Name: the use of primary human fibroblasts for monitoring mitochondrial phenotypes in the field of parkinson's disease
Uploaded: 2012-10-03T13:00:00
Description: Watch this Scientific Journal Video about The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease at JoVE.com

This work was supported by grants from the Fritz Thyssen Foundation (10.11.2.153 to R.K.), the German Research Council (DFG, KR2119/3-2 and KR2119/8-1 to R.K.), the Federal Ministry for Education and Research (BMBF, NGFNplus; 01GS08134 to R.K.) and by a doctoral scholarship from the charitable Hertie Foundation [to L.F.B]. We thank Carolin Obermaier and Julia Westermeier for their support during the video shoot.

1. Skin Biopsy and Culturing of Human Fibroblasts
<ol>
	<li>The skin biopsy has to be taken by an experienced physician. The procedure takes place under sterile conditions and requires local anesthesia. Typical sites used for biopsy are the inner side of the upper arm, shoulder or lower back.</li>
	<li>You can take 4x4mm or 6x6mm diameter specimen by punch biopsy to get enough tissue for culturing human fibroblasts.</li>
	<li>Cut the skin biopsy into equal small pieces under sterile conditions to separate them into 2-4...

<ol>
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Patient skin fibroblasts as ex vivo models represent an important tool to characterize disease-associated genetic defects. In addition, skin-derived fibroblasts are easily accessible and can be expanded upon culturing. Therefore, primary cells obtained from patients carrying PD-associated genetic mutations are preferable over the use of tumor cell lines as they contain not only the endogenous disease-causing gene, but the whole genetic background of the affected individual. Moreover, the fibroblasts have ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue no.</td>
 </tr>
 <tr>
 <td>Roswell Park Memorial Institute (RPMI) 1640 medium</td>
 <td>Invitrogen</td>
 <td>52400-025</td>
 </tr>
 <tr>
 <td>RPMI 1640 medium, no Phenol Red</td>
 <td>Invitrogen</td>
 <td>11835-063</td>
 </tr>
 <tr>
 <td>Dulbecco's Phosphate-Buffered Saline (DPBS)</td>
 <td>Invitrogen</td>
 <td>14190-094</td>
 </tr>
 <tr>
 <td>Fibroblast growth factor 2 (FGF2)</td>
 <td>PeproTech</td>
 <td>100-18B</td>
 </tr>
 <tr>
 <td>AccuMax (detachment solution)</td>
 <td>PAA</td>
 <td>L11-008</td>
 </tr>
 <tr>
 <td>Lab-TekTMII chambered coverglasses</td>
 <td>Nalge Nunc International</td>
 <td>115382</td>
 </tr>
 <tr>
 <td>Tetramethylrodamine ethyl ester (TMRE)</td>
 <td>Invitrogen</td>
 <td>T-669</td>
 </tr>
 <tr>
 <td>Mitotracker Green FM</td>
 <td>Invitrogen</td>
 <td>M-7514</td>
 </tr>
 <tr>
 <td>Mitotracker CM-H2XRos</td>
 <td>Invitrogen</td>
 <td>M-7513</td>
 </tr>
 <tr>
 <td>Lyostracker Red DND-99</td>
 <td>Invitrogen</td>
 <td>L-7528</td>
 </tr>
 <tr>
 <td>Hoechst 33342</td>
 <td>Invitrogen</td>
 <td>H-3570</td>
 </tr>
 </tbody>
</table>
Table 1. Specific reagents and equipment.

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.

Watch this Scientific Journal Video about The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease at JoVE.com

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

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 ...

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