Limits...
Regionalized pathology correlates with augmentation of mtDNA copy numbers in a patient with myoclonic epilepsy with ragged-red fibers (MERRF-syndrome).

Brinckmann A, Weiss C, Wilbert F, von Moers A, Zwirner A, Stoltenburg-Didinger G, Wilichowski E, Schuelke M - PLoS ONE (2010)

Bottom Line: However, mtDNA copy numbers were increased 3-7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen) and in skeletal muscle.Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs).We thus conclude that "futile" stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuropediatrics, Charité University Medical School, Berlin, Germany.

ABSTRACT
Human patients with myoclonic epilepsy with ragged-red fibers (MERRF) suffer from regionalized pathology caused by a mutation in the mitochondrial DNA (m.8344A→G). In MERRF-syndrome brain and skeletal muscles are predominantly affected, despite mtDNA being present in any tissue. In the past such tissue-specificity could not be explained by varying mtDNA mutation loads. In search for a region-specific pathology in human individuals we determined the mtDNA/nDNA ratios along with the mutation loads in 43 different post mortem tissue samples of a 16-year-old female MERRF patient and in four previously healthy victims of motor vehicle accidents. In brain and muscle we further determined the quantity of mitochondrial proteins (COX subunits II and IV), transcription factors (NRF1 and TFAM), and VDAC1 (Porin) as a marker for the mitochondrial mass. In the patient the mutation loads varied merely between 89-100%. However, mtDNA copy numbers were increased 3-7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen) and in skeletal muscle. Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs). Such mtDNA copy number increase was not paralleled by an augmentation of mitochondrial mass in some investigated tissues, predominantly in the most affected tissue regions of the brain. We thus conclude that "futile" stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome.

Show MeSH

Related in: MedlinePlus

Determination of protein abundance for structural mitochondrial proteins and for transcription factors.(A) Western blot of tissue samples from the putamen of three controls (C2-4) and the patient (individual 3_2 on Figure 1A). Protein abundance was determined semi-quantitatively through densitometry. β-Tubulin was used as loading control. (B) Results of the semi-quantitative analysis of the mitochondrial mass as represented by anti-Porin (VDAC1) immune reactivity. The whiskers represent the absolute (100%) range of all three control measurements. The result of the patient is represented as a black dot. Figures (C) and (D) represent the quantification of the transcription factors NRF1 and TFAM. (E) Relative abundance of the nuclear encoded subunit of cytochrome C oxidase (COIV) versus an mtDNA-encoded subunit (COII) of the same complex. The immune staining with both antibodies was done on the identical blot which secured the comparability. (F) Only in muscle the mtDNA-encoded COII subunit was much higher expressed (4.5×) than its nDNA encoded counterpart. Note: Due to the lack of sufficient autopsy material the loading of each lane could not be adjusted. However, these Western blots are only intended to illustrate an equal abundance of proteins encoded by the nuclear (COIV) versus the mtDNA (COII) and the absence of this balance in the muscle of the MERRF patient.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2958123&req=5

pone-0013513-g004: Determination of protein abundance for structural mitochondrial proteins and for transcription factors.(A) Western blot of tissue samples from the putamen of three controls (C2-4) and the patient (individual 3_2 on Figure 1A). Protein abundance was determined semi-quantitatively through densitometry. β-Tubulin was used as loading control. (B) Results of the semi-quantitative analysis of the mitochondrial mass as represented by anti-Porin (VDAC1) immune reactivity. The whiskers represent the absolute (100%) range of all three control measurements. The result of the patient is represented as a black dot. Figures (C) and (D) represent the quantification of the transcription factors NRF1 and TFAM. (E) Relative abundance of the nuclear encoded subunit of cytochrome C oxidase (COIV) versus an mtDNA-encoded subunit (COII) of the same complex. The immune staining with both antibodies was done on the identical blot which secured the comparability. (F) Only in muscle the mtDNA-encoded COII subunit was much higher expressed (4.5×) than its nDNA encoded counterpart. Note: Due to the lack of sufficient autopsy material the loading of each lane could not be adjusted. However, these Western blots are only intended to illustrate an equal abundance of proteins encoded by the nuclear (COIV) versus the mtDNA (COII) and the absence of this balance in the muscle of the MERRF patient.

Mentions: Further, we investigated whether the elevated mtDNA/nDNA ratios were solely due to enhanced mtDNA-replication or caused by a real increase of the number of mitochondrial organelles per cell ( = mitochondrial mass). We thus extracted total protein from brain specimens and pectoralis muscle of patient and controls and performed a semi-quantitative Western blot analysis using antibodies against Porin (VDAC1). This protein being highly expressed at the outer mitochondrial membrane [22] serves as a marker for mitochondrial mass [23]. Due to the small volumes of many biopsy specimens, we were limited in the number of tissues to be analyzed by this method but we were able to investigate hippocampus, putamen, and pectoralis muscle, representing tissues that are affected in MERRF-syndrome, and the occipital white matter and the pons, representing tissues that remain mostly unaffected. The mitochondrial mass of the patient's hippocampus and putamen was within the range of controls (Figure 4B). In contrast, a more than two-fold increase was found in pons, white matter, and skeletal muscle. Thus, we did not find a general correlation between increased mtDNA abundance and increased mitochondrial mass. On the contrary, in the investigated brain regions we either found an increase in mtDNA copy numbers or in mitochondrial mass. Only in skeletal muscle we found a parallel increase of both parameters.


