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. 2017 Nov 1;10(11):1343-1352.
doi: 10.1242/dmm.030502.

Early cerebellar deficits in mitochondrial biogenesis and respiratory chain complexes in the KIKO mouse model of Friedreich ataxia

Hong Lin  1 , Jordi Magrane  2 , Amy Rattelle  1 , Anna Stepanova  2   3 , Alexander Galkin  2   3 , Elisia M Clark  1   4 , Yi Na Dong  1 , Sarah M Halawani  1 , David R Lynch  5   4
Affiliations

Early cerebellar deficits in mitochondrial biogenesis and respiratory chain complexes in the KIKO mouse model of Friedreich ataxia

Hong Lin et al. Dis Model Mech. .

Erratum in

Abstract

Friedreich ataxia (FRDA), the most common recessive inherited ataxia, results from deficiency of frataxin, a small mitochondrial protein crucial for iron-sulphur cluster formation and ATP production. Frataxin deficiency is associated with mitochondrial dysfunction in FRDA patients and animal models; however, early mitochondrial pathology in FRDA cerebellum remains elusive. Using frataxin knock-in/knockout (KIKO) mice and KIKO mice carrying the mitoDendra transgene, we show early cerebellar deficits in mitochondrial biogenesis and respiratory chain complexes in this FRDA model. At asymptomatic stages, the levels of PGC-1α (PPARGC1A), the mitochondrial biogenesis master regulator, are significantly decreased in cerebellar homogenates of KIKO mice compared with age-matched controls. Similarly, the levels of the PGC-1α downstream effectors, NRF1 and Tfam, are significantly decreased, suggesting early impaired cerebellar mitochondrial biogenesis pathways. Early mitochondrial deficiency is further supported by significant reduction of the mitochondrial markers GRP75 (HSPA9) and mitofusin-1 in the cerebellar cortex. Moreover, the numbers of Dendra-labeled mitochondria are significantly decreased in cerebellar cortex, confirming asymptomatic cerebellar mitochondrial biogenesis deficits. Functionally, complex I and II enzyme activities are significantly reduced in isolated mitochondria and tissue homogenates from asymptomatic KIKO cerebella. Structurally, levels of the complex I core subunit NUDFB8 and complex II subunits SDHA and SDHB are significantly lower than those in age-matched controls. These results demonstrate complex I and II deficiency in KIKO cerebellum, consistent with defects identified in FRDA patient tissues. Thus, our findings identify early cerebellar mitochondrial biogenesis deficits as a potential mediator of cerebellar dysfunction and ataxia, thereby providing a potential therapeutic target for early intervention of FRDA.

Keywords: Cerebellum; Friedreich ataxia; Mitochondrial biogenesis; Neurodegenerative diseases; Respiratory chain complex.

