PKAN neurodegeneration and residual PANK2 activities in patient erythrocytes

Abstract Objective Pantothenate kinase 2‐associated neurodegeneration (PKAN) is a rare neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene. PKAN is associated with iron deposition in the basal ganglia and, occasionally, with the occurrence of misshaped erythrocytes (acanthocytes). The aim of this study was to assess residual activity of PANK2 in erythrocytes of PKAN patients and to correlate these data with the type of PANK2 mutations and the progression of neurodegeneration. Methods Residual PANK2 activities in erythrocytes of 14 PKAN patients and 14 related carriers were assessed by a radiometric assay. Clinical data on neurodegeneration included the Barry–Albright Dystonia Scale (BAD‐Scale) besides further general patient features. A molecular visualization and analysis program was used to rationalize the influence of the PKAN causing mutations on a molecular level. Results Erythrocytes of PKAN patients had markedly reduced or no PANK2 activity. However, patients with at least one allele of the c.1583C > T (T528M) or the c.833G > T (R278L) variant exhibited 12–56% of residual PANK2 activity. In line, molecular modeling indicated only minor effects on enzyme structure for these point mutations. On average, these patients with c.1583C > T or c.833G > T variant had lower BAD scores corresponding to lower symptom severity than patients with other PANK2 point mutations. Interpretation Residual erythrocyte PANK2 activity could be a predictor for the progression of neurodegeneration in PKAN patients. Erythrocytes are an interesting patient‐derived cell system with still underestimated diagnostic potential.


Introduction
Pantothenate kinase 2-associated neurodegeneration (PKAN) is a neurodegenerative disease caused by mutations in the pantothenate kinase 2 gene (PANK2), 1 and represents the most common genetic form of NBIA (neurodegeneration with brain iron accumulation). PKAN is characterized by progressive generalized dystonia, and imaging shows excess iron deposition in the basal ganglia, specifically the globus pallidus and the substantia nigra. Patients with an early onset before 6 years of age have been historically classified as classic PKAN (cPKAN), resulting in more severe disease. Atypical PKAN (aPKAN) has a later onset (>10 years) and progresses more slowly. 2 In some PKAN patients, misshaped erythrocytes with thorny protrusions (so-called "acanthocytes") are found in blood. This establishes a link to the neuroacanthocytoses (NA) syndromes, 3 such as McLeod syndrome, chorea-acanthocytosis, and Huntington's disease-like 2, which are also characterized by movement disorders due to neurodegeneration in the basal ganglia. While these acanthocytes are clinically irrelevant (e.g., not associated with anemia), they constitute an interesting cellular model system to study molecular mechanisms of the disease. [4][5][6][7][8][9] PANK2 encoded by the PANK2 gene is one of four isoforms of pantothenate kinases (PANK1-4) catalyzing the rate-limiting step in coenzyme A (CoA) biosynthesis. PANK2 has a mitochondrial targeting sequence and in neurons is localized to mitochondria. 10,11 More recently, both a nuclear localization and a nuclear export signal were also identified and complex subcellular targeting of PANK2 was reported. 12 Human PANK2 is inhibited by CoA and various acyl-CoA metabolites with IC 50 values in the high nanomolar range 13 and activated by palmitoylcarnitine at low micromolar concentrations 14 indicating that the finetuning of its activity is crucial for the regulation of the cellular lipid metabolism. PANK1 and PANK3 are assumed to be localized in the cytosol and their activities are distinctly affected by CoA and acyl-CoA metabolites. 15 A large variety of mutations have been identified in PANK2 in PKAN patients. 16,17 Interestingly, a considerable fraction of these are point mutations indicating that single amino acid substitutions might critically affect the function of PANK2 in the cellular context. Biochemical studies on recombinant wild type (WT) and mutant PANK2 revealed that only few of the tested point mutations strongly affected pantothenate kinase activity. 13 Some exhibited only mildly reduced and some even elevated activity as compared to WT PANK2. Similar results were obtained when PANK3 proteins with mutations corresponding to respective PKAN-associated PANK2 mutations were analyzed. 18 These studies suggest that some deleterious effects of point mutations only develop in the specific cellular context and may not become evident when respective mutant proteins are overexpressed in cell culture systems.
In this study we characterized endogenous mutant PANK2 protein in erythrocytes from 14 PKAN patients, assessed their residual PANK2 activity in the erythrocyte cytosol and correlated these with molecular models of the point mutations and clinical data of the patients.

