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. 2024 Nov 28;19(1):442.
doi: 10.1186/s13023-024-03453-x.

A therapeutic approach to pantothenate kinase associated neurodegeneration: a pilot study

Alessandra Pereira  1 , Carolina Fischinger Moura de Souza  2 , Mónica Álvarez-Córdoba  3 , Diana Reche-López  3 , José Antonio Sánchez-Alcázar  4
Affiliations

A therapeutic approach to pantothenate kinase associated neurodegeneration: a pilot study

Alessandra Pereira et al. Orphanet J Rare Dis. .

Abstract

Background: Neurodegeneration with brain iron accumulation (NBIA) is a group of genetic neurological 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 disorders. The diagnosis of PKAN is established with clinical features and the "eye of the tiger" sign identified on brain MRI and the identification of biallelic pantothenate kinase 2 (PANK2) pathogenic variants on molecular genetic testing. PANK2 catalyzes the first reaction of coenzyme A (CoA) biosynthesis, thus, altered PANK2 activity is expected to induce CoA deficiency as well as low levels of essential metabolic intermediates such as 4'-phosphopantetheine which is a necessary cofactor for critical proteins involved in cytosolic and mitochondrial pathways such as fatty acid biosynthesis, mitochondrial respiratory complex I assembly and lysine and tetrahydrofolate metabolism, among other metabolic processes.

Methods: In this manuscript, we examined the effect of a multitarget complex supplements (pantothenate, pantethine, omega-3 and vitamin E) on in vitro patient-derived cellular models and the clinical outcome of the adjuvant supplements in combination with the baseline neurological medication in three PKAN patients.

Results: Multitarget complex supplements significantly reduced iron accumulation and increased PANK2 and ACP expression levels in the cellular models derived from all three PKAN patients. In addition, the adjunct treatment to the standard neurological medication improved or stabilized the clinical symptoms of patients.

Conclusions: Our results suggest that multitarget complex supplements can be clinically useful as augmentation therapy for PKAN patients harboring pathogenic variants with residual enzyme levels.

Trial registration: CAAE: 58219522.6.0000.5330. Registered 25 May 2022-Retrospectively registered, https://plataformabrasil.saude.gov.br/visao/pesquisador/gerirPesquisa/gerirPesquisaAgrupador.jsf .

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: The study was approved by The Ethical Committee of Hospital Universitario Virgen del Rocío and Virgen Macarena of Seville, protocol code BRAINCURE16, following the Spanish laws, the principles of the Declaration of Helsinki, and the Guideline for Good Clinical Practices; and by the Ethics Committee of Moinhos de Vento Hospital (CAAE: 58219522.6.0000.5330) and all the parents or legal guardians of subjects signed the written informed consent forms before any procedure was done. Consent for publication: Consent for publication were obtained from all parents or legal guardians. Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
(A) P1: MRI (T2 flair and T2 FSE) showing signal hyperintensity due to gliosis in the antero-medial region of both globus pallidus, surrounded by hypointense areas secondary to iron deposits (“eye-of-the-tiger” sign). (B) P2: Presence of hypersignal in FLAIR sequence, affecting the globus pallidus bilaterally, delimited by a hyposignal halo, more evident in the sequence for magnetic susceptibility and diffusion, characterizing a "eye-of-the-tiger" appearance
Fig. 2
Fig. 2
Flow chart of the study design. The treatment period lasted for 24 weeks
Fig. 3
Fig. 3
Effect pantothenate, pantethine, omega-3 and vitamin E supplementation on iron accumulation in three mutant PANK2 cells. (A) Control (C1) and three PKAN fibroblast cell lines (P1, P2 and P3) were treated with 5 µM pantothenate, 5 µM pantethine, 5 µM vitamin E and 5 µM omega 3 for 10 days (+ T). Then, cells were stained with Prussian Blue as described in Material and Methods and examined by bright-field microscopy. Scale bar = 15 µm. (B) Quantification of Prussian Blue staining (C) Iron content determined by ICP-MS. Data represent the mean ± SD of three separate experiments. Significance between PKAN and control fibroblasts is represented as *p < 0.01 fibroblasts and ap < 0.05, aap < 0.01 between untreated and treated fibroblasts
Fig. 4
Fig. 4
Effect of pantothenate, pantethine, omega-3 and vitamin E supplementation on PANK2 and mtACP expression levels. (A) Expression levels of PANK2 and mtACP in Control (C1 and C2) and PKAN cells (P1, P2 and P3) assessed by Western blotting. (B) Quantification of PANK2 and mtACP expression levels. (C) Controls (C1) and patient P1 fibroblasts were treated with 5 µM pantothenate, 5 µM pantethine, 5 µM vitamin E and 5 µM omega 3 for 10 days (+ T). PANK2 and mtACP protein expression levels were analysed by Western blotting. Tubulin was used as loading control. (D) Densitometry of the Western blotting of PANK2 and mtACP under pantothenate, pantethine, omega-3 and vitamin E supplementation. (E) PANK2 transcripts were quantified by RT-qPCR. Data represent the mean ± SD of three separate experiments. *p < 0.01 between PKAN patients and controls. ap < 0.05 and aap < 0.01 between untreated and treated fibroblasts. A.U., arbitrary units. Unedited and uncut blots are shown in Supplementary Figs. 1 and 2
Fig. 5
Fig. 5
Effect of pantothenate, pantethine, omega-3 and vitamin E supplementation on lipid peroxidation. Control and PKAN fibroblasts (P1, P2 and P3) were treated with 5 µM pantothenate, 5 µM pantethine, 5 µM vitamin E and 5 µM omega 3 for 10 days (+ T). (A) Representative images of lipid peroxidation in treated and untreated control and PKAN cells using BODIPY® 581/591 C11 staining. Control cells treated with Luperox® (500 μM) for 15 min were used as a positive control of lipid peroxidation. Scale bar = 15 μm. (B) Ratio of the oxidized BODIPY-C11 signal (green) to reduced BODIPY-C11 signal (red). Data represent the mean ± SD of three separate experiments (50 cell images for each condition). *p < 0.01 between PKAN patients and controls. aap < 0.01, between untreated and treated fibroblasts. A.U., arbitrary units
Fig. 6
Fig. 6
Effect of pantothenate, pantethine, omega-3 and vitamin E supplementation on PDH activity. Control and PKAN fibroblasts (P1, P2, P3) were treated with 5 µM pantothenate, 5 µM pantethine, 5 µM vitamin E and 5 µM omega 3 for 10 days (+ T). (A) PDH activity in whole cellular extracts was measured as described in Material and Methods. (B) Quantification of PDH activity. Data represent the mean ± SD of three separate experiments. *p < 0.01 between PKAN patients and controls; aap < 0.01 between untreated and treated cells
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