政支援のために承認されています。

<ol>
	<li>Obeso, J. A., Olanow, C. W., Nutt, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levodopa+motor+complications+in+Parkinson's+disease.">Levodopa motor complications in Parkinson's disease.</a> Trends Neurosci. 23, S2-S7 (2000).</li><li>Caprioli, R. M., Farmer, T. B., Gile, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+biological+samples:+localization+of+peptides+and+proteins+using+MALDI-TOF+MS.">Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS.</a> MALDI-TOF MS. Anal. Chem. 69, 4751-4760 (1997).</li><li>Obeso, 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=The+evolution+and+origin+of+motor+complications+in+Parkinson's+disease.">The evolution and origin of motor complications in Parkinson's disease.</a> Neurology. 55, S13-S20 (2000).</li><li>O, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncommunicable+Diseases+and+Mental+Health+Cluster,+Noncommunicable+Disease+Prevention+and+Health+Promotion+Department,+Ageing+and+Life+Course.">Noncommunicable Diseases and Mental Health Cluster, Noncommunicable Disease Prevention and Health Promotion Department, Ageing and Life Course.</a> Active Ageing: A Policy framework. , (2002).</li><li>Schapira, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+disorders:+advances+in+cause+and+treatment.">Movement disorders: advances in cause and treatment.</a> Lancet Neurology. , 6-7 (2010).</li><li>Obeso, J. A., Rodriguez-Oroz, M. C., Rodriguez, M., DeLong, M. R., Olanow, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+levodopa-induced+dyskinesias+in+Parkinson's+disease:+problems+with+the+current+model.">Pathophysiology of levodopa-induced dyskinesias in Parkinson's disease: problems with the current model.</a> Ann. Neurol. 47, S22-S32 (2000).</li><li>Cenci, M. A., Lee, C. S., Bjorklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-DOPA-induced+dyskinesia+in+the+rat+is+associated+with+striatal+overexpression+of+prodynorphin-+and+glutamic+acid+decarboxylase+mRNA.">L-DOPA-induced dyskinesia in the rat is associated with striatal overexpression of prodynorphin- and glutamic acid decarboxylase mRNA.</a> Eur. J. Neurosci. 10, 2694-2706 (1998).</li><li>Andersson, M., Hilbertson, A., Cenci, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Striatal+fosB+expression+is+causally+linked+with+l-DOPA-induced+abnormal+involuntary+movements+and+the+associated+upregulation+of+striatal+prodynorphin+mRNA+in+a+rat+model+of+Parkinson's+disease.">Striatal fosB expression is causally linked with l-DOPA-induced abnormal involuntary movements and the associated upregulation of striatal prodynorphin mRNA in a rat model of Parkinson's disease.</a> Neurobiol Dis. 6, 461-474 (1999).</li><li>Hanrieder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+striatal+neuropeptides+revealed+by+imaging+mass+spectrometry.">Alterations of striatal neuropeptides revealed by imaging mass spectrometry.</a> Molecular &#38; Cellular Proteomics. , (2011).</li><li>Cornett, D. S., Reyzer, M. L., Chaurand, P., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+imaging+mass+spectrometry:+molecular+snapshots+of+biochemical+systems.">MALDI imaging mass spectrometry: molecular snapshots of biochemical systems.</a> Nat. Methods. 4, 828-833 (2007).</li><li>Ljungdahl, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Mass+Spectrometry+Reveals+Elevated+Nigral+Levels+of+Dynorphin+Neuropeptides+in+L-DOPA-Induced+Dyskinesia+in+Rat+Model+of+Parkinson's+Disease.">Imaging Mass Spectrometry Reveals Elevated Nigral Levels of Dynorphin Neuropeptides in L-DOPA-Induced Dyskinesia in Rat Model of Parkinson's Disease.</a> PLoS ONE. 6, e25653 (2011).</li><li>Groseclose, M. R., Andersson, M., Hardesty, W. M., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+proteins+directly+from+tissue:+in+situ+tryptic+digestions+coupled+with+imaging+mass+spectrometry.">Identification of proteins directly from tissue: in situ tryptic digestions coupled with imaging mass spectrometry.</a> J. Mass. Spectrom. 42, 254-262 (2007).</li><li>Andersson, M., Groseclose, M. R., Deutch, A. Y., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mass+spectrometry+of+proteins+and+peptides:+3D+volume+reconstruction.">Imaging mass spectrometry of proteins and peptides: 3D volume reconstruction.</a> Nat. Methods. 5, 101-108 (2008).</li><li>Deininger, S. -. O., Setou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging Mass Spectrometry. , 199-208 (2010).</li><li>Norris, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+MALDI+Mass+Spectra+to+Improve+Mass+Spectral+Direct+Tissue+Analysis.">Processing MALDI Mass Spectra to Improve Mass Spectral Direct Tissue Analysis.</a> Int. J. Mass. Spectrom. 260, 212-221 (2007).</li><li>Ihaka, R., Gentleman, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Language+for+Data+Analysis+and+Graphics.">A Language for Data Analysis and Graphics.</a> Journal of Computational and Graphical Statistics. 5, 299-314 (1996).</li><li>Tusher, V. G., Tibshirani, R., Chu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+analysis+of+microarrays+applied+to+the+ionizing+radiation+response.">Significance analysis of microarrays applied to the ionizing radiation response.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 5116-5121 (2001).</li><li>Bergstrom, L., Christensson, I., Folkesson, R., Stenstrom, B., Terenius, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ion+exchange+chromatography+and+radioimmunoassay+procedure+for+measuring+opioid+peptides+and+substance+P.">An ion exchange chromatography and radioimmunoassay procedure for measuring opioid peptides and substance P.</a> Life. Sci. 33, 1613-1619 (1983).</li><li>Falth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptidomics+strategies+for+specific+and+sensitive+identification+of+endogenous+peptides.">Neuropeptidomics strategies for specific and sensitive identification of endogenous peptides.</a> Mol. Cell. Proteomics. 6, 1188-1197 (2007).</li><li>Falth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SwePep,+a+database+designed+for+endogenous+peptides+and+mass+spectrometry.">SwePep, a database designed for endogenous peptides and mass spectrometry.</a> Mol. Cell. Proteomics. 5, 998-1005 (2006).</li></ol>

