Pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine treatment partially reduce iron accumulation and increase PANK2 and mtACP expression levels in mutant PKAN cells with residual PANK2 expression.
First, we identified six compounds able to eliminate intracellular iron accumulation and senescent cell morphology in patient P1 harboring a double heterozygous mutation (one allele harbors a stop codon mutation and the other a missense mutation) with residual levels of PANK2 protein. As shown in Figure 1a, 1b and 1c pantothenate, pantethine, vitamin E, omega 3, L-carnitine and thiamine at 5µM significantly reduced Prussian Blue staining and normalized cell morphology in P1 fibroblasts. Iron accumulation and elimination by positive compounds in mutant PANK2 cells were corroborate by ICP-MS assays (Figure 1d).
Next, to examine if any residual PANK2 enzyme could be stabilized by the beneficial compounds in PKAN fibroblasts, control and affected cells were treated with pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine and expression levels of PANK2 and mtACP were evaluated. As is illustrated in Figure 2a, 2b and 2c, all positive compounds correcting iron accumulation and cell morphology were also able to increase PANK2 and mtACP expression levels (Figure 2a, 2b and 2c).
Dose response effect of positive compounds (pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine) on PANK2 and mtACP expression levels.
We then examined the effect of a dose-response assay (1-100 mM) of the six positive compounds (pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine) on PANK2 and mtACP expression levels by Western blotting. In addition, as mtACP also participates in Fe/S cluster biosynthesis (20), we also explored the expression levels of NFS1 which participates in the mitochondrial Fe/S cluster synthesis complex.
The six selected compounds showed a dose response positive effect in PANK2, mtACP and NFS1 expression levels (Figure 3a, 3b, 4a, 4b, 5a and 5b; Supplementary figure 1, 2 and 3). The positive effect was noticeable since 1-5 mM and reached a maximum effect at 50-100 mM.
The effect of the different treatments (pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine) on control cells is shown in Supplementary figure 4a and 4b.
Positive compounds (pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine) up-regulate PANK2 gene transcription and increase the expression levels of essential transcription factors.
Furthermore, the favourable effect of the six positive compounds on PANK2 protein expression levels was associated with an increase in the steady-state levels of PANK2 transcripts (Figure 6a) suggesting that all six beneficial compounds up-regulated PANK2 gene expression or transcript stabilization. Indeed, the six compounds were able to increase the expression levels of well-known transcription factors that binds the PANK2 promoter such as NF-Y, FOXN4 and hnRNPA/B (Figure 6b and Supplementary figure 5) . These results support the hypothesis that favourable compounds increased the transcription of the PANK2 gene.
Also, we examined the protein expression levels of PGC-1alpha, PPGC1alpha and TFAM that are members of a family of transcription coactivators that play a central role in the regulation of cellular energy metabolism [24-26]. PGC-1alpha stimulates mitochondrial biogenesis and participates in the regulation of both carbohydrate and lipid metabolism while PPGC-1alpha is the active form of PGC-1alpha. TFAM plays a role in the determination of mitochondrial genome by regulating packaging, stability and replication. Therefore, disruption of TFAM could lead to mtDNA depletion and deficient mitochondrial bioenergetics. The six positive compounds were able to restore the decreased expression levels of PGC-1alpha, PPGC-1alpha and TFAM in P1 cells with residual PANK2 expression (Figure 6b and Supplementary figure 5).
Positive compounds (pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine) reduce lipid peroxidation of affected cells.
Literature supports the evidence of the relation between iron, ROS production and lipid peroxidation [27-30]. As consequence of increase intracellular iron in PKAN cells, the Fenton reaction may occur and generates high levels of ROS, which damage lipids through peroxidation . With the aim to confirm whether lipid peroxidation is a secondary pathological event in PKAN cells and to assess the effect of favorable compounds, treated and untreated mutant cells were stained with Bodipy, a fluorescent radio-probe for indexing lipid peroxidation and antioxidant efficacy in model membrane systems and living cells. As is shown in Figure 7a and 7b, PKAN cells showed increased levels of lipid peroxidation respect to control cells. Interestingly, all six positive compounds reduced lipid peroxidation in mutant cells.
Positive compounds (pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine) also correct PDH and complex I activity in PKAN cells with residual PANK2 levels.
Next, we focused in the pathological alterations potentially induced by mtACP deficiency. Thus, as mtACP is essential for lipoic acid synthesis by mitochondrial FAS II , which is a cofactor central to cellular metabolism [32, 33]. As a lysine posttranslational modification on particular components of enzymatic complexes, this functional group is required for the activities of these multimeric complexes [34, 35]. For example, the pyruvate dehydrogenase (PDH) and alpha-ketoglutarate (KDH) complexes regulate carbon entry points into the central metabolic pathway of the tricarboxylic acid cycle (TCA) . On both complexes, lipoylation is critical for proper enzyme function, and deficiency of this modification inhibits their activities.
As shown in Figure 7c and 7d, PDH activity was markedly reduced in PKAN fibroblasts. Interestingly, all six supplements (pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine at 5 mM) were able to restore partially PDH activity (Figure 7c and 7d) in responder mutant PANK2 fibroblasts.
As mtACP is also critically involved in the assembly of mitochondrial respiratory complex I , we next evaluated complex I activity in control and PANK2 mutant fibroblasts. The activity of complex I was significantly reduced in mutant fibroblasts (Figure 7d and 7f). The restoration of PANK2 and mtACP expression levels by the six positive compounds was also able to restore complex I enzymatic activity (Figure 7d and 7e).
Positive compounds (pantothenate, pantethine, vitamin E, omega 3, L-carnitine or thiamine) increased PANK2 and mtACP expression levels in several PKAN cell lines with residual PANK2 levels.
Next, we examined the effectiveness of beneficial compounds at 5 mM in three additional cell lines (P2, P3 and P4) carrying mutations with residual protein expression levels (Figure 8a, 8b, 8c, 8d, 8e, 8f) and in one cell line (P5) harboring a homozygous mutation causing a truncated PANK2 protein. As expected, positive compounds were able to increase the expression levels of PANK2 and mtACP in mutant cells lines with residual expression of PANK2 (Figure 8a, 8b, 8c, 8d, 8e, 8f) but had no effect in mutant cells with a truncated PANK2 protein (Figure 8g, 8h). Furthermore, the increased levels of PANK2 and mtACP induced by positive compounds were associated with reduced iron accumulation in mutant cells with residual PANK2 levels but not in mutant cells with truncated PANK2 (Supplementary figures 6, 7, 8 and 9).