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LETM1 haploinsufficiency causes mitochondrial defects in cells from humans with Wolf-Hirschhorn syndrome: implications for dissecting the underlying pathomechanisms in this condition.

Hart L, Rauch A, Carr AM, Vermeesch JR, O'Driscoll M - Dis Model Mech (2014)

Bottom Line: Growing evidence derived from several model organisms suggests that reduced LETM1 expression is associated with some element of mitochondrial dysfunction.Here, using a unique panel of WHS-patient-derived cell lines with deletions of differing sizes, incorporating LETM1 or not, we show, for the first time, that LETM1 expression is reduced in mitochondria isolated from WHS-patient cells.Our findings identify novel cellular phenotypes in WHS attributable to a 50% reduction in LETM1 expression level; these phenotypes could underlie and/or contribute to some of the core clinical features of this condition.

View Article: PubMed Central - PubMed

Affiliation: Human DNA Damage Response Disorders Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RQ, UK.

ABSTRACT
Wolf-Hirschhorn syndrome (WHS) represents an archetypical example of a contiguous gene deletion disorder - a condition comprising a complex set of developmental phenotypes with a multigenic origin. Epileptic seizures, intellectual disability, growth restriction, motor delay and hypotonia are major co-morbidities in WHS. Haploinsufficiency of LETM1, which encodes a mitochondrial inner-membrane protein functioning in ion transport, has been proposed as an underlying pathomechanism, principally for seizures but also for other core features of WHS, including growth and motor delay. Growing evidence derived from several model organisms suggests that reduced LETM1 expression is associated with some element of mitochondrial dysfunction. Surprisingly, LETM1-dependent mitochondrial functional deficits have not previously been described in cells from individuals with WHS. Here, using a unique panel of WHS-patient-derived cell lines with deletions of differing sizes, incorporating LETM1 or not, we show, for the first time, that LETM1 expression is reduced in mitochondria isolated from WHS-patient cells. Furthermore, we show that this is associated with distinct mitochondrial phenotypes, including altered intracellular [Ca(2+)] levels, dysfunctional mitochondrial transition-pore opening, hyperpolarization and superoxide leakage from resting mitochondria. Interestingly, we find that these phenotypes segregate with seizures in our WHS cohort. Our findings identify novel cellular phenotypes in WHS attributable to a 50% reduction in LETM1 expression level; these phenotypes could underlie and/or contribute to some of the core clinical features of this condition.

