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Start of funding 01.07.2008

Protein analysis of single neurons after Laser Capture Microdissection

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Neuropathologically, Parkinson disease (PD) is characterized by the accumulation of the protein α-synuclein in midbrain neurons. We have previously shown on the single cell level after Laser Capture Microdissection (LCM) that this is associated with increased levels of mitochondrial DNA mutations as well as with an impairment of energy metabolism. It is vital for our further understanding of the involved pathomechanisms that also protein analysis is carried out in the affected neurons. For this purpose we will stain frozen brain sections from a PD mouse modell in order to cut out 100 to 1000 affected neurons by LCM, using a semiautomatic imaging software. Downstream, we will extract proteins from these cells and develop a high-sensitive Western-Blot method to quantify levels of proteins involved in the mitochondrial respiratory chain. We believe the establishment of this protein-single-cell-technique has very powerful implications in the understanding and treatment of not only neurodegenerative disease, but any cell specific biological disorder.

Final report:
Mitochondrial abnormalities as well as protein accumulation seem to play an important role in the pathogenesis of parkinson´s disease (PD). We have previously shown that individual substantia nigra neurons of PD patients harbor high levels of mitochondrial DNA mutations. For this purpose, we employed laser capture microdissection (LCM) and quantitative PCR methods in order to analyze single neurons of post-mortem brain sections. In order to advance the analysis of the molecular pathomechanisms underlying PD, it is essential to not only look at the DNA but also at the protein level, specifically in affected cell populations. Therefore, in this project, we developed a molecular method to quantify protein expression on the single cell level using LCM and highsensitive westernblotting.

For this purpose, we used cryosections from transgenic PD mouse models, from human control subjects as well as from human muscle biopsies. Through the combination of LCM with optimized western blots, we were able to quantify protein levels of tyrosine hydroxylase (TH), the key enzyme of dopamine metabolism in pooled substantia nigra neurons. In order to show the specificity of the method, we also analyzed glia cells, which do not contain TH and lacked the specific band in the single cell blots. In muscle biopsies, we were able to quantify protein levels of the five complexes of the mitochondrial respiratory chain.

This project led to the development of a very sensitive method to quantify protein levels out of as little as 200 individual cells following LCM. In our view, this novel method is an important tool supporting the task of uncovering the molecular mechanisms at the basis of neurodegenerative diseases because it allows for a very specific analysis of those cell populations, which are affected the most.

Results of this project were recently published in an international journal:

Koob AO, Bruns L, Prassler C, Masliah E, Klopstock T, Bender A. Protein analysis through Western blot of cells excised individually from human brain and muscle tissue. Anal Biochem. 2012 Jun 15;425(2):120-4. doi:10.1016/j.ab.2012.02.034. Epub 2012 Mar 5. PubMed PMID: 22402104.