著者らは、開示することは何もありません。

神経ペプチドの研究ではMALDIイメージング質量分析を用いることのいくつかの利点があります。 MSデータの公平な分析は、特定の脳核を明らかにするか、線条体の背の部分は特定の病態生理学的状態に関連付けられているところここでしか示された結果のようにすることができます。空間情報を保持することにより、全体の脳切片の解析やペプチド抽出物を従来のペプチドミクス研究を使用した場合に比べて高い感度と低い変動と統計分析を実行するために関心領域を再定義することが可能である。また、MALDI IMSが容易に未知の翻訳後修飾を検出することができます実現するために重要であるが、構造解析では、変更された正確なアミノ酸の位置を決定するために従わなければなりません。 MALDI IMSデータを可視化する一般的な落とし穴がBLから線形光学スケールの最大ピーク強度のマッピングを含む代わりに、100％すべてのセクションの最大ピーク強度である共通の絶対的なスケールに、すべてのセクションをマッピングの実験シリーズ内の各セクション（図3）の色（100％）（0％）、ACK（図5） 。後者の方法は、グループデータと治療群間差の可視化の比較が可能になります。 MALDI IMS分析の主要な障害は、特定の質量ピークのペプチドの割り当てです。上の組織タンデム質量分析法は、可能な場合もありますが、多くの場合、13,14は非常に困難証明しています。我々は、逆相LC-MS/MSに続いて強陽イオン交換クロマトグラフィーで分取画など、多くの伝統的なアプローチが成功したシーケンスの多くの神経ペプチド、特にオピオイドペプチドに使用することができることがわかります。それはまだそのようなマスコットとして一般的な検索エンジンを使用して、任意のデータベースのエントリと一致しない良質のMS / MSスペクトルを取得することも珍しくありません。これらのケースでは手動でのde novoシーケンシングは、ONLです。yオプション。ピーク身元の最終的な証拠は、適切なノックアウトマウスからの組織切片のMALDI IMSすることによって得ることができる、しかし、これは常に可能か可能ではありません。代わりに、西部のイムノブロッティングや免疫組織化学によって、例えば、直径方向に異なる方法で結果を検証することです。これは多くの場合、抗体と新しい抗体を検証する作業のかなりの量を高めることを含めることができます。 このプロトコルで提示一般的な戦略は、いくつかのセクションに、実験条件を含む大規模な神経ペプチドMALDI IMS実験のために最適化されています。痛みのメカニズムと依存症の薬物に対する内因性の応答を含む研究の多様な分野で採用されるようにプロトコルは、特にオピオイドペプチドのために最適化されており、今後の研究に大きな影響を与えます。