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Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Levels of the mitochondrial biogenesis master regulator PGC-1α are significantly decreased in the cerebellum of frataxin-deficient KIKO mice at asymptomatic and symptomatic ages. Western blotting of cerebellar homogenates (30 μg per lane) showing frataxin (A,B) and PGC-1α (C,D) levels, as well as actin as an internal control, in the cerebellum of KIKO mice and age-matched controls at postnatal days P30, P90, P180 and P270 (n=3-8 for KIKO and control mice per time point; #P<0.05, **P<0.01, ***P<0.001; two-tailed, unpaired Student's t-test). Blots in Fig. 1 were stripped and reprobed with multiple antibodies in Figs 2 and 6; α-actin served as the loading control for each.
Fig. 2.
Fig. 2.
Levels of PGC-1α effectors NRF1 and Tfam are significantly decreased in KIKO cerebellum at asymptomatic and symptomatic ages. Western blotting and quantification of cerebellar homogenates (30 μg per lane) showing NRF1 (A,B) and Tfam (C,D) levels, as well as actin as an internal control, in the cerebellum of KIKO mice and controls at P30, P90, P180 and P270 (n=3-8 for KIKO and control mice per time point; #P<0.05, *P<0.05, **P<0.01; two-tailed, unpaired Student's t-test). Blots in Fig. 2 were stripped and reprobed with multiple antibodies in Figs 1, 3 and 6; α-actin served as the loading control for each.
Fig. 3.
Fig. 3.
Levels of the mitochondrial markers GRP75 and MFN1 are significantly decreased in KIKO cerebellum at symptomatic and symptomatic ages. Western blotting and quantification of cerebellar homogenates (30 μg per lane) showing GRP75 (A,B) and MFN1 (C,D) levels, as well as actin as an internal control, in the cerebellum of KIKO mice and controls at P30, P90, P180 and P270 (n=3-8 for KIKO and control mice per time point; *P<0.05, **P<0.01 ***P<0.001; two-tailed, unpaired Student's t-test). Blots in Fig. 3 were stripped and reprobed with multiple antibodies in Figs 2 and Fig 6; α-actin served as the loading control for each.
Fig. 4.
Fig. 4.
Reduction of GRP75 and MFN1 in the cerebellar cortex of asymptomatic mitoDendra-KIKO mice. (A-F) Confocal images of mitoDendra (green), GRP75 (red) and merged images with DAPI-stained nuclei, showing a reduction in the overall levels of mitoDendra and GRP75 in the cerebellar cortex of mitoDendra-KIKO mice (D-F) compared with control mice (A-C) at P90. (G-L) Confocal images of mitoDendra (green), MFN1 (red) and merged images with DAPI-stained nuclei, showing a reduction in the overall levels of MFN1 and mitoDendra in the cerebellar cortex of mitoDendra-KIKO mice (J-L) compared with control mice (G-I) at P90. GL, granular layer; ML, molecular layer; PL, Purkinje layer. Scale bars: 50 μm.
Fig. 5.
Fig. 5.
Levels and number of mitoDendra are significantly reduced in the cerebellar cortex of asymptomatic mitoDendra-KIKO mice. (A-D) Confocal images of mitoDendra (green) merged images with DAPI-stained nuclei, showing marked reduction in the number of Dendra-labeled mitochondria in the cerebellar ML (B) and GL (D) of P90 KIKO mice compared with control mice (A,C). Insets are higher magnification confocal images, showing marked reduction of Dendra-labeled mitochondrial puncta in the cerebellar ML (B) and GL (D) of mitoDendra-KIKO mice compared with control mice (A,C). (E,F) Quantification of levels and number of mitoDendra puncta per μm2, showing a significant decrease in the levels and number of mitoDendra in the cerebellar cortex of mitoDendra-KIKO mice compared with control mice (E) (n=5 sections per animal; three animals per group; ***P<0.001, two-tailed, unpaired Student's t-test). Scale bars: 25 μm (5 μm in insets).
Fig. 6.
Fig. 6.
Mitochondrial complex I and II subunits levels are significantly reduced in the cerebellum of asymptomatic KIKO mice. Western blotting and quantification of cerebellar homogenates (30 μg per lane), showing complex II subunit SDHA (A,B) and complex I-V markers NDUFB8, SDHB, UQCRC2, MTCO1 and ATP5A (C,D) levels, as well as actin as an internal control, in the cerebellum of KIKO mice and controls at P30, P90, P180 and P270 (n=3-8 for KIKO and control mice per time point; *P<0.05, **P<0.01, ***P<0.001; two-tailed, unpaired Student's t-test). Blots in Fig. 6 were stripped and reprobed with multiple antibodies in Figs 1-3; α-actin served as the loading control for each.
Fig. 7.
Fig. 7.
Mitochondrial complex I and II activities are compromised in the cerebellar homogenates of asymptomatic KIKO mice. Complex I and II activity assay in the cerebellar homogenates of KIKO mice compared with control mice at P90. All activity values are expressed as percentages of mean control values (CI, n=4 mice per group; CII, n=3 mice per group; *P<0.05, two-tailed, unpaired Student's t-test).
Fig. 8.
Fig. 8.
Reduction of complex I, II and IV activities in isolated mitochondria from the cerebellum of asymptomatic KIKO mice. Mitochondria were isolated from the cerebellum of P90 mice. Activities of several mitochondrial respiratory chain complexes in the cerebellum of KIKO and control mice were measured. All activity values were normalized to citrate synthase and expressed as percentages of those of control mice (n=3 mice per group; *P<0.05, **P<0.01, two-tailed, unpaired Student's t-test).
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