Redesigning therapies for pantothenate kinase-associated neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN) is an incurable rare genetic disorder of children and young adults caused by mutations in the PANK2 gene, which encodes an enzyme critical for the biosynthesis of coenzyme A. Although PKAN affects only a small number of patients, it shares several hallmarks of more common neurodegenerative diseases of older adults such as Alzheimer's disease and Parkinson's disease. Advances in etiological understanding and treatment of PKAN could therefore have implications for our understanding of more common diseases and may shed new lights on the physiological importance of coenzyme A, a cofactor critical for the operation of various cellular metabolic processes. The large body of knowledge that accumulated over the years around PKAN pathology, including but not limited to studies of various PKAN models and therapies, has contributed not only to progress in our understanding of the disease but also, importantly, to the crystallization of key questions that guide future investigations of the disease. In this review, we will summarize this knowledge and demonstrate how it forms the backdrop to new avenues of research.

Proton magnetic resonance spectroscopy detects cerebral metabolic derangement in a mouse model of brain coenzyme a deficiency.

BACKGROUND: Pantothenate kinase (PANK) is the first and rate-controlling enzymatic step in the only pathway for cellular coenzyme A (CoA) biosynthesis. PANK-associated neurodegeneration (PKAN), formerly known as Hallervorden-Spatz disease, is a rare, life-threatening neurologic disorder that affects the CNS and arises from mutations in the human PANK2 gene. Pantazines, a class of small molecules containing the pantazine moiety, yield promising therapeutic effects in an animal model of brain CoA deficiency. A reliable technique to identify the neurometabolic effects of PANK dysfunction and to monitor therapeutic responses is needed. METHODS: We applied 1H magnetic resonance spectroscopy as a noninvasive technique to evaluate the therapeutic effects of the newly developed Pantazine BBP-671. RESULTS: 1H MRS reliably quantified changes in cerebral metabolites, including glutamate/glutamine, lactate, and N-acetyl aspartate in a neuronal Pank1 and Pank2 double-knockout (SynCre+ Pank1,2 dKO) mouse model of brain CoA deficiency. The neuronal SynCre+ Pank1,2 dKO mice had distinct decreases in Glx/tCr, NAA/tCr, and lactate/tCr ratios compared to the wildtype matched control mice that increased in response to BBP-671 treatment. CONCLUSIONS: BBP-671 treatment completely restored glutamate/glutamine levels in the brains of the mouse model, suggesting that these metabolites are promising clinically translatable biomarkers for future therapeutic trials.

PANK2 p.A170fs：a novel pathogenetic mutation, compound with PANK2 p.R440P, causing pantothenate kinase Associated neurodegeneration in a Chinese family.

AIM: Pantothenate kinase associated neurodegeneration (PKAN) is a severe autosomal recessive rare disease and characterized by iron accumulation in the basal ganglia. To investigate the pathogenesis of this disease in two sibling patients with PANK in a Chinese family, whole-exome variant detection and functional analysis were performed. MATERIALS AND METHODS: Clinical and radiographic investigations were performed in the two brother patients. Whole exome sequencing (WES) was used in mutation detection, and the mutations were confirmed by Sanger sequencing. A longevity cohort genetic database was applied as Chinese urban controls. Bioinformatic analysis was performed to predict the pathogenicity. RESULTS: Compound heterozygous mutations of PANK2 were detected in two sibling brothers with PKAN in a Chinese family: c.510_522del (p.A170fs) and c.1319G > C (p.R440P) in the transcript NM_153638. PANK2: c.510_522del (p.A170fs) was absent in public data and the Chinese urban controls. Bioinformatics analysis showed that the above two variants were pathogenicity. CONCLUSIONS: We identified a rare compound heterozygous combination of PANK2 mutations found in a Chinese family in which two sibling brothers suffered from PKAN. PANK2 c.510_522del (p.A170fs) was the first reported to be a PKAN pathogenic variant.

Bi-Allelic Mutations in Zebrafish pank2 Gene Lead to Testicular Atrophy and Perturbed Behavior without Signs of Neurodegeneration.

