Pantethine treatment is effective in recovering the disease phenotype induced by ketogenic diet in a pantothenate kinase-associated neurodegeneration mouse model

Abstract

Pantothenate kinase-associated neurodegeneration, caused by mutations in the PANK2 gene, is an autosomal recessive disorder characterized by dystonia, dysarthria, rigidity, pigmentary retinal degeneration and brain iron accumulation. PANK2 encodes the mitochondrial enzyme pantothenate kinase type 2, responsible for the phosphorylation of pantothenate or vitamin B5 in the biosynthesis of co-enzyme A. A Pank2 knockout (Pank2(-/-)) mouse model did not recapitulate the human disease but showed azoospermia and mitochondrial dysfunctions. We challenged this mouse model with a low glucose and high lipid content diet (ketogenic diet) to stimulate lipid use by mitochondrial beta-oxidation. In the presence of a shortage of co-enzyme A, this diet could evoke a general impairment of bioenergetic metabolism. Only Pank2(-/-) mice fed with a ketogenic diet developed a pantothenate kinase-associated neurodegeneration-like syndrome characterized by severe motor dysfunction, neurodegeneration and severely altered mitochondria in the central and peripheral nervous systems. These mice also showed structural alteration of muscle morphology, which was comparable with that observed in a patient with pantothenate kinase-associated neurodegeneration. We here demonstrate that pantethine administration can prevent the onset of the neuromuscular phenotype in mice suggesting the possibility of experimental treatment in patients with pantothenate kinase-associated neurodegeneration.

Keywords: ketogenic diet; mitochondria; pantethine; pantothenate kinase-associated neurodegeneration (PKAN).

Figure 1

Mice phenotype. (A) Body weight of Pank2^+/+ and Pank2^−/− mice fed a standard diet (SD). (B) Body weight of Pank2^+/+ and Pank2^−/− mice on a ketogenic diet (KD). (C) Kyphosis with hunched position; (D) feet clasping in ketogenic diet-fed Pank2^−/− (arrow); (E) whitening of the fur and rigidity of the tail in ketogenic diet-fed Pank2^−/−; (F) dystonic hindlimb position. (G) Body weight of Pank2^−/− mice was similar to that of Pank2^+/+ fed a ketogenic diet with pantethine administration (KD + P). (H) No sign of kyphosis or feet clasping were present in Pank2^−/− mice on a ketogenic diet with pantethine administration (arrow). (I) Comparison of Pank2^−/− body size in mice on standard (SD), ketogenic (KD) and ketogenic + pantethine (KD + P) diets (J) Survival curves for different diet conditions: Pank2^−/− mice fed a ketogenic diet died after 2 months (dotted green line); Pank2^+/+ mice fed a ketogenic (dotted yellow line); Pank2^+/+ mice fed a ketogenic diet with pantethine (dotted pink line); Pank2^−/− mice fed a ketogenic diet with pantethine administration prolonged their survival by up to 5 months (orange line); Pank2^−/− mice fed a standard diet (dotted brown line); and Pank2^+/+ mice fed a standard diet (blue line). In A, B and G, the red symbols indicate Pank2^−/−; blue symbols indicate Pank2^+/+.

Figure 2

Motor performance evaluation. (A) Activity cage: no differences were observed in Pank2^+/+ and Pank2^−/− mice on a standard diet (SD) or with pantethine (SD + P); Pank2^+/+mice were more active with pantethine treatment (*P < 0.05, two-tailed, unpaired Student’s t-test). Pank2^−/− mice fed a ketogenic diet were lethargic and showed a significant reduction of spontaneous movements as compared with Pank2^+/+ mice on a ketogenic diet (**P < 0.001, two-tailed, unpaired Student’s t-test). Pantethine treatment (KD+P) rescues the reduced movements. (B) Treadmill test: no differences in the distance travelled by Pank2^+/+ and Pank2^−/− mice on a standard diet (SD) or with pantethine (SD + P). Pank2^−/− mice on a ketogenic diet ran only 50 m as compared to 180 m of Pank2^+/+ mice (**P < 0.001, two-tailed, unpaired Student’s t-test). Pantethine treatment (KD + P) restored the running capability in Pank2^−/− mice and increased the performance in Pank2^+/+ mice (*P < 0.05, two-tailed, unpaired Student’s t-test). (C) Footprint pattern: Pank2^−/− mice fed a ketogenic diet showed shorter stride lengths and hind paw width, and an irregular gait as compared with Pank2^+/+ mice fed a ketogenic diet. Pantethine treatment (KD + P) abolishes the movement disorders. In A and B, the red bars indicate Pank2^−/− mice and blue bars indicate Pank2^+/+ mice, respectively.