Blood sample collection
Blood was collected within the Global NBIA Biobank (Technical University of Munich) and/or from the Muscle Tissue Culture Collection (Friedrich-Baur-Institute, Klinikum of Ludwig-Maximilians-University of Munich). Informed consent was obtained from each participant (IRB-ID: TUM 5578-12s or KUM 45-14). This study was also approved by the ethics committee of the Medical University of Vienna (EK-Nr. 1148/2015). Nine milliliter of blood from 14 patients, 14 carriers and a healthy control (HC1) in Munich were collected by venipuncture into EDTA vacutainers and sent to Vienna overnight at 4-8°C. There, freshly drawn blood from local donors (HC2-6) was used to control transport-induced alterations.

Genetic and clinical evaluation of the patients
Genetic and clinical data have been collected within the Global NBIA Registry (IRB-ID 187-12) and shared for the purposes of this study in an anonymized way (Table 1). Data shared included the Barry-Albright Dystonia (BAD) score, a scale to rate severity of dystonia in eight different body regions (eyes, mouth, neck, trunk, both arms, and both legs) with total scores ranging from 0 (no dystonia) to 32 (severe generalized dystonia). Within the Global NBIA registry, only the threshold of 6 years of age at the onset of motor symptoms has been chosen as definition for discrimination between cPKAN and aPKAN, without taking into account any other phenotypic features of the patients (e.g., disease progression). Thus, the Registry clinical diagnosis does not reflect the course of the disease. The variable BAD score / yr was calculated (BAD score divided by the age in years) to provide a rough measure for the progression of neurodegeneration.
Pantothenate kinase (PANK) activity assay PANK activity was determined as described 19 with modifications. Washed erythrocytes were hypotonically lysed, ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association centrifuged (16,000g, 20 min, 4°C), and total protein in the supernatant was determined at 280 nm (NanoDrop 2000C, ThermoFisherScientific). Two hundred and fifty microgram of extract were mixed with 11.25 µmol/L 14 Clabeled D-pantothenic acid (50 mCi/mmol; American Radiolabeled Chemicals), 2.5 mmol/L ATP, 10 mmol/L MgCl 2 and 0.1 mol/L Tris/Cl pH = 7.5 in 40 µL and incubated at 37°C. The reaction mixture was transferred onto an ion-exchange filter (GE Whatman) and immersed in 95% ethanol/1% acetic acid. The filter was suspended in 3 mL scintillation liquid (PerkinElmer) and the radioactivity quantified in a scintillation counter (TRI-CARB 2100TR, PerkinElmer).

Statistical analyses
Data were analyzed with SPSS (version 25) (IBM) and GraphPad Prism (version 6) (GraphPad Software) using methods of descriptive statistics.

Results
PANK activity could be measured in crude cytosolic preparations of erythrocytes thereby confirming earlier data. 21 Since the activities of the PANK isoforms are feedback inhibited by CoA and some of its metabolites 13,15 we tested the effect of acetyl-CoA on erythrocyte pantothenate kinase activity. There was a dose-dependent inhibition of PANK activity, with an IC 50 of 0.26µM acetyl-CoA The DNA mutations refer to the NCBI Reference Sequence: NM_153638.3 (pantothenate kinase 2 mRNA transcript variant 1). The c.1235 + 5G > A mutation of patient P11 is located in the intron 5 bases downstream the splice site after exon 3 and changes the consensus sequence from GTAAGT to GTAAAT which likely results in improper splicing.
( Fig. 1A). This conforms well to the IC 50 of PANK2 13 and is clearly below that of PANK3 and PANK1b (1 and 5 µmol/L, respectively). 15 Moreover, palmitoyl-L-carnitine increased erythrocyte PANK activity to the same extent and concentration range as reported for PANK2 14 (Fig. 1B). Furthermore, a hemoglobin-depleted extract of the erythrocyte cytosol was prepared and subjected to Western analysis. A polyclonal PANK2 antibody revealed a specific band of about 49 kD (Fig. 1C) which is in accordance with the size of the mature PANK2 protein. 11 This band was (strongly) reduced in extracts of PKAN patients (Fig. 1D). In line with these data, the presence of PANK2 and the absence of other PANK isoforms in erythrocytes were found by proteomic analyses. 22,23 We analyzed erythrocyte PANK activity of 14 PKAN patients, 14 related healthy carriers (father and/or mother of patients) and 6 healthy control donors. Healthy control donor HC1 donated blood at the same date and place (Munich) as the patients. Intra-donor variation of PANK activity of the HC1 samples was within the range of intraand inter-donor variation of freshly drawn samples from healthy donors (HC2-HC6) in Vienna where the assays were performed indicating that sample shipment did not affect PANK activity. HC1 therefore served as reference for normalization. Patients P1 to P7 had very low or absent PANK activities (<5% of control). This again strongly indicates that PANK2 is the only PANK isoform in human erythrocytes. In contrast, patients P8 to P14 had considerable residual activities (>10% of control); P13 and P14 had 40% and 56% of control, respectively ( Fig. 2 and Table 2). Six of the patients with PANK activities >10% were homo-or heterozygous for the T528M mutation and P12 was homozygous for the R278L mutation. Carrier blood samples showed reduced PANK activity (except CR11). Expectedly, their PANK activities were higher than those of the related patients ( Fig. 2 (families are grouped in panels) and Fig. 3).