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.

Atypical pantothenate kinase-associated neurodegeneration with PANK2 mutations : clinical description and a review of the literature.

Panthothenate kinase-associated neurodegeneration (PKAN) is arare neurodegeneration caused by mutations in the pantothenate kinase (PANK2) gene, which is located on chromosome 20p13. These mutations result in iron accumulation in the brain basal ganglia leading to parkinsonism, dysarthria, spasticity, cognitive impairment, and retinopathy. Herein, we report acase of adult-onset PKAN who presented with young-onset action tremor, bradykinesia, dysarthria, and bilateral interossei atrophy.  Neuroimaging demonstrated "eye-of-the-tiger signs". Through analyzing PANK2 gene, PANK2 NM_153638:c.1133A>G (p.Asp378 Gly) and PANK2 NM_153638:c.1502 T > A (p.lle501Asn), were detected. In addition, we reviewed the clinical and genetic features and therapeutic strategies for patients with PKAN.

Harmful Iron-Calcium Relationship in Pantothenate kinase Associated Neurodegeneration.

Pantothenate Kinase-associated Neurodegeneration (PKAN) belongs to a wide spectrum of diseases characterized by brain iron accumulation and extrapyramidal motor signs. PKAN is caused by mutations in PANK2, encoding the mitochondrial pantothenate kinase 2, which is the first enzyme of the biosynthesis of Coenzyme A. We established and characterized glutamatergic neurons starting from previously developed PKAN Induced Pluripotent Stem Cells (iPSCs). Results obtained by inductively coupled plasma mass spectrometry indicated a higher amount of total cellular iron in PKAN glutamatergic neurons with respect to controls. PKAN glutamatergic neurons, analyzed by electron microscopy, exhibited electron dense aggregates in mitochondria that were identified as granules containing calcium phosphate. Calcium homeostasis resulted compromised in neurons, as verified by monitoring the activity of calcium-dependent enzyme calpain1, calcium imaging and voltage dependent calcium currents. Notably, the presence of calcification in the internal globus pallidus was confirmed in seven out of 15 genetically defined PKAN patients for whom brain CT scan was available. Moreover, we observed a higher prevalence of brain calcification in females. Our data prove that high amount of iron coexists with an impairment of cytosolic calcium in PKAN glutamatergic neurons, indicating both, iron and calcium dys-homeostasis, as actors in pathogenesis of the disease.

Abnormal Vasculature Development in Zebrafish Embryos with Reduced Expression of Pantothenate Kinase 2 Gene.

Mutations in pank2 gene encoding pantothenate kinase 2 determine a pantothenate kinase-associated neurodegeneration, a rare disorder characterized by iron deposition in the globus pallidus. To extend our previous work, we performed microinjections of a new pank2-specific morpholino to zebrafish embryos and thoroughly analyzed vasculature development. Vessels development was severely perturbed in the head, trunk, and tail, where blood accumulation was remarkable and associated with dilation of the posterior cardinal vein. This phenotype was specific as confirmed by p53 expression analysis and injection of the same morpholino in pank2-mutant embryos. We can conclude that pank2 gene is involved in vasculature development in zebrafish embryos. The comprehension of the underlining mechanisms could be of relevance for understanding of pantothenate kinase-associated neurodegeneration.

Novel compound heterozygous PANK2 gene mutations in a Chinese patient with atypical pantothenate kinase-associated neurodegeneration.

AIM: Pantothenate-kinase-associated neurodegeneration (PKAN), which is characterised by iron accumulation in the basal ganglia, is a rare autosomal recessive neurodegenerative disorder caused by pantothenate kinase 2 (PANK2) gene mutations. The PANK2 gene is located on chromosome 20p13 and encodes pantothenate kinase. Herein, we identified one patient with PKAN who had mutations in the PANK2 gene. MATERIALS AND METHODS: We performed clinical and radiographic investigations, and diagnosed this disease at the clinical and genetic levels. RESULTS: It is worth mentioning that the patient displayed an eye-of-the-tiger sign. Through scanning the exons and flanking intronic sequences of PANK2 in patient and control subjects, we report a compound heterozygote c. 260A > G (NM_001324191) and c.405dupC (NM_153638) for PANK2 mutations in a Chinese patient with clinical manifestation of progressive prosopospasm, dysarthria and gait disturbance. Bioinformatics analysis showed that two variants exhibited highly conserved residues across species. CONCLUSION: we reported a patient presenting with atypical PKAN, and identified novel compound heterozygous PANK2 gene mutations..

