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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.

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Morphometric analysis of neuronal cell density in the insular cortex of the index patient.(A) Density of cortical neurons using Abercrombie correction. The open bar from the patient represents the neuronal density from 130,000 µm2 of cross-sectional plane per cortical layer. (B) Ratio between the numbers of glial versus neuronal cells. Black bars, control individuals from the literature (reference provided); open bars, MERRF patient; the whiskers depict the SEM; n.a., data not available.
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pone-0013513-g002: Morphometric analysis of neuronal cell density in the insular cortex of the index patient.(A) Density of cortical neurons using Abercrombie correction. The open bar from the patient represents the neuronal density from 130,000 µm2 of cross-sectional plane per cortical layer. (B) Ratio between the numbers of glial versus neuronal cells. Black bars, control individuals from the literature (reference provided); open bars, MERRF patient; the whiskers depict the SEM; n.a., data not available.

Mentions: A tissue sample from the patient's insular cortex (Brodman area 43) including all neuronal layers and the white matter was fixed for >4 weeks in phosphate-buffered 4% formaldehyde, dehydrated and subsequently embedded in paraffin. Microtome sections of 5 µm were mounted on glass slides, deparaffinized, stained with anti-NeuN and subsequently with Cy3-labeled fluorescent secondary antibodies. The cell nuclei were counterstained with DAPI. An unbiased counting frame of 186.2×139.6 µm2 was then systematically moved through the cortical layers of interest and photographs were taken on the UV and Cy3-channel through a 20× oil immersion objective (Leica Microsystems, Wetzlar, Germany) with a SPOT3 cooled CCD camera (Visitron, Puchheim, Germany). From each cortical layer we analyzed five such frames which thus represented 130,000 µm2 of cross-sectional plane per respective cortical layer. The two images from the DAPI and the Cy3-channel were superimposed with false colors and on each frame we separately counted the total number of DAPI positive nuclei versus those nuclei that co-localized with a NeuN-signal representing neuronal cells that had been cut in the plane of the nucleus. All 2D-counts were corrected with the Abercrombie formula [10] taking into consideration the thickness of the section (5 µm) and the diameter of the cell nuclei. The Ferret's diameter of the nuclei was 14±5 µm and had been determined in 50 random neuronal nuclei at their largest optical cross-sectional diameter with a 63× oil immersion objective. The Abercrombie-corrected cell densities were then compared to published reference values that had been obtained by the same staining and 2D-counting method [11], [12]. The final results were then depicted as neurons/mm2 and as glia/neuron ratio (Figure 2A,B).


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)

Morphometric analysis of neuronal cell density in the insular cortex of the index patient.(A) Density of cortical neurons using Abercrombie correction. The open bar from the patient represents the neuronal density from 130,000 µm2 of cross-sectional plane per cortical layer. (B) Ratio between the numbers of glial versus neuronal cells. Black bars, control individuals from the literature (reference provided); open bars, MERRF patient; the whiskers depict the SEM; n.a., data not available.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013513-g002: Morphometric analysis of neuronal cell density in the insular cortex of the index patient.(A) Density of cortical neurons using Abercrombie correction. The open bar from the patient represents the neuronal density from 130,000 µm2 of cross-sectional plane per cortical layer. (B) Ratio between the numbers of glial versus neuronal cells. Black bars, control individuals from the literature (reference provided); open bars, MERRF patient; the whiskers depict the SEM; n.a., data not available.
Mentions: A tissue sample from the patient's insular cortex (Brodman area 43) including all neuronal layers and the white matter was fixed for >4 weeks in phosphate-buffered 4% formaldehyde, dehydrated and subsequently embedded in paraffin. Microtome sections of 5 µm were mounted on glass slides, deparaffinized, stained with anti-NeuN and subsequently with Cy3-labeled fluorescent secondary antibodies. The cell nuclei were counterstained with DAPI. An unbiased counting frame of 186.2×139.6 µm2 was then systematically moved through the cortical layers of interest and photographs were taken on the UV and Cy3-channel through a 20× oil immersion objective (Leica Microsystems, Wetzlar, Germany) with a SPOT3 cooled CCD camera (Visitron, Puchheim, Germany). From each cortical layer we analyzed five such frames which thus represented 130,000 µm2 of cross-sectional plane per respective cortical layer. The two images from the DAPI and the Cy3-channel were superimposed with false colors and on each frame we separately counted the total number of DAPI positive nuclei versus those nuclei that co-localized with a NeuN-signal representing neuronal cells that had been cut in the plane of the nucleus. All 2D-counts were corrected with the Abercrombie formula [10] taking into consideration the thickness of the section (5 µm) and the diameter of the cell nuclei. The Ferret's diameter of the nuclei was 14±5 µm and had been determined in 50 random neuronal nuclei at their largest optical cross-sectional diameter with a 63× oil immersion objective. The Abercrombie-corrected cell densities were then compared to published reference values that had been obtained by the same staining and 2D-counting method [11], [12]. The final results were then depicted as neurons/mm2 and as glia/neuron ratio (Figure 2A,B).

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