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Related in: MedlinePlus

Mitochondrial [O2−] and total cellular ROS levels in WHS-patient LCLs. (A) Representative fluorescence profiles following treatment of WT and WHS-patient LCLs with MitoTracker-SOX (250 nM for 15 minutes) as obtained from the FACS Canto platform. Elevated levels of mitochondrial-derived ROS (O2−) are indicated by the black triangle. The WHS LCLs with LETM1 haploinsufficiency show marked peaks towards the right of each profile (elevated fluorescence) compared with WT LCLs and those from the WHS patients with normal LETM1 copy number [C(355) and FN4367]. A control experiment using thalidomide, a drug known to produce reactive oxygen species, is shown in supplementary material Fig. S1C. (B) Mean relative fluorescence of MitoTracker-SOX in WHS LCLs is shown relative to that of wild-type (WT) normal LCLs, the latter illustrated by the horizontal dashed line. WHS LCLs A(83), D(78) and E(88) exhibit elevated MitoTracker-SOX fluorescence indicative of elevated O2− production from resting mitochondria, compared with WT LCLs (dashed line) and those from patients C(355) and FN4367 (P<0.05, Student’s t-test). Furthermore, a MERRF (myoclonus epilepsy associate with ragged-red fibres) patient-derived LCL also exhibited a similarly significantly elevated MitoTracker-SOX fluorescence to that of the LETM1+/− WHS LCLs. Data represent the mean ± s.d. from three separate experiments (PE-A: area under the curve). (C) Mean relative fluorescence of CellROX-Red in WHS LCLs is shown relative to that of wild-type (WT) normal LCLs, the latter illustrated by the horizontal dashed line. CellROX-Red fluorescence is a measure of total cellular ROS. Pre-treatment of WT LCLs with the oxidant tert-Butyl hydroperoxide (tBOOH; 100 μM, 1 hour) resulted in an ~fivefold increase in CellROX-Red mean fluorescence relative to untreated WT LCLs. All of the WHS LCLs, irrespective of LETM1 copy number, exhibit a comparable level of CellROX-Red fluorescence compared with WT. Data represent the mean ± s.d. from three separate experiments (PE-A: area under the curve).
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f5-0070535: Mitochondrial [O2−] and total cellular ROS levels in WHS-patient LCLs. (A) Representative fluorescence profiles following treatment of WT and WHS-patient LCLs with MitoTracker-SOX (250 nM for 15 minutes) as obtained from the FACS Canto platform. Elevated levels of mitochondrial-derived ROS (O2−) are indicated by the black triangle. The WHS LCLs with LETM1 haploinsufficiency show marked peaks towards the right of each profile (elevated fluorescence) compared with WT LCLs and those from the WHS patients with normal LETM1 copy number [C(355) and FN4367]. A control experiment using thalidomide, a drug known to produce reactive oxygen species, is shown in supplementary material Fig. S1C. (B) Mean relative fluorescence of MitoTracker-SOX in WHS LCLs is shown relative to that of wild-type (WT) normal LCLs, the latter illustrated by the horizontal dashed line. WHS LCLs A(83), D(78) and E(88) exhibit elevated MitoTracker-SOX fluorescence indicative of elevated O2− production from resting mitochondria, compared with WT LCLs (dashed line) and those from patients C(355) and FN4367 (P<0.05, Student’s t-test). Furthermore, a MERRF (myoclonus epilepsy associate with ragged-red fibres) patient-derived LCL also exhibited a similarly significantly elevated MitoTracker-SOX fluorescence to that of the LETM1+/− WHS LCLs. Data represent the mean ± s.d. from three separate experiments (PE-A: area under the curve). (C) Mean relative fluorescence of CellROX-Red in WHS LCLs is shown relative to that of wild-type (WT) normal LCLs, the latter illustrated by the horizontal dashed line. CellROX-Red fluorescence is a measure of total cellular ROS. Pre-treatment of WT LCLs with the oxidant tert-Butyl hydroperoxide (tBOOH; 100 μM, 1 hour) resulted in an ~fivefold increase in CellROX-Red mean fluorescence relative to untreated WT LCLs. All of the WHS LCLs, irrespective of LETM1 copy number, exhibit a comparable level of CellROX-Red fluorescence compared with WT. Data represent the mean ± s.d. from three separate experiments (PE-A: area under the curve).

Mentions: Mitochondrial dysfunction is often associated with elevated superoxide (O2−) leakage (Kang and Pervaiz, 2012; Pieczenik and Neustadt, 2007). Using MitoTracker-SOX fluorescence as a direct measure of mitochondrial [O2−], we found a striking increase (~1.4-to 1.6-fold) in mitochondrial ROS in WHS-patient LCLs with LETM1 haploinsufficiency, specifically A(83), D(78) and E(88) patient LCLs, in contrast to WHS-patient cells with normal LETM1 copy number [i.e. C(355), FN4367] and to WT LCLs (Fig. 5A,B). In fact, the elevated level of mitochondrial O2− production in these LCLs was comparable to that of an LCL derived from a patient with myoclonus epilepsy with ragged-red fibres (MERRF) (Fig. 5B). MERFF is a primary mitochondrial disorder. Interestingly, elevated mitochondrially derived O2− was not associated with an overall elevated level of intracellular ROS in these cells, as determined by CellROX-Red fluorescence (Fig. 5C). These data further indicate that LETM1 haploinsufficiency in the context of WHS is associated with specific mitochondrial dysfunctions.


