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Attention deficit-hyperactivity disorder suffers from mitochondrial dysfunction

View Article: PubMed Central - PubMed

ABSTRACT

Background: Pathophysiology of attention-deficit hyperactivity disorder (ADHD) is not known, and therefore the present study investigated mitochondrial defects, if any in cybrids created from patients and control population.

Methods: To investigate mitochondrial pathology in ADHD, cybrids cell lines were created from ADHD probands and controls by fusing their platelets with ρ0-cells prepared from SH-SY5Y neuroblastoma cell line. Cellular respiration, oxidative stress, mitochondrial membrane potential and morphology were evaluated employing oxygraph, mitochondria-specific fluorescence staining and evaluation by FACS, and immunocytochemistry. HPLC-electrochemical detection, quantitative RT-PCR and Blue Native PAGE were employed respectively for assays of serotonin, mitochondrial ATPase 6/8 subunits levels and complex V activity.

Results: Significantly low cellular and mitochondrial respiration, ATPase6/8 transcripts levels, mitochondrial complex V activity and loss of mitochondrial membrane potential and elevated oxidative stress were observed in ADHD cybrids. Expression of monoamine oxidizing mitochondrial enzymes, MAO-A and MAO-B levels remained unaffected. Two-fold increase in serotonin level was noted in differentiated cybrid-neurons.

Conclusions: Since cybrids are shown to replicate mitochondrial defects seen in post-mortem brains, these observed defects in ADHD cybrids strongly suggest mitochondrial pathology in this disorder.

General significance: Mitochondrial defects are detected in ADHD cybrids created from patients' platelets, implying bioenergetics crisis in the mitochondria could be a contributory factor for ADHD pathology and/or phenotypes.

No MeSH data available.


Related in: MedlinePlus

Long-template PCR analysis and PicoGreen®staining of cybrids, ρ0-cells and SH-SY5Y cells: (A) Genomic DNA from SH-SY5Y, ρ0-cells, control and ADHD cybrids were amplified using both internal and external primers and subjected to electrophoresis on 0.6% agarose gel. Amplicons are absent in the PCR using external primers for ρ0 cells. (B) Punctated cytoplasm and nuclei are fluorescent in SH-SY5Y cells, control and ADHD cybrids, whereas in ρ0 cells only the nuclei exhibited green fluorescence by Pico-green staining. C6, C9, C10 and C11 are control cybrids, ADHD1, 5 and 6 are cybrids prepared using platelets of ADHD probands. Scale bar represents 10 μm.
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f0005: Long-template PCR analysis and PicoGreen®staining of cybrids, ρ0-cells and SH-SY5Y cells: (A) Genomic DNA from SH-SY5Y, ρ0-cells, control and ADHD cybrids were amplified using both internal and external primers and subjected to electrophoresis on 0.6% agarose gel. Amplicons are absent in the PCR using external primers for ρ0 cells. (B) Punctated cytoplasm and nuclei are fluorescent in SH-SY5Y cells, control and ADHD cybrids, whereas in ρ0 cells only the nuclei exhibited green fluorescence by Pico-green staining. C6, C9, C10 and C11 are control cybrids, ADHD1, 5 and 6 are cybrids prepared using platelets of ADHD probands. Scale bar represents 10 μm.

Mentions: The inheritance of mitochondria was tested in ADHD samples employing long-template PCR method. The primer sets were designed for PCR so that the internal primers amplified a 5.8 kb sequence present in both the nuclear (chromosome 1) and mitochondrial genomes and its amplicon was nested in that of the external primers, which amplified a 5.9 kb mtDNA-specific sequence (Fig. 1A). In case of SH-SY5Y, control and ADHD cybrids PCR product of external and internal primers is obtained, while in ρ0 cells lacking mitochondria, only nuclear sequence amplified by internal primers was observed. PicoGreen dye binds to minor groove of double stranded DNA and gives green fluorescence, so we can see the punctate mitochondria in the cytoplasm of SH-SY5Y cells. The punctate fluorescence is absent in ρ0 cells, but show only nuclear fluorescence because they do not contain mtDNA (Fig. 1B). The punctates observed in control, ADHD cybrids confirm their inheritance of mitochondria from platelets of healthy individuals and patients.