Each patient had at least one mutant allele resulting in a single amino acid exchange in the PANK2 sequence (Fig. 4A). To rationalize the considerable variation in residual PANK activity in patients, structural models of the point mutants were analyzed. PANK2 is a homodimer with each monomer consisting of two conserved domains, A (residues 208-356) and B (residues 357-566), and a 200 amino acid long N-terminal region. Dimerization is mediated by long a-helices (residues 486-513) and two reciprocal interactions between an extended loop (residues 446-471) and another helix (residues 414-426). The ATP binding site is located between domain A (residues 219-221) and domain B (residues 520-522) with the active site (E338) being located in domain A (Fig. 5A). Mutation R278L (patient P12): Residue R278 is at the surface of domain A and forms a salt bridge with E237 (Fig. 5B). Interestingly, the recombinant PANK3 mutant R78 corresponding to the PANK2 R278L mutant exhibited increased activity compared to WT PANK3. Erythrocytes of patient P12 (homozygous for R278M) have considerable PANK2 activity (27.7%). Mutation D457Y (patient P5): D457Y is in a pantothenate-binding loop (domain B) and likely affects binding of the substrate (Fig. 5C). Mutations P288R and E554K (patients P2, P7, P4, and P6): P288 is in the interior of domain A next to the ATP binding groove, E554 is near the ATP binding site in domain B. Both mutations probably impair ATP binding (Fig. 5D). Mutation L425P (patient P1): Residue L425 is mapped on a helix at the dimerization interface. PANK proteins are highly cooperative allosteric enzymes exploiting structural changes at the dimer interface to coordinately switch between active and inactive conformations. 20 Since proline is a potent helixbreaker, the L425P mutant likely exhibits an impaired activation-inactivation cycle (Fig. 5E). Mutation G521R (patients P9, P10, P5, and P3): G521 is located in the short ATP binding helix, probably Figure 2. Phosphopantothenate generation is strongly reduced in the erythrocyte cytosol of PKAN patients. Generation of radiolabeled phosphopantothenate in erythrocyte cytosolic extracts was assessed as described in Materials and Methods. The relative amounts of phosphopantothenate were determined by normalization to the amount generated within 40 min in the extract of HC1 (which was set to 100%) and respective time courses are given. Patients with identical mutations are shown in the same panels together with respective carriers as indicated. Figure 3. Comparison of erythrocyte PANK2 activity between control donors, carriers, and patients. Erythrocyte PANK2 activities are plotted for control donors (black circles), carriers (blue squares), and patients (orange and red triangles with or without at least one allele harboring a T528M or R278L, respectively). For numerical data, compare to Table 2. Statistical analyses (unpaired student's t-test) revealed the significance of the difference between the mean values of the groups (**** <0.001) as indicated. impairing ATP binding (Fig. 5F). Recombinant G521R PANK2 mutant had no activity. 13 In line, erythrocytes of P3 (homozygous for G521R) exhibit virtually no PANK activity (Fig. 2). Mutation T528M (P8, P9, P11, P10, P14, and P13): T528 is located on the surface of domain B with the amino acid pointing toward the solvent (Fig. 5G). Interestingly, both recombinant mutants of PANK2 (T528M) and PANK3 (T328M corresponding to the PANK2 T528M mutant) had increased activity compared to wild-type PANK2. 13,20 Conversely, functional testing of PANK2 mutants in an E. coli complementation assay revealed that the clone transformed with the p.T528M allele grew significantly better than any other tested mutant, but less well than the clone with the wild-type allele. 17 Erythrocytes of patients P13 and P14 (both homozygous for the T528M mutation) had residual PANK2 of 39.8% and 55.9%, respectively (Fig. 2, Table 2). Interestingly, the amino acid of PANK1 corresponding to T528 of PANK2 is actually a methionine (M553) (Fig. 4). Phylogenetic comparison of the sequences around this site shows a nearby serine conserved in all PANK isoforms and in all species except for the mammalian PANK2. The T528M mutation in PANK2 therefore abrogates the only potential phosphorylation site in this region (Fig. 4B). Hence, hypothetically, the detrimental effect of this mutation is due to aberrant posttranscriptional regulation rather than to instability of the mutant protein.