1.1サンプルの収集と準備<ol><li>イソフルラン（Apoteksbolaget AB、スウェーデン） </li><li>手術ツール、はさみの2ペア、骨鉗子、へら</li><li>細かく粉砕ドライアイス</li><li>ストレージ用のアルミ箔とビニール袋</li><li>クライオスタット（マイクロモル、ハイデルベルク、ドイツ） </li><li>インジウム - スズ酸化物（ITO）でコーティングされたMALDIスライド（ブルカーダルト、ドイツ） </li><li>最適な切削温度媒体（OCT）（サクラファインテックヨーロッパ、オランダ） </li><li>真空デシケーター</li></ol> 1.2マトリックスアプリケーション<ol><li>のアセトニトリル（ACN）、メタノール（MeOH）、エタノール（EtOH）プロ分析グレード（メルク、ドイツ）。 </li><li>トリフルオロ酢酸（TFA）（シグマアルドリッチ、セントルイス、MO）。 </li><li>水のMilli-Q精製システム（ミリポア、ベッドフォード、MA）で精製した。 </li><li> CHIP-1000（島津製作所、日本）またはPortrait 630スポッター（LabCyte、サニーベール、カリフォルニア州） </li></ol> 1.3質量分析<ol><li> ULtraflex II（ブルカーダルト） </li><li> FlexImagingソフトウェア（V 2.0、ダルトニクス） </li><li>ペプチドまたはタンパク質校正標準（ブルカーダルト） </li></ol> 1.4データ処理<ol><li>原点v.8.1（OriginLabの社、Norhampton、MA） </li><li> Matlabのバージョン7.9.0.529（MathWorks社、ネーティック、マサチューセッツ州） </li><li> pBin（ <a href="http://www.vicc.org/biostatistics/software.php" target="_blank">http://www.vicc.org/biostatistics/software.php</a> ） </li></ol> 1.5ニューロ識別<ol><li>超音波超音波</li><li> 10kDaの分子量カットオフフィルター（ミリポア、ベッドフォード、MA） </li><li> SPセファデックスC 25ゲル（​​Sigma社、ドイツ） </li><li> Hypersilカラム（サーモフィッシャー） </li><li>ピリジン、プロの分析グレードのギ酸（FA）、アセトニトリル（ACN）、メタノール（MeOH）、エタノール（EtOH）（メルク、ドイツ）。 </li><li>トリフルオロ酢酸（TFA）、シグマアルドリッチ（セントルイス、MO、米国）。 </li><li> Agilent 1100シリーズナノフローLC、microWPS、C18カラム（0.075x150mm） </li><li> Agilent 1100シリーズマイクロfractioncollector、Prespotted anchorchipターゲットプレート（ブルカーダルト） </li><li> BioToolsソフトウェア（バージョン3.1 SR2、ダルトニクス） </li><li>バイオワークスソフトウェア（V.3.3、サーモフィッシャー。 <a href="http://www.thermoscientific.com" target="_blank">www.thermoscientific.com</a> ） </li><li>マスコットソフトウェア（V 2.2、MatrixScience。 <a href="http://www.matrixscience.com" target="_blank">www.matrixscience.com</a> ） </li><li> Swepepデータベース（ <a href="http://www.swepep.org" target="_blank">www.swepep.org</a> ） </li></ol>