Attention deficit-hyperactivity disorder suffers from mitochondrial dysfunction
Long-template PCR analysis and PicoGreen®staining of cybrids, ρ0-cells and SH-SY5Y cells: (A) Genomic DNA from SH-SY5Y, ρ0-cells, control and ADHD cybrids were amplified using both internal and external primers and subjected to electrophoresis on 0.6% agarose gel. Amplicons are absent in the PCR using external primers for ρ0 cells. (B) Punctated cytoplasm and nuclei are fluorescent in SH-SY5Y cells, control and ADHD cybrids, whereas in ρ0 cells only the nuclei exhibited green fluorescence by Pico-green staining. C6, C9, C10 and C11 are control cybrids, ADHD1, 5 and 6 are cybrids prepared using platelets of ADHD probands. Scale bar represents 10 μm.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121149&req=5

f0005: Long-template PCR analysis and PicoGreen®staining of cybrids, ρ0-cells and SH-SY5Y cells: (A) Genomic DNA from SH-SY5Y, ρ0-cells, control and ADHD cybrids were amplified using both internal and external primers and subjected to electrophoresis on 0.6% agarose gel. Amplicons are absent in the PCR using external primers for ρ0 cells. (B) Punctated cytoplasm and nuclei are fluorescent in SH-SY5Y cells, control and ADHD cybrids, whereas in ρ0 cells only the nuclei exhibited green fluorescence by Pico-green staining. C6, C9, C10 and C11 are control cybrids, ADHD1, 5 and 6 are cybrids prepared using platelets of ADHD probands. Scale bar represents 10 μm.
Mentions: The inheritance of mitochondria was tested in ADHD samples employing long-template PCR method. The primer sets were designed for PCR so that the internal primers amplified a 5.8 kb sequence present in both the nuclear (chromosome 1) and mitochondrial genomes and its amplicon was nested in that of the external primers, which amplified a 5.9 kb mtDNA-specific sequence (Fig. 1A). In case of SH-SY5Y, control and ADHD cybrids PCR product of external and internal primers is obtained, while in ρ0 cells lacking mitochondria, only nuclear sequence amplified by internal primers was observed. PicoGreen dye binds to minor groove of double stranded DNA and gives green fluorescence, so we can see the punctate mitochondria in the cytoplasm of SH-SY5Y cells. The punctate fluorescence is absent in ρ0 cells, but show only nuclear fluorescence because they do not contain mtDNA (Fig. 1B). The punctates observed in control, ADHD cybrids confirm their inheritance of mitochondria from platelets of healthy individuals and patients.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Pathophysiology of attention-deficit hyperactivity disorder (ADHD) is not known, and therefore the present study investigated mitochondrial defects, if any in cybrids created from patients and control population.

Methods: To investigate mitochondrial pathology in ADHD, cybrids cell lines were created from ADHD probands and controls by fusing their platelets with ρ0-cells prepared from SH-SY5Y neuroblastoma cell line. Cellular respiration, oxidative stress, mitochondrial membrane potential and morphology were evaluated employing oxygraph, mitochondria-specific fluorescence staining and evaluation by FACS, and immunocytochemistry. HPLC-electrochemical detection, quantitative RT-PCR and Blue Native PAGE were employed respectively for assays of serotonin, mitochondrial ATPase 6/8 subunits levels and complex V activity.

Results: Significantly low cellular and mitochondrial respiration, ATPase6/8 transcripts levels, mitochondrial complex V activity and loss of mitochondrial membrane potential and elevated oxidative stress were observed in ADHD cybrids. Expression of monoamine oxidizing mitochondrial enzymes, MAO-A and MAO-B levels remained unaffected. Two-fold increase in serotonin level was noted in differentiated cybrid-neurons.

Conclusions: Since cybrids are shown to replicate mitochondrial defects seen in post-mortem brains, these observed defects in ADHD cybrids strongly suggest mitochondrial pathology in this disorder.

General significance: Mitochondrial defects are detected in ADHD cybrids created from patients' platelets, implying bioenergetics crisis in the mitochondria could be a contributory factor for ADHD pathology and/or phenotypes.

No MeSH data available.


Related in: MedlinePlus