LETM1 haploinsufficiency causes mitochondrial defects in cells from humans with Wolf-Hirschhorn syndrome: implications for dissecting the underlying pathomechanisms in this condition.

Hart L, Rauch A, Carr AM, Vermeesch JR, O'Driscoll M - Dis Model Mech (2014)

Mitochondrial [O2−] and total cellular ROS levels in WHS-patient LCLs. (A) Representative fluorescence profiles following treatment of WT and WHS-patient LCLs with MitoTracker-SOX (250 nM for 15 minutes) as obtained from the FACS Canto platform. Elevated levels of mitochondrial-derived ROS (O2−) are indicated by the black triangle. The WHS LCLs with LETM1 haploinsufficiency show marked peaks towards the right of each profile (elevated fluorescence) compared with WT LCLs and those from the WHS patients with normal LETM1 copy number [C(355) and FN4367]. A control experiment using thalidomide, a drug known to produce reactive oxygen species, is shown in supplementary material Fig. S1C. (B) Mean relative fluorescence of MitoTracker-SOX in WHS LCLs is shown relative to that of wild-type (WT) normal LCLs, the latter illustrated by the horizontal dashed line. WHS LCLs A(83), D(78) and E(88) exhibit elevated MitoTracker-SOX fluorescence indicative of elevated O2− production from resting mitochondria, compared with WT LCLs (dashed line) and those from patients C(355) and FN4367 (P<0.05, Student’s t-test). Furthermore, a MERRF (myoclonus epilepsy associate with ragged-red fibres) patient-derived LCL also exhibited a similarly significantly elevated MitoTracker-SOX fluorescence to that of the LETM1+/− WHS LCLs. Data represent the mean ± s.d. from three separate experiments (PE-A: area under the curve). (C) Mean relative fluorescence of CellROX-Red in WHS LCLs is shown relative to that of wild-type (WT) normal LCLs, the latter illustrated by the horizontal dashed line. CellROX-Red fluorescence is a measure of total cellular ROS. Pre-treatment of WT LCLs with the oxidant tert-Butyl hydroperoxide (tBOOH; 100 μM, 1 hour) resulted in an ~fivefold increase in CellROX-Red mean fluorescence relative to untreated WT LCLs. All of the WHS LCLs, irrespective of LETM1 copy number, exhibit a comparable level of CellROX-Red fluorescence compared with WT. Data represent the mean ± s.d. from three separate experiments (PE-A: area under the curve).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4007405&req=5

