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Pantothenate Rescues Iron Accumulation in Pantothenate Kinase-Associated Neurodegeneration Depending on the Type of Mutation

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Abstract

Neurodegeneration with brain iron accumulation (NBIA) is a group of inherited neurologic disorders in which iron accumulates in the basal ganglia resulting in progressive dystonia, spasticity, parkinsonism, neuropsychiatric abnormalities, and optic atrophy or retinal degeneration. The most prevalent form of NBIA is pantothenate kinase-associated neurodegeneration (PKAN) associated with mutations in the gene of pantothenate kinase 2 (PANK2) which is essential for coenzyme A (CoA) synthesis. There is no cure for NBIA, nor is there a standard course of treatment. In the current work, we describe that fibroblasts derived from patients harbouring PANK2 mutations can reproduce many of the cellular pathological alterations found in the disease such as intracellular iron and lipofuscin accumulation, increased oxidative stress and mitochondrial dysfunction. Furthermore, mutant fibroblasts showed a characteristic senescent morphology. Treatment with pantothenate, the PANK2 enzyme substrate, was able to correct all pathological alterations in responder mutant fibroblasts with residual PANK2 enzyme expression. However, pantothenate had no effect on mutant fibroblasts with truncated/incomplete protein expression. The positive effect of pantothenate in particular mutations was also confirmed in induced neurons obtained by direct reprograming of mutant fibroblasts. Our results suggest that pantothenate treatment can stabilize the expression levels of PANK2 in selected mutations. These results encourage us to propose our screening model as a quick and easy way to detect pantothenate-responder patients with PANK2 mutations. The existence of residual enzyme expression in some affected individuals raises the possibility of treatment using high dose of pantothenate. FULL TEXT: https://rdcu.be/5FHU
Pantothenate rescues iron accumulation in Pantothenate Kinase-
associated neurodegeneration depending on the type of mutation
Mónica Álvarez-Córdoba, Aida Fernández Khoury, Marina Villanueva-Paz,Carmen
Gómez-Navarro, Irene Villalón-García, Juan M. Suárez-Rivero, Suleva Povea-Cabello,
Mario de la Mata, David Cotán, Marta Talaverón-Rey, Antonio J. Pérez-Pulido, Joaquín
J. Salas, Eva Mª Pérez-Villegas, Antonio Díaz-Quintana, José A. Armengol and José A.
Sánchez-Alcázar.
Molecular Neurobiology, 2018 in press
Highlights
- Mutant PANK2 fibroblasts derived from patients show accumulation of iron and
lipofuscin (age pigment). Furthermore, mutant fibroblasts show a characteristic
senescent morphology.
- Paradoxically, impaired mitochondrial iron metabolism in patient cells induces
cytosolic iron deficiency and a vicious cycle with increased iron uptake which is
accumulated in lipofuscin granules.
- Pantothenate can correct pathological alterations depending on the type of
mutation in mutant fibroblasts.
- Expression levels of mutant PANK2 can be restored by pantothenate in particular
mutations.
- For the first time, iron accumulation is demonstrated in induced neuronsobtained by
direct reprograming of mutant fibroblasts.
- The positive effect of pantothenate is also confirmed in induced neurons.
- Residual enzyme expression raises the possibility of treatment with pantothenate in
selected mutations.
- The methodological strategy described in this manuscript can be also applied to
other NBIA subtypes such as PLAN, BPAN or MPAN.
Electron microscopy image of a PANK2 mutant fibroblast with lipofuscin granules
... The category of Neurodegeneration with Brain Iron Accumulation (NBIA) includes rare, hereditary disorders sharing a complex neurological phenotype with progressive motor dysfunction and the eponymous sign of iron deposition in the cerebral parenchyma, particularly in the basal ganglia [1]. To date, 15 NBIA subtypes have been genetically defined [2]. Pantothenate Kinase Associated Neurodegeneration (PKAN, OMIM # 234200) is one of the most common forms [3] and accounts for a lifetime risk of 0.24/100,000 [4]. ...
... Nonetheless, we have an extensive description of the distinct biological perturbations induced by PANK2 deficiency and contributing to the neurodegenerative process. Many studies performed in animal [11,12] and cellular models [13][14][15][16] highlighted the presence of mitochondrial abnormalities and dysfunction, including increased fragmentation, structural swelling and damaged cristae, altered mitochondrial membrane potential, and reduced oxygen consumption; this was constantly associated with significant changes in the cellular oxidative status. The direct connection with CoA biosynthesis was confirmed by the rescue capacity of molecules such as pantethine, CoA, fosmetpantotenate, and 4 -phosphopantetheine [11,[17][18][19][20], capable of directly or indirectly bypassing the blockage in pantothenate phosphorylation. ...