Regionalized pathology correlates with augmentation of mtDNA copy numbers in a patient with myoclonic epilepsy with ragged-red fibers (MERRF-syndrome).

Brinckmann A, Weiss C, Wilbert F, von Moers A, Zwirner A, Stoltenburg-Didinger G, Wilichowski E, Schuelke M - PLoS ONE (2010)

Determination of protein abundance for structural mitochondrial proteins and for transcription factors.(A) Western blot of tissue samples from the putamen of three controls (C2-4) and the patient (individual 3_2 on Figure 1A). Protein abundance was determined semi-quantitatively through densitometry. β-Tubulin was used as loading control. (B) Results of the semi-quantitative analysis of the mitochondrial mass as represented by anti-Porin (VDAC1) immune reactivity. The whiskers represent the absolute (100%) range of all three control measurements. The result of the patient is represented as a black dot. Figures (C) and (D) represent the quantification of the transcription factors NRF1 and TFAM. (E) Relative abundance of the nuclear encoded subunit of cytochrome C oxidase (COIV) versus an mtDNA-encoded subunit (COII) of the same complex. The immune staining with both antibodies was done on the identical blot which secured the comparability. (F) Only in muscle the mtDNA-encoded COII subunit was much higher expressed (4.5×) than its nDNA encoded counterpart. Note: Due to the lack of sufficient autopsy material the loading of each lane could not be adjusted. However, these Western blots are only intended to illustrate an equal abundance of proteins encoded by the nuclear (COIV) versus the mtDNA (COII) and the absence of this balance in the muscle of the MERRF patient.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958123&req=5

pone-0013513-g004: Determination of protein abundance for structural mitochondrial proteins and for transcription factors.(A) Western blot of tissue samples from the putamen of three controls (C2-4) and the patient (individual 3_2 on Figure 1A). Protein abundance was determined semi-quantitatively through densitometry. β-Tubulin was used as loading control. (B) Results of the semi-quantitative analysis of the mitochondrial mass as represented by anti-Porin (VDAC1) immune reactivity. The whiskers represent the absolute (100%) range of all three control measurements. The result of the patient is represented as a black dot. Figures (C) and (D) represent the quantification of the transcription factors NRF1 and TFAM. (E) Relative abundance of the nuclear encoded subunit of cytochrome C oxidase (COIV) versus an mtDNA-encoded subunit (COII) of the same complex. The immune staining with both antibodies was done on the identical blot which secured the comparability. (F) Only in muscle the mtDNA-encoded COII subunit was much higher expressed (4.5×) than its nDNA encoded counterpart. Note: Due to the lack of sufficient autopsy material the loading of each lane could not be adjusted. However, these Western blots are only intended to illustrate an equal abundance of proteins encoded by the nuclear (COIV) versus the mtDNA (COII) and the absence of this balance in the muscle of the MERRF patient.
Mentions: Further, we investigated whether the elevated mtDNA/nDNA ratios were solely due to enhanced mtDNA-replication or caused by a real increase of the number of mitochondrial organelles per cell ( = mitochondrial mass). We thus extracted total protein from brain specimens and pectoralis muscle of patient and controls and performed a semi-quantitative Western blot analysis using antibodies against Porin (VDAC1). This protein being highly expressed at the outer mitochondrial membrane [22] serves as a marker for mitochondrial mass [23]. Due to the small volumes of many biopsy specimens, we were limited in the number of tissues to be analyzed by this method but we were able to investigate hippocampus, putamen, and pectoralis muscle, representing tissues that are affected in MERRF-syndrome, and the occipital white matter and the pons, representing tissues that remain mostly unaffected. The mitochondrial mass of the patient's hippocampus and putamen was within the range of controls (Figure 4B). In contrast, a more than two-fold increase was found in pons, white matter, and skeletal muscle. Thus, we did not find a general correlation between increased mtDNA abundance and increased mitochondrial mass. On the contrary, in the investigated brain regions we either found an increase in mtDNA copy numbers or in mitochondrial mass. Only in skeletal muscle we found a parallel increase of both parameters.

Bottom Line: However, mtDNA copy numbers were increased 3-7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen) and in skeletal muscle.Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs).We thus conclude that "futile" stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuropediatrics, Charité University Medical School, Berlin, Germany.

ABSTRACT
Human patients with myoclonic epilepsy with ragged-red fibers (MERRF) suffer from regionalized pathology caused by a mutation in the mitochondrial DNA (m.8344A→G). In MERRF-syndrome brain and skeletal muscles are predominantly affected, despite mtDNA being present in any tissue. In the past such tissue-specificity could not be explained by varying mtDNA mutation loads. In search for a region-specific pathology in human individuals we determined the mtDNA/nDNA ratios along with the mutation loads in 43 different post mortem tissue samples of a 16-year-old female MERRF patient and in four previously healthy victims of motor vehicle accidents. In brain and muscle we further determined the quantity of mitochondrial proteins (COX subunits II and IV), transcription factors (NRF1 and TFAM), and VDAC1 (Porin) as a marker for the mitochondrial mass. In the patient the mutation loads varied merely between 89-100%. However, mtDNA copy numbers were increased 3-7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen) and in skeletal muscle. Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs). Such mtDNA copy number increase was not paralleled by an augmentation of mitochondrial mass in some investigated tissues, predominantly in the most affected tissue regions of the brain. We thus conclude that "futile" stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome.

Show MeSH
Related in: MedlinePlus