First successful trial of preimplantation genetic diagnosis for pantothenate kinase-associated neurodegeneration.

PURPOSE: We aim to present a case of a healthy infant born after intracytoplasmic sperm injection-in vitro fertilization (ICSI-IVF) with a preimplantation genetic diagnosis (PGD) for pantothenate kinase-associated neurodegeneration (PKAN) due to PANK2 mutation. METHODS: ICSI-IVF was performed on a Thai couple, 34-year-old female and 33-year-old male, with a family history of PKAN in their first child. Following fertilization, each of the embryos were biopsied in the cleavage stage and subsequently processed for whole-genome amplification. Genetic status of the embryos was diagnosed by linkage analysis and direct mutation testing using primer extension-based mini-sequencing. Comprehensive chromosomal aneuploidy screening was performed using a next-generation sequencing-based strategy. RESULTS: Only a single cycle of ICSI-IVF was processed. There were seven embryos from this couple-two were likely affected, three were likely carriers, one was likely unaffected, and one failed in target genome amplification. Aneuploidy screening was performed before making a decision on embryo transfer, and only one unaffected embryo passed the screening. That embryo was transferred in a frozen thawed cycle, and the pregnancy was successful. The diagnosis was confirmed by amniocentesis, which presented with a result consistent with PGD. At 38 weeks of gestational age, a healthy male baby was born. Postnatal genetic confirmation was also consistent with PGD and the prenatal results. At the age of 24 months, the baby presented with normal growth and development lacking any neurological symptoms. CONCLUSIONS: We report the first successful trial of PGD for PKAN in a developing country using linkage analysis and mini-sequencing in cleavage stage embryos.

Knock-down of pantothenate kinase 2 severely affects the development of the nervous and vascular system in zebrafish, providing new insights into PKAN disease.

Pantothenate Kinase Associated Neurodegeneration (PKAN) is an autosomal recessive disorder with mutations in the pantothenate kinase 2 gene (PANK2), encoding an essential enzyme for Coenzyme A (CoA) biosynthesis. The molecular connection between defects in this enzyme and the neurodegenerative phenotype observed in PKAN patients is still poorly understood. We exploited the zebrafish model to study the role played by the pank2 gene during embryonic development and get new insight into PKAN pathogenesis. The zebrafish orthologue of hPANK2 lies on chromosome 13, is a maternal gene expressed in all development stages and, in adult animals, is highly abundant in CNS, dorsal aorta and caudal vein. The injection of a splice-inhibiting morpholino induced a clear phenotype with perturbed brain morphology and hydrocephalus; edema was present in the heart region and caudal plexus, where hemorrhages with reduction of blood circulation velocity were detected. We characterized the CNS phenotype by studying the expression pattern of wnt1 and neurog1 neural markers and by use of the Tg(neurod:EGFP/sox10:dsRed) transgenic line. The results evidenced that downregulation of pank2 severely impairs neuronal development, particularly in the anterior part of CNS (telencephalon). Whole-mount in situ hybridization analysis of the endothelial markers cadherin-5 and fli1a, and use of Tg(fli1a:EGFP/gata1a:dsRed) transgenic line, confirmed the essential role of pank2 in the formation of the vascular system. The specificity of the morpholino-induced phenotype was proved by the restoration of a normal development in a high percentage of embryos co-injected with pank2 mRNA. Also, addition of pantethine or CoA, but not of vitamin B5, to pank2 morpholino-injected embryos rescued the phenotype with high efficiency. The zebrafish model indicates the relevance of pank2 activity and CoA homeostasis for normal neuronal development and functioning and provides evidence of an unsuspected role for this enzyme and its product in vascular development.