... Less clear is the connection between PANK2, CoA biosynthesis, and iron accumulation, probably because this feature is rarely reproduced in the available experimental systems. While signs of iron dyshomeostasis were commonly detected in in vitro models of the disease [14,22,23], the presence of iron deposits was observed occasionally in fibroblasts [15] and more recently in γ-aminobutyric acid (GABA)ergic neurons and astrocytes [24] obtained from PKAN induced-pluripotent stem cells (iPSc). Nor signs of iron imbalance or deposits of the metal were found in PKAN animal models, such as drosophila [25] or mice [12,26], indicating a major limit of these models in the investigation of the origin and the role of iron depositions in neurodegeneration, particularly in PKAN. ...
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Coenzyme A (CoA) is an essential cofactor in all living organisms, being involved in a large number of chemical reactions. Sequence variations in pantothenate kinase 2 (PANK2), the first enzyme of CoA biosynthesis, are found in patients affected by Pantothenate Kinase Associated Neurodegeneration (PKAN), one of the most common forms of neurodegeneration, with brain iron accumulation. Knowledge about the biochemical and molecular features of this disorder has increased a lot in recent years. Nonetheless, the main culprit of the pathology is not well defined, and no treatment option is available yet. In order to contribute to the understanding of this disease and facilitate the search for therapies, we explored the potential of the zebrafish animal model and generated lines carrying biallelic mutations in the pank2 gene. The phenotypic characterization of pank2-mutant embryos revealed anomalies in the development of venous vascular structures and germ cells. Adult fish showed testicular atrophy and altered behavioral response in an anxiety test but no evident signs of neurodegeneration. The study suggests that selected cell and tissue types show a higher vulnerability to pank2 deficiency in zebrafish. Deciphering the biological basis of this phenomenon could provide relevant clues for better understanding and treating PKAN.
... Western blotting was performed using standard protocols [26]. Membranes were incubated with primary antibodies diluted between 1:500 and 1:1000 overnight. ...
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Mutations in several genes involved in the epigenetic regulation of gene expression have been considered risk alterations to different intellectual disability (ID) syndromes associated with features of autism spectrum disorder (ASD). Among them are the pathogenic variants of the lysine-acetyltransferase 6A (KAT6A) gene, which causes KAT6A syndrome. The KAT6A enzyme participates in a wide range of critical cellular functions, such as chromatin remodeling, gene expression, protein synthesis, cell metabolism, and replication. In this manuscript, we examined the pathophysiological alterations in fibroblasts derived from three patients harboring KAT6A mutations. We addressed survival in a stress medium, histone acetylation, protein expression patterns, and transcriptome analysis, as well as cell bioenergetics. In addition, we evaluated the therapeutic effectiveness of epigenetic modulators and mitochondrial boosting agents, such as pantothenate and L-carnitine, in correcting the mutant phenotype. Pantothenate and L-carnitine treatment increased histone acetylation and partially corrected protein and transcriptomic expression patterns in mutant KAT6A cells. Furthermore, the cell bioenergetics of mutant cells was significantly improved. Our results suggest that pantothenate and L-carnitine can significantly improve the mutant phenotype in cellular models of KAT6A syndrome.
... Western blotting was performed using standard methods described in previous manuscripts of the research group [26]. Membranes were incubated with primary antibodies diluted between 1:500 and 1:1000 overnight. ...