Figure 3

Histology and immunohistochemistry of brains from Pank2^−/− mice fed a ketogenic diet and a ketogenic diet with pantethine. The analysed animals were 3 months old. Round, eosinophilic, periodic acid Schiff-positive cytoplasmic inclusions were present in neurons of Pank2^−/− mice fed a ketogenic diet (A–C, arrows in B and C). Cytoplasmic bodies are markedly positive on ubiquitin immunostain (D and F, arrows). Scattered neurons showed a finely granular cytoplasmic stain for ubiquitin (D and E, asterisks). There were large degenerating neurons with a diffuse positivity for ubiquitin (E, arrowhead). Ubiquitin stain was barely detected in neurons from Pank2^−/− mice fed a ketogenic diet with pantethine (G), which show histological and immunohistochemical features similar to their wild-type littermates (H). Scale bars = 25 µm.

Figure 4

Electron microscopy of basal ganglia in Pank2^+/+ and Pank2^−/− mice under different diet conditions. The analysed animals were 3 months old. Pank2^+/+ mice on a standard diet (SD), a standard diet + pantethine (SD + P), ketogenic diet (KD) and ketogenic diet + pantethine (KD + P) show normal mitochondria. Pank2^−/− mice on a standard diet show mitochondria with swollen cristae (asterisks and insert). With a ketogenic diet the mitochondrial morphology is worsened with focal loss of cristae (arrow and insert). Pank2^−/− mice on standard diet with pantethine were similar to wild-type littermates; with a ketogenic diet with pantethine there is a prevalence of mitochondria with normal cristae morphology, although a few mitochondria still showed swollen cristae.

Figure 5

Electron microscopy of peripheral nerve in Pank2^+/+ and Pank2^−/− mice under different diet conditions. The analysed animals were 3 months old. Sciatic nerve axons of Pank2^+/+ mice on a standard diet (SD), standard diet + pantethine (SD + P), ketogenic diet (KD) and ketogenic diet + pantethine (KD + P) contain morphologically normal mitochondria. Sciatic nerve axons of Pank2^−/− mice on a standard diet and ketogenic diet show swollen mitochondria with altered cristae (arrows). Sciatic nerve axons of Pank2^−/− mice on a standard diet + pantethine and ketogenic diet + pantethine show a high prevalence of normally-shaped mitochondria with regular cristae organization (arrowheads). Scale bars = 500 µm.

Figure 6

Membrane potential in Pank2^+/+ and Pank2^−/− neurons derived from mice under different diet conditions. Neurons derived from Pank2^+/+ animals treated with a standard (SD) or ketogenic diet (KD), with or without pantethine (P) presented red fluorescent aggregates indicating the preservation of the mitochondrial membrane potential. Neurons derived from Pank2^−/− mice treated with a standard or ketogenic diet presented with a diffuse green fluorescence confirming the presence of a defective mitochondrial membrane potential. Neurons derived from Pank2^−/− mice with standard and ketogenic treatment plus the addition of pantethine, showed predominantly red fluorescence aggregates, indicating preservation of mitochondrial membrane potential. Scale bars = 100 µm.

Figure 7

Evaluation of mitochondrial bioenergetics status. Oxygen consumption rate (OCR) in mitochondria isolated from brains of Pank2^+/+ and Pank2^−/− mice treated with pantethine and compared with untreated mice. We measured basal oxygen consumption rate (OCR-B) after ADP addition (OCR-ADP), and after oligomycin addition (OCR-O). Pantethine was able to significantly increase oxygen consumption rate in both Pank2^+/+ and Pank2^−/− mitochondria, especially ADP-induced respiration. Black, white and grey histograms indicate OCR-B, -ADP, and -O, respectively. Bars indicate the standard deviation (SD). **P < 0.01; *** P < 0.001 (unpaired, two-tail Student’s t-test). WT = wild-type; KO = knock-out.

Figure 8

Muscle COX (cytochrome c oxidase) histochemical reaction and electron microscopy. (A) Pank2^−/− mice on a ketogenic diet. (B) Patient with PKAN revealed the same peculiar staining pattern as mice fed a ketogenic diet. (C) Electron microscopy highlighted the presence of giant mitochondria spanning the sarcomere between two neighbouring Z-lines and showing irregularly shaped cristae (×20 000).

Figure 9

Gomori trichrome (TG) and COX histochemical reactions in muscle of Pank2^+/+ and Pank2^−/− mice under different diet conditions. No alterations were present in Pank2^+/+ mice. Ketogenic diet (KD) induced muscle histological abnormalities in Pank2^−/− mice (see also Fig. 8A) characterized by the presence of enlarged mitochondria. These alterations were rescued by pantethine administration (KD + P). Magnification × 400. SD = standard diet.