Correction of a genetic deficiency in pantothenate kinase 1 using phosphopantothenate replacement therapy.

Coenzyme A (CoA) is a ubiquitous cofactor involved in numerous essential biochemical transformations, and along with its thioesters is a key regulator of intermediary metabolism. Pantothenate (vitamin B5) phosphorylation by pantothenate kinase (PanK) is thought to control the rate of CoA production. Pantothenate kinase associated neurodegeneration is a hereditary disease that arises from mutations that inactivate the human PANK2 gene. Aryl phosphoramidate phosphopantothenate derivatives were prepared to test the feasibility of using phosphopantothenate replacement therapy to bypass the genetic deficiency in the Pank1(-/-) mouse model. The efficacies of candidate compounds were first compared by measuring the ability to increase CoA levels in Pank1(-/-) mouse embryo fibroblasts. Administration of selected candidate compounds to Pank1(-/-) mice corrected their deficiency in hepatic CoA. The PanK bypass was confirmed by the incorporation of intact phosphopantothenate into CoA using triple-isotopically labeled compound. These results provide strong support for PanK as a master regulator of intracellular CoA and illustrate the feasibility of employing PanK bypass therapy to restore CoA levels in genetically deficient mice.

Pallidal neuronal apolipoprotein E in pantothenate kinase-associated neurodegeneration recapitulates ischemic injury to the globus pallidus.

Pantothenate kinase-associated neurodegeneration (PKAN) is a progressive movement disorder that is due to mutations in PANK2. Pathologically, it is a member of a class of diseases known as neurodegeneration with brain iron accumulation (NBIA) and features increased tissue iron and ubiquitinated proteinaceous aggregates in the globus pallidus. We have previously determined that these aggregates represent condensed residue derived from degenerated pallidal neurons. However, the protein content, other than ubiquitin, of these aggregates remains unknown. In the present study, we performed biochemical and immunohistochemical studies to characterize these aggregates and found them to be enriched in apolipoprotein E that is poorly soluble in detergent solutions. However, we did not determine a significant association between APOE genotype and the clinical phenotype of disease in our database of 81 cases. Rather, we frequently identified similar ubiquitin- and apolipoprotein E-enriched lesions in these neurons in non-PKAN patients in the penumbrae of remote infarcts that involve the globus pallidus, and occasionally in other brain sites that contain large γ-aminobutyric acid (GABA)ergic neurons. Our findings, taken together, suggest that tissue or cellular hypoxic/ischemic injury within the globus pallidus may underlie the pathogenesis of PKAN.

Skin fibroblasts from pantothenate kinase-associated neurodegeneration patients show altered cellular oxidative status and have defective iron-handling properties.

Pantothenate kinase-associated neurodegeneration (PKAN) is a neurodegenerative disease belonging to the group of neurodegeneration with brain iron accumulation disorders. It is characterized by progressive impairments in movement, speech and cognition. The disease is inherited in a recessive manner due to mutations in the Pantothenate Kinase-2 (PANK2) gene that encodes a mitochondrial protein involved in Coenzyme A synthesis. To investigate the link between a PANK2 gene defect and iron accumulation, we analyzed primary skin fibroblasts from three PKAN patients and three unaffected subjects. The oxidative status of the cells and their ability to respond to iron were analyzed in both basal and iron supplementation conditions. In basal conditions, PKAN fibroblasts show an increase in carbonylated proteins and altered expression of antioxidant enzymes with respect to the controls. After iron supplementation, the PKAN fibroblasts had a defective response to the additional iron. Under these conditions, ferritins were up-regulated and Transferrin Receptor 1 (TfR1) was down-regulated to a minor extent in patients compared with the controls. Analysis of iron regulatory proteins (IRPs) reveals that, with respect to the controls, PKAN fibroblasts have a reduced amount of membrane-associated mRNA-bound IRP1, which responds imperfectly to iron. This accounts for the defective expression of ferritin and TfR1 in patients' cells. The inaccurate quantity of these proteins produced a higher bioactive labile iron pool and consequently increased iron-dependent reactive oxygen species formation. Our results suggest that Pank2 deficiency promotes an increased oxidative status that is further enhanced by the addition of iron, potentially causing damage in cells.