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Autism Spectrum disorder (ASD) and intellectual disability (ID) are the most frequent develop-mental disorders with a prevalence between 3% and 5% of the population. In addition, both ASD and ID can be found in the same patient. Mutations in several genes involved in the epigenetic regulation of gene expression have been linked to different ID associated with ASD features including alterations of the ly-sine-acetyltransferase 6A (KAT6A) gene in KAT6A syndrome. KAT6A enzyme participates in a wide range of critical cellular functions such as chromatin remodeling, gene expression, protein synthesis, cell metabolism, and replication. In this manuscript, we examined the pathophysiolog-ical alterations in fibroblasts derived from three patients harboring KAT6A mutations. We ad-dressed survival in stress medium, histone acetylation, protein expression patterns and tran-scriptome analysis as well as cell bioenergetics. In addition, we evaluated the therapeutic effec-tiveness of epigenetic modulators and mitochondrial boosting agents such as pantothenate and L-carnitine in correcting the mutant phenotype. Pantothenate and L-carnitine treatment increased histone acetylation and corrected protein and transcriptomic expression patterns in mutant KAT6A cells. Furthermore, cell bioenergetics of mutant cells was significantly improved. Our results suggest that pantothenate and L-carnitine can significantly correct the mutant phe-notype in cellular models of KAT6A syndrome. https://www.preprints.org/manuscript/202211.0273/v1
... Abnormal iron metabolism is common in most neurological diseases. The pathology most directly related to iron accumulation is neurodegeneration with brain iron accumulation (NBIA), a group of inherited neurologic disorders in which iron accumulates in the basal ganglia, resulting in progressive dystonia, spasticity, parkinsonism, neuropsychiatric abnormalities, and optic atrophy or retinal degeneration [9]. ...
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The alteration of iron homeostasis related to the aging process is responsible for increased iron levels, potentially leading to oxidative cellular damage. Iron is modulated in the Central Nervous System in a very sensitive manner and an abnormal accumulation of iron in the brain has been proposed as a biomarker of neurodegeneration. However, contrasting results have been presented regarding brain iron accumulation and the potential link with other factors during aging and neurodegeneration. Such uncertainties partly depend on the fact that different techniques can be used to estimate the distribution of iron in the brain, e.g., indirect (e.g., MRI) or direct (post-mortem estimation) approaches. Furthermore, recent evidence suggests that the propensity of brain cells to accumulate excessive iron as a function of aging largely depends on their anatomical location. This review aims to collect the available data on the association between iron concentration in the brain and aging, shedding light on potential mechanisms that may be helpful in the detection of physiological neurodegeneration processes and neurodegenerative diseases such as Alzheimer’s disease.
... Supplementation of coenzyme A or, in the case of partially preserved PANK2 activity, pantothenate, rescued the phenotype in cellular and animal models of PKAN [178,185,186]. Iron accumulation was also reduced after pantothenate supplementation suggesting that coenzyme A depletion is directly associated with Fe accumulation [187]. The role of coenzyme A deficiency was further supported by showing that patients with biallelic pathogenic variants in Coenzyme A synthase have phenotype and MRI abnormalities like PKAN [188] and that zebrafish models of coenzyme A synthase and PANK2 deficiency exhibit similar phenotypes [189]. ...
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Disruption of cerebral iron regulation appears to have a role in aging and in the pathogenesis of various neurodegenerative disorders. Possible unfavorable impacts of iron accumulation include reactive oxygen species generation, induction of ferroptosis, and acceleration of inflammatory changes. Whole-brain iron-sensitive magnetic resonance imaging (MRI) techniques allow the examination of macroscopic patterns of brain iron deposits in vivo, while modern analytical methods ex vivo enable the determination of metal-specific content inside individual cell-types, sometimes also within specific cellular compartments. The present review summarizes the whole brain, cellular, and subcellular patterns of iron accumulation in neurodegenerative diseases of genetic and sporadic origin. We also provide an update on mechanisms, biomarkers, and effects of brain iron accumulation in these disorders, focusing on recent publications. In Parkinson’s disease, Friedreich’s disease, and several disorders within the neurodegeneration with brain iron accumulation group, there is a focal siderosis, typically in regions with the most pronounced neuropathological changes. The second group of disorders including multiple sclerosis, Alzheimer’s disease, and amyotrophic lateral sclerosis shows iron accumulation in the globus pallidus, caudate, and putamen, and in specific cortical regions. Yet, other disorders such as aceruloplasminemia, neuroferritinopathy, or Wilson disease manifest with diffuse iron accumulation in the deep gray matter in a pattern comparable to or even more extensive than that observed during normal aging. On the microscopic level, brain iron deposits are present mostly in dystrophic microglia variably accompanied by iron-laden macrophages and in astrocytes, implicating a role of inflammatory changes and blood–brain barrier disturbance in iron accumulation. Options and potential benefits of iron reducing strategies in neurodegeneration are discussed. Future research investigating whether genetic predispositions play a role in brain Fe accumulation is necessary. If confirmed, the prevention of further brain Fe uptake in individuals at risk may be key for preventing neurodegenerative disorders.
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Abstract Blackground: 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.
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