f5-0070535: Mitochondrial [O2−] and total cellular ROS levels in WHS-patient LCLs. (A) Representative fluorescence profiles following treatment of WT and WHS-patient LCLs with MitoTracker-SOX (250 nM for 15 minutes) as obtained from the FACS Canto platform. Elevated levels of mitochondrial-derived ROS (O2−) are indicated by the black triangle. The WHS LCLs with LETM1 haploinsufficiency show marked peaks towards the right of each profile (elevated fluorescence) compared with WT LCLs and those from the WHS patients with normal LETM1 copy number [C(355) and FN4367]. A control experiment using thalidomide, a drug known to produce reactive oxygen species, is shown in supplementary material Fig. S1C. (B) Mean relative fluorescence of MitoTracker-SOX in WHS LCLs is shown relative to that of wild-type (WT) normal LCLs, the latter illustrated by the horizontal dashed line. WHS LCLs A(83), D(78) and E(88) exhibit elevated MitoTracker-SOX fluorescence indicative of elevated O2− production from resting mitochondria, compared with WT LCLs (dashed line) and those from patients C(355) and FN4367 (P<0.05, Student’s t-test). Furthermore, a MERRF (myoclonus epilepsy associate with ragged-red fibres) patient-derived LCL also exhibited a similarly significantly elevated MitoTracker-SOX fluorescence to that of the LETM1+/− WHS LCLs. Data represent the mean ± s.d. from three separate experiments (PE-A: area under the curve). (C) Mean relative fluorescence of CellROX-Red in WHS LCLs is shown relative to that of wild-type (WT) normal LCLs, the latter illustrated by the horizontal dashed line. CellROX-Red fluorescence is a measure of total cellular ROS. Pre-treatment of WT LCLs with the oxidant tert-Butyl hydroperoxide (tBOOH; 100 μM, 1 hour) resulted in an ~fivefold increase in CellROX-Red mean fluorescence relative to untreated WT LCLs. All of the WHS LCLs, irrespective of LETM1 copy number, exhibit a comparable level of CellROX-Red fluorescence compared with WT. Data represent the mean ± s.d. from three separate experiments (PE-A: area under the curve).
Mentions: Mitochondrial dysfunction is often associated with elevated superoxide (O2−) leakage (Kang and Pervaiz, 2012; Pieczenik and Neustadt, 2007). Using MitoTracker-SOX fluorescence as a direct measure of mitochondrial [O2−], we found a striking increase (~1.4-to 1.6-fold) in mitochondrial ROS in WHS-patient LCLs with LETM1 haploinsufficiency, specifically A(83), D(78) and E(88) patient LCLs, in contrast to WHS-patient cells with normal LETM1 copy number [i.e. C(355), FN4367] and to WT LCLs (Fig. 5A,B). In fact, the elevated level of mitochondrial O2− production in these LCLs was comparable to that of an LCL derived from a patient with myoclonus epilepsy with ragged-red fibres (MERRF) (Fig. 5B). MERFF is a primary mitochondrial disorder. Interestingly, elevated mitochondrially derived O2− was not associated with an overall elevated level of intracellular ROS in these cells, as determined by CellROX-Red fluorescence (Fig. 5C). These data further indicate that LETM1 haploinsufficiency in the context of WHS is associated with specific mitochondrial dysfunctions.

Bottom Line: Growing evidence derived from several model organisms suggests that reduced LETM1 expression is associated with some element of mitochondrial dysfunction.Here, using a unique panel of WHS-patient-derived cell lines with deletions of differing sizes, incorporating LETM1 or not, we show, for the first time, that LETM1 expression is reduced in mitochondria isolated from WHS-patient cells.Our findings identify novel cellular phenotypes in WHS attributable to a 50% reduction in LETM1 expression level; these phenotypes could underlie and/or contribute to some of the core clinical features of this condition.

View Article: PubMed Central - PubMed

Affiliation: Human DNA Damage Response Disorders Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RQ, UK.

ABSTRACT
Wolf-Hirschhorn syndrome (WHS) represents an archetypical example of a contiguous gene deletion disorder - a condition comprising a complex set of developmental phenotypes with a multigenic origin. Epileptic seizures, intellectual disability, growth restriction, motor delay and hypotonia are major co-morbidities in WHS. Haploinsufficiency of LETM1, which encodes a mitochondrial inner-membrane protein functioning in ion transport, has been proposed as an underlying pathomechanism, principally for seizures but also for other core features of WHS, including growth and motor delay. Growing evidence derived from several model organisms suggests that reduced LETM1 expression is associated with some element of mitochondrial dysfunction. Surprisingly, LETM1-dependent mitochondrial functional deficits have not previously been described in cells from individuals with WHS. Here, using a unique panel of WHS-patient-derived cell lines with deletions of differing sizes, incorporating LETM1 or not, we show, for the first time, that LETM1 expression is reduced in mitochondria isolated from WHS-patient cells. Furthermore, we show that this is associated with distinct mitochondrial phenotypes, including altered intracellular [Ca(2+)] levels, dysfunctional mitochondrial transition-pore opening, hyperpolarization and superoxide leakage from resting mitochondria. Interestingly, we find that these phenotypes segregate with seizures in our WHS cohort. Our findings identify novel cellular phenotypes in WHS attributable to a 50% reduction in LETM1 expression level; these phenotypes could underlie and/or contribute to some of the core clinical features of this condition.

Show MeSH
Related in: MedlinePlus