Die Rolle mitochondrialer Mutationen bei neurodegenerativen Erkrankungen
Molecular Biology and Physiology of Neurons and Glial Cells
Final Report Abstract
Background: Respiratory chain (RC) deficiencies are found in primary mtDNA diseases. Focal RC defects are also associated with ageing and neurodegenerative disorders, e.g. in substantia nigra (SN) neurons from idiopathic Parkinson’s disease (IPD) patients. In mitochondrial disease and ageing, mtDNA mutational loads vary considerably between neurons necessitating single cell-based assessment of RC deficiencies. Evaluating the full extent of RC deficiency within SN neurons is challenging because their size precludes investigations in serial sections. The standard histochemical method to investigate mitochondrial dysfunction - the cytochrome c oxidase/succinate dehydrogenase assay - measures complex IV (CIV) and CII activities. To also study CI in a patient, immunohistology in additional sections, i.e. in different neurons, is required. Here, we developed a method that allows correlation of the abundance of different RC complex subunits or mtDNA- associated proteins and mitochondrial mass at a single cell level. This method in combination with mtDNA analysis in single neurons was then used to investigate the molecular underpinnings of CI deficiency in IPD. Methods: We used primary antibodies against subunits of CI-IV, or mitochondrial transcription factor A (TFAM) and B2 (TFB2M) in combination with antibodies against the mitochondrial mass marker porin and the dopaminergic marker tyrosine hydroxylase. Primary antibodies were detected with IgG subtype-specific fluorescent-labeled secondary antibodies. The abundance of each target protein was determined densitometrically in single neurons by means of the image processing software ImageJ and target protein-to-mitochondrial mass ratios were calculated. To assess the link between RC deficiencies in IPD and mtDNA integrity in midbrain dopaminergic neurons, cells were isolated with laser capture microdissection from frozen tissue after immunohistochemistry. MtDNA deletions and copy number in single neurons were quantified with a real-time PCR approach. Results: Applying quadruple immunofluorescent labelling to postmortem midbrain sections from patients with mtDNA disease and known RC deficiencies consistently showed the expected loss of CI and/or CIV. In single dopaminergic neurons from IPD patients (n=10), we detected a significant reduction of the CI:porin expression ratios when compared to age-matched controls (n=10). A subset of patients additionally showed reduced CIV:porin ratios. Moreover, mtDNA-associated TFAM and TFB2M were present at low levels in CI-deficient cells. Analysis of minor/major arc mtDNA deletions and copy number in neurons with normal versus low CI abundance identified mtDNA depletion as cause of CI deficiency in IPD. Finally, the assessment of RC complexes in control brains showed an association of CI and CIV abundance with age at death. Conclusions: Taken together, we conclude that (i) quantitative quadruple-label immunofluorescence is a reliable tool to measure RC deficiencies in individual neurons. (ii) RC CI deficiency in dopaminergic SN neurons of IPD patients is reflected by a loss of CI protein. (iii) MtDNA depletion is the cause of CI deficiency in IPD midbrain neurons. (iv) CI deficiency is likely preceding CIV and CIII deficiency in the time course of neurodegeneration. (v) Loss of CI and CIV abundance are age-related phenomena also occurring in control brains. Future plans: To further study the relevance of mtDNA depletion in IPD, a cellular IPD model system will be required. In this context, we assessed the behavior of the mitochondrial genome during reprogramming of control fibroblasts into iPS cells and differentiation into dopaminergic neurons. This analysis showed an (expected) increase in mtDNA copy number in dopaminergic neurons compared to the iPS cell and fibroblast stage. Furthermore, the transformation did not impact on mtDNA integrity as indicated by consistently low deletion levels throughout the procedure. According to these preliminary results, iPS-derived neurons may indeed serve as a tool to study the relevance of the mitochondrial genome in the development of IPD in vivo.
Publications
- Joint loss of respiratory chain complex I and IV expression in substantia nigra neurons from Parkinson’s disease patients. GeoPD 2013
Grünewald A, Reeve AK, Rocha M, Hepplewhite PD, Rygiel K, Klein C, Turnbull DM
- A new tool to assess mitochondrial respiratory chain dysfunction in single muscle fibres. EuroMit 2014
Rocha MC, Grady J, Grünewald A, Taylor R, Turnbull D, Rygiel K
- Investigating the cause of mitochondrial dysfunction in single substantia nigra neurons from idiopathic Parkinson’s disease patients. DGN 2014; abstract: V38
Grünewald A, Lax NZ, Reeve AK, Turnbull DM
- Loss of respiratory chain complex I in substantia nigra neurons from Parkinson’s disease patients coincides with reduced abundance of complex IV. MDS 2014; poster: 33
Grünewald A, Reeve AK, Lax N, Hepplewhite PD, Klein C, Turnbull DM
- PINK1 Loss of Function Mutations Affect Mitochondrial Complex I Activity via NdufA10 Ubiquinone Uncoupling. Science 2014; 344:203-7
Morais VA, Haddad D, Craessaerts K, De Bock PJ, Swerts J, Vilain S, Aerts L, Overbergh L, Grünewald A, Seibler P, Klein C, Gevaert K, Verstreken P, De Strooper B
(See online at https://doi.org/10.1126/science.1249161) - Quantitative quadruple-label immunofluorescence of mitochondrial and cytoplasmic proteins in single neurons from human midbrain tissue. J Neurosci Meth 2014; 232C:143-149
Grünewald A, Lax NZ, Rocha MC, Reeve AK, Hepplewhite PD, Rygiel K, Taylor RW, Turnbull DM
(See online at https://doi.org/10.1016/j.jneumeth.2014.05.026) - The alloantigenicity of mitochondria in mouse somatic cell nucleus transfer-derived stem cells. Cell Stem Cell 2014; Epub ahead of print (20.11.14)
Deuse T, Wang D, Stubbendorff M, Itagaki R, Grabosch A, Greaves LC, Alawi M, Grünewald A, Hu X, Hua X, Velden J, Reichenspurner H, Robbins RC, Jaenisch R, Weissman IL, Schrepfer S