Pantothenate kinase-associated neurodegeneration is not a synucleinopathy.

AIMS: Mutations in the pantothenate kinase 2 gene (PANK2) are responsible for the most common type of neurodegeneration with brain iron accumulation (NBIA), known as pantothenate kinase-associated neurodegeneration (PKAN). Historically, NBIA is considered a synucleinopathy with numerous reports of NBIA cases with Lewy bodies and Lewy neurites and some cases reporting additional abnormal tau accumulation. However, clinicopathological correlations in genetically proven PKAN cases are rare. We describe the clinical, genetic and neuropathological features of three unrelated PKAN cases. METHODS: All three cases were genetically screened for the PANK2 gene mutations using standard Sanger polymerase chain reaction sequencing. A detailed neuropathological assessment of the three cases was performed using histochemical and immunohistochemical preparations. RESULTS: All cases had classical axonal swellings and Perls' positive iron deposition in the basal ganglia. In contrast to neuroaxonal dystrophies due to mutation of the phospholipase A2, group VI (PLA2G6) gene, in which Lewy body pathology is widespread, no α-synuclein accumulation was detected in any of our PKAN cases. In one case (20-year-old male) there was significant tau pathology comprising neurofibrillary tangles and neuropil threads, with very subtle tau pathology in another case. CONCLUSIONS: These findings indicate that PKAN is not a synucleinopathy and, hence the cellular pathways implicated in this disease are unlikely to be relevant for the pathomechanism of Lewy body disorders.

Excess iron harms the brain: the syndromes of neurodegeneration with brain iron accumulation (NBIA).

Regulation of iron metabolism is crucial: both iron deficiency and iron overload can cause disease. In recent years, our understanding of the syndromes of Neurodegeneration with Brain Iron Accumulation (NBIA) continues to grow considerably. These are characterized by excessive iron deposition in the brain, mainly the basal ganglia. Pantothenate kinase-associated neurodegeneration (PKAN, NBIA1) and PLA2G6-associated neurodegeneration (PLAN, NBIA2) are the core syndromes, but several other genetic causes have been identified (including FA2H, C19orf12, ATP13A2, CP and FTL). These conditions show a wide clinical and pathological spectrum, with clinical overlap between the different NBIA disorders and other diseases including spastic paraplegias, leukodystrophies, and neuronal ceroid lipofuscinosis. Lewy body pathology was confirmed in some clinical subtypes (C19orf12-associated neurodegeneration and PLAN). Research aims at disentangling the various NBIA genes and their related pathways to move towards pathogenesis-targeted therapies. Until then treatment remains symptomatic. Here we will introduce the group of NBIA syndromes and review the main clinical features and investigational findings.

Phenotypes and genotypes of patients with pantothenate kinase-associated neurodegeneration in Asian and Caucasian populations: 2 cases and literature review.

OBJECTIVES: Pantothenate kinase-associated neurodegeneration (PKAN) is a rare disease caused by pantothenate kinase 2 (PANK2, OMIM 606157) mutations. This study is aimed to investigate clinical presentations, pathologies, and genetics in patients with PKAN. METHODS: Two patients with PKAN were reported. We reviewed the literature to include additional 19 patients with PKAN in Eastern Asia. These patients were divided into classic and atypical groups by the age of onset. We compared the data on PKAN patients of Asian and Caucasian populations. RESULTS: We found iron deposits in the globus pallidus in our Patient 1 and a heterozygous truncating mutation (c.1408insT) in Patient 2. Literature review shows that generalized dystonia and bulbar signs are more common in classic PKAN patients, whereas segmental dystonia and tremors are more specific to atypical ones. Asian patients have less complex presentations--lower prevalence of pyramidal signs, mental impairment, and parkinsonism--than Caucasians. D378G in exon 3 is the most frequent mutation (28%) in Asians. CONCLUSIONS: Our study demonstrates that the distribution of dystonia is the major distinction between subgroups of PKAN. Caucasian patients have more complex presentations than Asians. Exon 3 and 4 are hot spots for screening PANK2 mutations in Asian patients.

