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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

Mitotracker Green®,TMRM and MitoSOX™ staining of control and ADHD cybrids: (A) Confocal images of Mitotracker Green® staining of the cybrids. (B) The intensity of Mitotracker Green® fluorescence quantified by Image J software. (C & D) The graphs showing the percent change of TMRM and MitoSOX™ fluorescence staining measured by fluorimetry and (E & G) FACS analysis, in ADHD as compared to the control cybrids. (F & H) are the representative scan of cybrids. Data are presented as Mean ± SEM. Values of p* ≤ 0.05 are considered significant. C6 and C9 are control cybrids, ADHD1, 5 and 6 are cybrids prepared using platelets of ADHD probands. Scale bar represents 10 μm.
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f0010: Mitotracker Green®,TMRM and MitoSOX™ staining of control and ADHD cybrids: (A) Confocal images of Mitotracker Green® staining of the cybrids. (B) The intensity of Mitotracker Green® fluorescence quantified by Image J software. (C & D) The graphs showing the percent change of TMRM and MitoSOX™ fluorescence staining measured by fluorimetry and (E & G) FACS analysis, in ADHD as compared to the control cybrids. (F & H) are the representative scan of cybrids. Data are presented as Mean ± SEM. Values of p* ≤ 0.05 are considered significant. C6 and C9 are control cybrids, ADHD1, 5 and 6 are cybrids prepared using platelets of ADHD probands. Scale bar represents 10 μm.

Mentions: Mitotracker Green enters mitochondrial matrix where it covalently reacts with free thiol groups of cysteine residues of mitochondrial proteins and produce green fluorescence which allows visualization of the mitochondria. SH-SY5Y cells are showing elongated mitochondria and ρ0 cells have fragmented mitochondria (Fig. 2A). Bar diagrams represents the fluorescence intensity in the control and ADHD cybrids measured using Image J software which shows significant reduction in ADHD cybrids compared to controls (Fig. 2B). Mitochondrial membrane potential was examined in cybrid neurons with TMRM staining followed by spectrofluorimetric (Fig. 2C) quantification as well as flow cytometric (Fig. 2E and F) procedure. Significantly low TMRM fluorescence observed in ADHD cybrid-neurons implied higher mitochondrial depolarisation in these cells. Mitochondrial membrane potential was also analysed in differentiated cybrid neurons [20] by fluorimetric and FACS analysis and we observed similar results (data are not shown) as in undifferentiated neurons.


Attention deficit-hyperactivity disorder suffers from mitochondrial dysfunction
Mitotracker Green®,TMRM and MitoSOX™ staining of control and ADHD cybrids: (A) Confocal images of Mitotracker Green® staining of the cybrids. (B) The intensity of Mitotracker Green® fluorescence quantified by Image J software. (C & D) The graphs showing the percent change of TMRM and MitoSOX™ fluorescence staining measured by fluorimetry and (E & G) FACS analysis, in ADHD as compared to the control cybrids. (F & H) are the representative scan of cybrids. Data are presented as Mean ± SEM. Values of p* ≤ 0.05 are considered significant. C6 and C9 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

f0010: Mitotracker Green®,TMRM and MitoSOX™ staining of control and ADHD cybrids: (A) Confocal images of Mitotracker Green® staining of the cybrids. (B) The intensity of Mitotracker Green® fluorescence quantified by Image J software. (C & D) The graphs showing the percent change of TMRM and MitoSOX™ fluorescence staining measured by fluorimetry and (E & G) FACS analysis, in ADHD as compared to the control cybrids. (F & H) are the representative scan of cybrids. Data are presented as Mean ± SEM. Values of p* ≤ 0.05 are considered significant. C6 and C9 are control cybrids, ADHD1, 5 and 6 are cybrids prepared using platelets of ADHD probands. Scale bar represents 10 μm.
Mentions: Mitotracker Green enters mitochondrial matrix where it covalently reacts with free thiol groups of cysteine residues of mitochondrial proteins and produce green fluorescence which allows visualization of the mitochondria. SH-SY5Y cells are showing elongated mitochondria and ρ0 cells have fragmented mitochondria (Fig. 2A). Bar diagrams represents the fluorescence intensity in the control and ADHD cybrids measured using Image J software which shows significant reduction in ADHD cybrids compared to controls (Fig. 2B). Mitochondrial membrane potential was examined in cybrid neurons with TMRM staining followed by spectrofluorimetric (Fig. 2C) quantification as well as flow cytometric (Fig. 2E and F) procedure. Significantly low TMRM fluorescence observed in ADHD cybrid-neurons implied higher mitochondrial depolarisation in these cells. Mitochondrial membrane potential was also analysed in differentiated cybrid neurons [20] by fluorimetric and FACS analysis and we observed similar results (data are not shown) as in undifferentiated neurons.

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