Together, in contrast to the surface mutations T528M and R278L, the L425P, D457Y, G521R and the E554K mutations are likely associated with major structural and/ or functional impairment. Correspondingly, patients with the R278L or the T528M mutation in at least one of their PANK2 alleles had residual PANK2 activities >10% (Fig. 3, Table 2). For further analyzes, we segregated our cohort of patients in a T528M/R278L-positive (P8-P14) and T528M/R278L-negative (P1-P7) group. These groups have nearly identical age at onset but significantly different means in PANK2 activity (Figs. 3 and 6C).
To evaluate the clinical relevance of these analyses, we assessed the link between PANK2 activity and the BAD score of the patients or an age-normalized version thereof (BAD score / yr). PANK2 activity correlated with both the BAD score and the BAD score / yr with a Pearson's correlation coefficient of À0.473 (P = 0.088) and À0.520 (P = 0.057), respectively. Interestingly, the BAD score and BAD score / yr correlated better with residual PANK2 activity in erythrocytes than with the age of onset (À0.166 (P = 0.571) and À0.408 (P = 0.147)) ( Figs. 3 and 6A and B). Moreover, the means of the BAD score and of the BAD score / yr were lower in the group of T528M/R278L positive patients (Fig. 6C). Thus, erythrocyte PANK2 activity may be a predictor for the progression of neurodegeneration in PKAN.
Finally, we addressed whether T528M PANK2 mutants with residual activity remained responsive to physiologic regulation. Erythrocyte cytosol from a second blood donation of patients P9 and P10 (both T528M/G521R) revealed residual PANK2 activities of 11% and 12%, respectively. Similar to control (HC3), these activities were completely blocked by 5µM acetyl-CoA but increased by palmitoylcarnitine (data not shown). Thus, the T528M PANK2 mutant is accessible to metabolic regulators. Erythrocyte extracts of healthy control donors (HC), carriers (CR) and PKAN patients (P) were assayed for phospho-pantothenate generation as described in the Materials and Methods section and the specific PANK2 activities per total protein were determined. Relative PANK2 activities were calculated from the specific activities by normalization to the specific activity of donor HC1 that served as WT control in each experiment (activity denotes mean values of relative PANK2 activity in %; StDev denotes standard deviation; n denotes the number of data points).

Discussion
Here we show that PANK activity in erythrocytes is mediated by PANK2, assess the activity of PANK2 mutants in PKAN erythrocytes and suggest that erythrocyte PANK2 could serve as a predictor for the progression of PKAN neurodegeneration.
CoA is a known co-factor for maintaining plasma membrane lipid metabolism in erythrocytes. 24 Hence CoA biosynthesis is likely essential for erythrocyte homeostasis. This study provides evidence that it is the PANK isoform 2 that is required for CoA biosynthesis in erythrocytes: (1) The characteristics of PANK activation and inhibition by palmitoylcarnitine and acetyl-CoA, respectively, are in line with the known features of the PANK2 isoform (Fig. 1). 13,14 (2) Western blot analysis of erythrocyte cytosolic extracts reveals the characteristic 49 kDa band of the mature PANK2 protein (Fig. 1C). (3) Most significantly, erythrocytes of PKAN patients have strongly reduced or absent PANK activity (Figs. 1D and 2, Table 2). In line with proteomic data, 22,23 this corroborates that PANK2 is the only PANK isoform in erythrocytes.
Mitochondrial localization and processing of PANK2 was described previously. 10,11 Mature PANK2 (49 kDa) is generated by two proteolytic events on the full-length translation product (63 kDa) by the mitochondrial processing peptidase (MPP). It is puzzling that erythrocytes contain PANK2, since these cells lose mitochondria during terminal erythropoiesis. Two scenarios can be envisaged: (1) PANK2 is leaking from mitochondria to the cytosolic compartment during erythrocyte maturation. (2) An alternative in-frame start AUG site (Met-124) generates a cytosolic version of the PANK2 protein (Uniprot database as Q9BZ23-3). In line with this scenario, dual targeting of proteins to mitochondria and the cytosolic compartment has been described. 25 While we have no proof yet for the presence of this PANK2 sequence variant in the erythrocytes, the possibility that in neurons both a mitochondrial and a cytosolic version of PANK2 may be generated from the same mRNA transcript, has implications for the interpretation of PKAN pathology. CoA biosynthesis can take place independently at various subcellular locations within the topologically distinct cytosolic compartments of neurons. Subcellular CoA biosynthesis could be specifically regulated by local metabolic constraints necessitating the non-redundant properties of cytosolic PANK1, PANK3, and PANK2 regarding their specific modes of activation by CoA metabolites. Thus, not only mitochondrial but also cytosolic PANK2-specific aberrations may be causative factors for PKAN neurodegeneration.