maldi imaging mass spectrometry of neuropeptides in parkinson's disease

MALDIイメージングマススペクトロメトリー（IMS）は、生体組織サンプル2（図1）の分子種の空間分析を容易にする強力なアプローチです。 12μmの薄い組織切片は、通常、MALDI TOF / TOF質量分析計を用いて、質量分析器で検出することができ無傷のペプチドおよびタンパク質の脱離とイオン化を容易にするMALDIマトリックスで覆われています。一般的にはピークの何百ものは、単一のラット脳組織切片で評価することができる。一般的に使用されるイメージング技術とは対照的に、このアプローチは、目的の分子の事前知識を必要とせず、高い分子特異性と感度2を維持しながら、複数の分子種の教師と包括的な分析が可能になります。ここでは、動物モデル、パーキンソン病（PD）のラット脳内の神経ペプチドの地域に固有のディストリビューションプロファイルを解明するためのMALDI IMSベースのアプローチを説明します。 PDは、年齢3,4の65歳以上の人々のための1％の有病率が一般的な神経変性疾患である。最も一般的な対症療法は、L-DOPA 5を使用してドーパミンの交換に基づいています。ただし、これは不随意異常な動き、と呼ばれるL-DOPA誘発ジスキネジア（LID）1,3,6を含む重篤な副作用が付属しています。 LIDの中で最も著名な分子変化の一つは、オピオイド前駆prodynorphin mRNAの7のレギュレーションです。ダイノルフィンペプチドは本質的に移動制御7,8に関与している脳領域で神経伝達を調節する。しかしながら、神経ペプチド前駆体の処理に由来する正確なオピオイドペプチドを最新の状態に特徴付けされていない。したがって、我々は実験的パーキンソン病とL-DOPA誘発ジスキネジアの動物モデルでMALDI IMSを利用した。 MALDIイメージング質量分析法は、神経ペプチド特性評価に関して特に有利であることが判明る、一般的に使用される抗体ベースのアプローチから知られたペプチド配列と以前に観察された翻訳後修飾を対象としています。これとは対照的にMALDI IMSは、新規ペプチドの加工製品を解明するため、神経伝達の神経変調の新たな分子メカニズムを明らかにすることができます。神経ペプチドの絶対量はMALDI IMSによって決定することはできませんが、ペプチドイオンの相対量は、健康と病気のレベルを変更する方法について洞察を与えて、質量スペクトルから、線引きすることができます。ここで示す例では、ダイノルフィンB、α-neoendorphinとサブスタンスPのピーク強度が大幅に背に増加することが認められたが、重度の運動障害を持つ動物の背内側、線条体（図顔面、体幹と口舌の筋肉を伴わない5）。また、MALDI IMSは、機能不活性化の以前に未知のメカニズムを表す、ジスキネジアの重症度とDES-チロシンα-neoendorphinのレベルとの相関関係を明らかにしたN末端 ​​のチロシンの除去などの線条体におけるダイノルフィンのリザベーションがダイノルフィンのオピオイド受容体結合能力9を減らすことができます 。これは、MALDI IMSを使用して、LIDの神経ペプチドの特性に関する最初の研究であるとの結果は、生物医学研究のあらゆる分野のアプリケーションのための技術の潜在能力を強調表示します。

Watch this Scientific Journal Video about MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease at JoVE.com

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson&#039;s Disease

L-DOPAを用いたドーパミン補充薬物療法は、パーキンソン病の最も一般的に使用される対症療法ですが、不随意の異常な動き、と呼ばれる運動障害などの副作用が付属しています<sup&gt; 1</sup&gt;。ここでは、MALDIイメージング質量分析のためのプロトコルは、運動障害に関連したラット脳の神経ペプチドレベルの変化を検出し、その提示されます。

パーキンソン病における神経ペプチドのMALDIイメージングマススペクトロメトリー

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

maldi-imaging-mass-spectrometry-neuropeptides-parkinson-s

Research

JoVE Journal

Medicine

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

25.8K ビュー.  Uppsala University. L-DOPAを用いたドーパミン補充薬物療法は、パーキンソン病の最も一般的に使用される対症療法ですが、不随意の異常な動き、と呼ばれる運動障害などの副作用が付属しています 1。ここでは、MALDIイメージング質量分析のためのプロトコルは、運動障害に関連したラット脳の神経ペプチドレベルの変化を検出し、その提示されます。

ビデオ: MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

パーキンソン病の単方向、病変6-OHDAモデルマウスの開発

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

医学

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

プライマリーヒト線維芽細胞の導出のために皮膚パンチ生検植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

パーキンソン病の分野におけるミトコンドリアの表現型を監視するための主要ヒト線維芽細胞の使用

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson&#039;s Disease

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

パーキンソン病における感受性メカニズムのマスク解除を行う遺伝子環境相互作用モデル

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Controlling Parkinson&#039;s Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

アダプティブ脳深部刺激とパーキンソン病の防除

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

骨格筋疾患の定量的磁気共鳴イメージング

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

液体および固形腫瘍由来の癌細胞のプロテオームの定量的質量分析プロファイリング

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

組換えアデノ随伴ウイルスベクターの定位注射を介して、パーキンソン病のα-シヌクレインベースのラットモデルの開発

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

誘導結合プラズマ -   - レーザーアブレーションによる脳組織におけるイメージング金属質量分析法（LA-ICP-MS）

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&amp;#8212;Application in Premanifest Huntington&#039;s Disease

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

全体脳セグメンテーションと解剖学的脳 MRI の変化点分析-Premanifest ハンチントン病のアプリケーション

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...