Therapeutic approach with commercial supplements for pantothenate kinase-associated neurodegeneration with residual PANK2 expression levels

Abstract

Background: Neurodegeneration with brain iron accumulation (NBIA) is a group of rare neurogenetic disorders frequently associated with iron accumulation in the basal nuclei of the brain characterized by progressive spasticity, dystonia, muscle rigidity, neuropsychiatric symptoms, and retinal degeneration or optic nerve atrophy. Pantothenate kinase-associated neurodegeneration (PKAN) is one of the most widespread NBIA subtypes. It is caused by mutations in the gene of pantothenate kinase 2 (PANK2) that result in dysfunction in PANK2 enzyme activity, with consequent deficiency of coenzyme A (CoA) biosynthesis, as well as low levels of essential metabolic intermediates such as 4'-phosphopantetheine, a necessary cofactor for essential cytosolic and mitochondrial proteins.

Methods: In this manuscript, we examined the therapeutic effectiveness of pantothenate, panthetine, antioxidants (vitamin E and omega 3) and mitochondrial function boosting supplements (L-carnitine and thiamine) in mutant PANK2 cells with residual expression levels.

Results: Commercial supplements, pantothenate, pantethine, vitamin E, omega 3, carnitine and thiamine were able to eliminate iron accumulation, increase PANK2, mtACP, and NFS1 expression levels and improve pathological alterations in mutant cells with residual PANK2 expression levels.

Conclusion: Our results suggest that several commercial compounds are indeed able to significantly correct the mutant phenotype in cellular models of PKAN. These compounds alone or in combinations are of common use in clinical practice and may be useful for the treatment of PKAN patients with residual enzyme expression levels.

Keywords: Acyl carrier protein; Carnitine; Coenzyme A; Omega 3; Pantethine; Pantothenate; Pantothenate kinase; Pantothenate kinase-associated neurodegeneration; Thiamine; Vitamin E.

Fig. 1

Effect of pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine treatment on iron accumulation and cell morphology in mutant PANK2 cells with residual PANK2 expression levels. a Control (C1) and PKAN fibroblasts (P1) were treated with pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine at 5 μM for 20 days. Then, cells were stained with Prussian Blue as described in Material and Methods and examined by bright-field microscopy. Scale bar = 15μm. b Quantification of Prussian Blue staining Images were analyzed by the Image J software. c Cell perimeter of untreated and treated Control and PKAN fibroblasts (P1). Images were analyzed by the Image J software. d Total iron content of untreated and treated control and PKAN cells was determined by ICP-MS as described in Material and Methods. Data represent the mean ± SD of three separate experiments. *p < 0.01 between Control and PKAN fibroblasts. ^ap < 0.01 between untreated and treated fibroblasts. A.U., arbitrary units

Fig. 2

Effect of pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine treatment on PANK 2 and mtACP expression levels in mutant PANK2 cells with residual PANK2 expression levels. a Control (C1) and PKAN fibroblasts (P1) were treated with pantothenate (Pant), pantethine (Pantethi), vitamin E (Vit E), omega 3 (O3), L-carnitine (L-carnit) or thiamine (Tiam) at 5 μM for 20 days. Protein extracts (50 μg) were separated on a SDS polyacrylamide gel and immunostained with antibodies against PANK2 and mtACP. Actin was used as a loading control. b Densitometry of the Western blotting of PANK2. c Densitometry of the Western blotting of mtACP. Data represent the mean ± SD of three separate experiments. *p < 0.01 between PKAN patients and controls. ^ap < 0.01 between untreated and treated fibroblasts. A.U., arbitrary units

Fig. 3

Dose response effect of pantothenate or pantethine treatment on PANK2, mtACP and NFS1 expression levels in mutant PANK2 cells with residual PANK2 expression levels. PKAN fibroblasts (P1) were treated with increasing concentrations of pantothenate a or pantethine b at 1, 5, 10, 50 and 100 μM for 20 days. Protein extracts (50 μg) were separated on a SDS polyacrylamide gel and immunostained with antibodies against PANK2, mtACP and NFS1. Actin was used as a loading control

Fig. 4

Dose response effect of vitamin E or omega 3 treatment on PANK2, mtACP and NFS1 expression levels in mutant PANK2 cells with residual PANK2 expression levels. PKAN fibroblasts (P1) were treated with increasing concentrations of vitamin E a or omega 3 b at 1, 5, 10, 50 and 100 μM for 20 days. Protein extracts (50 μg) were separated on a SDS polyacrylamide gel and immunostained with antibodies against PANK2, mtACP and NFS1. Actin was used as a loading control

Fig. 5

Dose response effect of carnitine or thiamine treatment on PANK2, mtACP and NFS1 expression levels in mutant PANK2 cells with residual PANK2 expression levels. PKAN fibroblasts (P1) were treated with increasing concentrations of carnitine a or thiamine (a) at 1, 5, 10, 50 and 100 μM for 20 days. Protein extracts (50 μg) were separated on a SDS polyacrylamide gel and immunostained with antibodies against PANK2, mtACP and NFS1. Actin was used as a loading control

Fig. 6

Effect of pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine treatment on PANK2 transcripts. a Control (C1) and patient P1 fibroblasts were treated with pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine at 5 μM for 20 days. PANK2 transcripts were quantified by qPCR as described in Material and Methods. b Protein expression levels of transcription factors NF-Y, FOXN4, hnRNPA/B, PGC-1alpha, PPGC-1 alpha and TFAM assessed by Western blotting. Data represent the mean ± SD of three separate experiments. *p < 0.01 between PKAN patients and controls. ^ap < 0.01 between untreated and treated fibroblasts. A.U., arbitrary units

Fig. 7

Effect of pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine treatment on lipid peroxidation, PDH activity and mitochondrial complex I activity in mutant PANK2 cells with residual PANK2 expression levels. P1 fibroblasts were treated with pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine at 5 μM for 20 days. a Lipid peroxidation was assessed by BODIPY staining and fluorescence microscopy analysis. b Quantification of BODIPY signal. c PDH activity in whole cellular extracts was determined as described in Material and Methods. d Quantification of PDH activity. e Mitochondrial complex I activity in whole cellular extracts was determined as described in Material and Methods. f Quantification of Complex I activity Data represent the mean ± SD of three separate experiments. *p < 0.01 between PKAN patients and controls; ^ap < 0.01 between untreated and treated cells

Fig. 8

Effect of pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine treatment on PANK 2 and mtACP expression levels in mutant PANK2 cells with residual PANK2 expression levels and mutant cells with expression of truncated PANK2. PKAN fibroblasts were treated with pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine at 5 μM for 20 days. Protein extracts (50 μg) were separated on a SDS polyacrylamide gel and immunostained with antibodies against PANK2 and mtACP. Actin was used as a loading control. a Western blotting of mutant of P2 fibroblasts. b Densitometry of Western blotting of mutant P2 fibroblasts. c Western blotting of mutant of P3 fibroblasts. d Densitometry of Western blotting of mutant P3 fibroblasts. e Western blotting of mutant of P4 fibroblasts. f Densitometry of Western blotting of mutant P4 fibroblasts. g Western blotting of mutant of P5 fibroblasts. h Densitometry of Western blotting of mutant P5 fibroblasts. Data represent the mean ± SD of three separate experiments. *p < 0.01 between PKAN patients and controls. ^ap < 0.01 between untreated and treated fibroblasts. A.U., arbitrary units