We show that PANK2 activity in the erythrocytes of the patients negatively correlates with the severity of dystonia as estimated by an age-normalized BAD score (BAD score / yr in Fig. 6B and C). In our cohort, this correlation is stronger than that between the age of onset and the severity of dystonia. Thus, early determination of erythrocyte PANK activity at the onset of the disease might be a prognostic marker for the progression of neurodegeneration. To confirm this, a prospective study with repetitive evaluation of BAD scores will be necessary. Patient P5 (G521R/D457Y) and patients P2 and P7 (P288R/S191fs) show the atypical form of PKAN and relatively low age-normalized BAD scores despite of a (near) total lack of PANK2 activity in their erythrocytes ( Fig. 6A and B). Possibly, the P288R and the D457Y mutants have higher stability and specific activity in neurons as compared to erythrocytes. Neuron-specific chaperones, 26 absent in erythrocytes, may well account for a rescue of activity and stability of these mutants in neurons and confer slower disease progression.
Our study reveals the potential of erythrocytes as patient-derived cell system to study molecular aberrations in PKAN pathology. Being easily available, erythrocytes could turn out to be useful in studies on the cellular effects of novel PKAN drugs. 27,28 More generally, since recent analyses revealed a yet unrecognized Figure 4. Conservation of PANK2 point mutations sites between the isoforms and phylogenetic variation within a partial sequence corresponding to the region around T528 of human PANK2. (A) Sequence alignment of the three human PANK isoforms was performed with the CLUSTALW algorithm using the Uniprot sequences of human PANK2 (Q9BZ23), PANK1 (Q8TE04), and PANK3 (Q9H999) with * (asterisk) and: (colon) indicating full conservation and conservation between groups of amino acids with highly similar properties, respectively. The seven PANK2 point mutations relevant for this study are indicated above the respective positions in red and amino acid conservation is highlighted in yellow. (B) Phylogenetic comparisons of PANK2 sequences around T528 between two mammalian, one reptile, one amphibian, and one fish species (species names and sequence identifiers below the alignments) are shown for the three PANK isoforms as indicated. Please note that the respective threonine corresponding to T528 of human PANK2 (highlighted in red) is conserved in PANK2 and PANK3 isoforms while it shifted to methionine (green) early in the evolution of the PANK1 protein. Conversely, the alanine at position 530 of human PANK2 is only found in mammalian PANK2 isoforms while phylogenetically distant species have a serine at this position (highlighted in yellow), which is also conserved in PANK1 and PANK3 isoforms.
complexity of the erythrocyte proteome and metabolome, erythrocytes may also be a valuable source for studying other disease-causing mutations and/or pathological molecular mechanisms of congenital diseases. Lacking transcriptional and translational adaptation mechanisms, erythrocytes maintain their protein-and lipid-based regulatory network and homeostatic features for considerable times after removal from the blood circuit so that proper analyses can unravel donor-specific characteristics and even lifestyle-and diet-dependent intra-donor variations (unpublished observations). Thus, erythrocytes may be used as reporters on molecular effects of treatments and/or alimentation. Sophisticated tests on erythrocytes as a whole and/or analyses of erythrocyte-derived samples may generate patient-specific information with diagnostic and prognostic potential and thereby emerge as a new methodological approach in the field of personalized medicine not only in the context of rare congenital diseases. Figure 6. Negative correlation between residual erythrocyte PANK2 activity in patients and their score of neurodegeneration. Residual erythrocyte PANK2 activity is plotted against the BAD score (A) or an age-normalized version thereof (B). Data points are identified by the patient IDs and either designated by orange circles (patients with at least one allele harboring a T528M or R278L mutation) or dark red circles (patients without T528M or 278L mutation), respectively. (C) Patients were grouped whether or not they have at least one allele of the c.1583C > T or c.833G > T mutation in their PANK2 gene (T528M/R278L positive or negative). Mean values and standard error (SE) of the indicated parameters within these subgroups are given in percent of control (activity see also Table 2) or absolute numbers (BAD score and BAD score / yr, see also Table 1). An unpaired student's t-test was applied to assess the significance (P-value) of the difference between respective means.