Pantothenate kinase-associated neurodegeneration: altered mitochondria membrane potential and defective respiration in Pank2 knock-out mouse model

Abstract

Neurodegeneration with brain iron accumulation (NBIA) comprises a group of neurodegenerative disorders characterized by high brain content of iron and presence of axonal spheroids. Mutations in the PANK2 gene, which encodes pantothenate kinase 2, underlie an autosomal recessive inborn error of coenzyme A metabolism, called pantothenate kinase-associated neurodegeneration (PKAN). PKAN is characterized by dystonia, dysarthria, rigidity and pigmentary retinal degeneration. The pathogenesis of this disorder is poorly understood and, although PANK2 is a mitochondrial protein, perturbations in mitochondrial bioenergetics have not been reported. A knock-out (KO) mouse model of PKAN exhibits retinal degeneration and azoospermia, but lacks any neurological phenotype. The absence of a clinical phenotype has partially been explained by the different cellular localization of the human and murine PANK2 proteins. Here we demonstrate that the mouse Pank2 protein localizes to mitochondria, similar to its human orthologue. Moreover, we show that Pank2-defective neurons derived from KO mice have an altered mitochondrial membrane potential, a defect further corroborated by the observations of swollen mitochondria at the ultra-structural level and by the presence of defective respiration.

Figure 1.

Mitochondrial localization of murine Pank2 protein. (A) Western blot analysis of different sub-cellular fractions derived from mouse brain: +/+ indicates Pank2^+/+ animals; −/− indicates Pank2^−/− animals. The same filter was incubated with anti-PANK2, anti-NDUFA9 and anti-SDH70 antibodies. (B) Western blot analysis on different mitochondrial compartments isolated from WT mouse brain. (C) Western blot analysis on different mitochondrial compartments isolated from WT mouse fibroblasts. In (B and C) the filters were sequentially incubated with anti-PANK2, anti-NDUFA9, anti-SDH70 and anti-ETHE1 antibodies.

Figure 2.

Evaluation of mitochondrial bioenergetics status. (A) OCR in Pank2^+/+ and Pank2^−/− mouse brain mitochondria. Black, white and grey histograms indicate OCR-B, -ADP and -O, respectively. Bars indicate the standard deviation (SD). **P < 0.01 (unpaired, two-tail Student's t-test). (B) Analysis of OCR-B, -ADP, -O and -F. The z-score values (dots) for OCR-B, -ADP, -O and -F are shown for Pank2^+/+ versus Pank2^−/−. Data are expressed as the means (dots) and 95% confidence intervals, Confidence interval (bars). Student's t-test for Pank2^+/+ versus Pank2^−/−: diamond, P = 1.0E−07; square, P = 1.3E−04; triangle, P = 1.0E−07; asterisk, P = 1.2E−09. Three Pank2^+/+ and three Pank2^−/− mice of 4-week-old were analysed.

Figure 3.

Mitochondrial morphology evaluated with the Mitotracker-red. (A) Neurons derived from hair bulge from Pank2^+/+ mice. (B) Neurons derived from hair bulge from Pank2^−/− mice. (C) Neurons derived from sciatic nerve of Pank2^+/+ mice. (D) Neurons derived from sciatic nerve of Pank2^−/− mice. (E) Control fibroblasts. (F) Control fibroblasts treated with valinomycin. (G) Histogram reporting the quantification of Mitotracker diffusion in Pank2^+/+ and Pank2^−/− neurons using the RawIntDen parameter (see Materials and Methods). Bars indicated SD. *P = 3.37E−23 (unpaired, two-tail Student's t-test).

Figure 4.

Membrane potential and electron microscopy in Pank2^+/+ and Pank2^−/− neurons. (A and B) JC1 staining of neurons derived from hair bulge. (C and D) JC1 staining of neurons derived from sciatic nerve. (E and F) JC1 staining of neonatal neuron derived from hippocampus. Blue fluorescence indicates DAPI staining of nuclei. Green fluorescence indicates an alteration in mitochondrial ΔΨ; red fluorescence indicates the preservation of a normal mitochondrial ΔΨ. (G and H) Electron microscopy of neurons derived from sciatic nerve (semithin section Toluidin Blue). (I–L) Electron microscopy of neurons derived from sciatic nerve (ultrastructure uranyl acetate and lead citrate). Normal appearing mitochondria in Pank2^+/+ neurons (I–K) and few normal mitochondria in Pank2^−/− neurons (black arrows in J–L); aberrant mitochondria with disrupted matrix and cristae (red arrows in J–L); lipid filled vesicle (asterisk in J).

Figure 5.

Evaluation of neurodegeneration of peripheral nervous systems with optic and electron microscopy. Sciatic nerve semithin section (Toluidin Blue) of (A) 24-week-old and (C) 48-week-old Pank2^+/+ mice; (B) 24-week-old and (D) 48-week-old Pank2^−/− mice. Fibre density is not clearly different, whereas the axonal diameter seems to be reduced in Pank2^−/−. Electron micrographs of 48-week-old mice sciatic nerve in Pank2^+/+ mice (E) and Pank2^−/− mice (F). No sign of un-myelinated fibre loss in the form of collagen pockets or denervated Schwann cells were evident; in Pank2^−/− mice, some enlarged mitochondria were detected (arrows in F).

Figure 6.

Electron microscopy of peripheral and CNS of 24- and 48-week-old animals. (A–C) Sciatic nerve of Pank2^+/+ mice show normal mitochondria (arrows). (B–D) Sciatic nerve of Pank2^−/− mice show swollen mitochondria with altered cristae (arrows). (E–G) Basal ganglia derived from 48-week-old Pank2^+/+ mice show normally shaped mitochondria. (F–H) Basal ganglia derived from 48-week-old Pank2^−/− mice show altered cristae and enlarged cisternae.