Syndromes of neurodegeneration with brain iron accumulation (NBIA): an update on clinical presentations, histological and genetic underpinnings, and treatment considerations.

In recent years, understanding of the syndromes of neurodegeneration with brain iron accumulation (NBIA) has grown considerably. In addition to the core syndromes of pantothenate kinsase-associated neurodegeneration (PKAN, NBIA1) and PLA2G6-associated neurodegeneration (PLAN, NBIA2), several other genetic causes have been identified. The acknowledged clinical spectrum has broadened, age-dependent presentations have been recognized, and we are becoming aware of overlap between the different NBIA disorders as well as with other diseases. Autopsy examination of genetically confirmed cases has demonstrated Lewy bodies and/or tangles in some subforms, bridging the gap to more common neurodegenerative disorders such as Parkinson's disease. NBIA genes map into related pathways, the understanding of which is important as we move toward mechanistic therapies. Our aim in this review is to provide an overview of not only the historical developments, clinical features, investigational findings, and therapeutic results but also the genetic and molecular underpinnings of the NBIA syndromes.

Missense PANK2 mutation without "eye of the tiger" sign: MR findings in a large group of patients with pantothenate kinase-associated neurodegeneration (PKAN).

PURPOSE: To present some unusual MR findings in a group of patients from the south-west of the Dominican Republic suffering from Pantothenate Kinase Associated Neurodegeneration (PKAN). MATERIALS AND METHODS: Twenty patients and one preclinical case homozygous for the PANK2 mutation, 13 heterozygous gene carriers and 14 healthy volunteers were scanned prospectively using a 3 Tesla system. RESULTS: All patients showed the typical signal reduction within the globus pallidus and the substantia nigra. A surprising finding was the absence of the bright spot ("tiger's eye") in the medial part of the pallidum in 6 patients, but not in the preclinical case. Both fractional anisotropy (FA) and mean diffusivity (MD) were increased with high significance in the globus pallidus, whereas a reduction of FA in the anterior parts of the internal capsule was accompanied by an elevation of MD. CONCLUSION: Our findings support the hypothesis that the absence of the "tiger's eye" in PKAN might be secondary, probably caused by an increased accumulation of iron. This could artificially increase FA and MD values and change fiber tracking results. Except for the fronto-basal tracts, white matter was preserved well. This encouraging finding might support efforts to develop further therapeutic strategies in this devastating dystonia.

Novel histopathologic findings in molecularly-confirmed pantothenate kinase-associated neurodegeneration.

Pantothenate kinase-associated neurodegeneration is a form of neurodegeneration with brain iron accumulation, characterized by a progressive movement disorder and prominent iron deposition in the globus pallidus. Formerly referred to as Hallervorden-Spatz syndrome, the disorder was renamed pantothenate kinase-associated neurodegeneration after discovery of the causative gene, PANK2. Although the pathological features of clinically characterized Hallervorden-Spatz syndrome have been described, the literature is confounded by the historical use of this term for nearly all conditions with prominent basal ganglia iron accumulation and by the fact that this term encompasses a genetically heterogeneous group of disorders, now referred to as 'neurodegeneration with brain iron accumulation'. As a result, interpreting reports that precede molecular characterization of specific forms of neurodegeneration with brain iron accumulation is problematic. In the present studies, we describe neuropathological findings in six cases of molecularly confirmed pantothenate kinase-associated neurodegeneration. We identify prominent ubiquinated deposits in pantothenate kinase-associated neurodegeneration. We also characterize two distinct origins of spheroid bodies and delineate histological features of iron deposition. In so doing, we characterize fundamental features of the disease and redefine its nosological relationship to other neurodegenerative disorders.