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Article
40-Phosphopantetheine corrects CoA, iron, and
dopamine metabolic defects in mammalian models
of PKAN
Suh Young Jeong
1
, Penelope Hogarth
1,2
, Andrew Placzek
3
, Allison M Gregory
1
, Rachel Fox
1
,
Dolly Zhen
1
, Jeffrey Hamada
1
, Marianne van der Zwaag
4
, Roald Lambrechts
4
, Haihong Jin
3
,
Aaron Nilsen
3
, Jared Cobb
5
, Thao Pham
5
, Nora Gray
2
, Martina Ralle
1
, Megan Duffy
1
, Leila
Schwanemann
1
, Puneet Rai
1
, Alison Freed
1
, Katrina Wakeman
1
, Randall L Woltjer
5
,
Ody CM Sibon
4
& Susan J Hayflick
1,2,6,*
Abstract
Pantothenate kinase-associated neurodegeneration (PKAN) is an
inborn error of CoA metabolism causing dystonia, parkinsonism,
and brain iron accumulation. Lack of a good mammalian model
has impeded studies of pathogenesis and development of rational
therapeutics. We took a new approach to investigating an existing
mouse mutant of Pank2and found that isolating the disease-
vulnerable brain revealed regional perturbations in CoA metabo-
lism, iron homeostasis, and dopamine metabolism and functional
defects in complex I and pyruvate dehydrogenase. Feeding mice a
CoA pathway intermediate, 40-phosphopantetheine, normalized
levels of the CoA-, iron-, and dopamine-related biomarkers as well
as activities of mitochondrial enzymes. Human cell changes also
were recovered by 40-phosphopantetheine. We can mechanistically
link a defect in CoA metabolism to these secondary effects via the
activation of mitochondrial acyl carrier protein, which is essential
to oxidative phosphorylation, iron–sulfur cluster biogenesis, and
mitochondrial fatty acid synthesis. We demonstrate the fidelity of
our model in recapitulating features of the human disease. More-
over, we identify pharmacodynamic biomarkers, provide insights
into disease pathogenesis, and offer evidence for 40-phosphopan-
tetheine as a candidate therapeutic for PKAN.
Keywords 40-phosphopantetheine; coenzyme A; NBIA; PANK2; PKAN
Subject Categories Neuroscience; Pharmacology & Drug Discovery
DOI 10.15252/emmm.201910489 | Received 19 February 2019 | Revised 7
August 2019 | Accepted 14 August 2019 | Published online 29 October 2019
EMBO Mol Med (2019)11:e10489
See also: RA Lambrechts et al (December 2019)
Introduction
Pantothenate kinase-associated neurodegeneration (PKAN) is an
autosomal recessive movement disorder affecting children and
adults. This profoundly disabling disorder manifests with severe,
painful dystonia, young-onset parkinsonism, globus pallidus iron
accumulation, and blindness from pigmentary retinopathy (Hayflick
et al, 2003). PKAN is one of the neurodegeneration with brain iron
accumulation (NBIA) disorders and though ultra-rare, is readily
identifiable in the clinical setting by its distinctive brain MRI pattern,
the “eye of tiger” sign. As disease advances, affected persons lose
control of movement yet retain substantial intellectual function.
Currently, no disease-modifying therapeutic is available.
Pantothenate kinase-associated neurodegeneration is an inborn
error of coenzyme A (CoA) metabolism that may be amenable to a
therapeutic approach in which the enzymatic defect is bypassed by
supplying a “downstream” pathway intermediate (Fig 1A; Zhou
et al, 2001). Pantothenate kinase catalyzes the first step in de novo
CoA synthesis starting from vitamin B
5
(pantothenate, Fig 1A), a
function in mammals that is shared by four isozymes (Leonardi
et al, 2005). PKAN is caused by mutations in PANK2, encoding
pantothenate kinase 2 (Zhou et al, 2001), which is the only isozyme
that localizes to mitochondria (Hortnagel et al, 2003; Johnson et al,
2004; Kotzbauer et al, 2005). CoA is essential for hundreds of meta-
bolic reactions including the tricarboxylic acid cycle, fatty acid
oxidation and synthesis, amino acid metabolism, and neurotransmit-
ter synthesis (Strauss, 2010). Moreover, the ratio of CoA to acetyl-
CoA is a central determinant in coordinating cellular metabolism
with gene regulation (Pietrocola et al, 2015). Despite its importance
at the nexus of intermediary metabolism, our knowledge of CoA
1Department of Molecular & Medical Genetics, Oregon Health & Science University, Portland, OR, USA
2Department of Neurology, Oregon Health & Science University, Portland, OR, USA
3Medicinal Chemistry Core, Oregon Health & Science University, Portland, OR, USA
4Department of Cell Biology, University Medical Center Groningen, Groningen, the Netherlands
5Department of Pathology, Oregon Health & Science University, Portland, OR, USA
6Department of Pediatrics, Oregon Health & Science University, Portland, OR, USA
*Corresponding author. Tel: +1(503)494 7703; E-mail: hayflick@ohsu.edu
ª2019 The Authors. Published under the terms of the CC BY 4.0license EMBO Molecular Medicine 11:e10489 |2019 1of 17
synthesis, homeostasis, and transport is incomplete, as is our knowl-
edge of the mechanism by which loss of pantothenate kinase 2 leads
to neurodegeneration and iron accumulation. The canonical CoA
synthesis pathway (Fig 1A) was thought to be the only source of
cellular CoA until recent work revealed an alternate mechanism to
synthesize CoA within cells from the extracellular delivery of the
pathway intermediate 40-phosphopantetheine (Srinivasan et al,
2015). Srinivasan et al further reasoned that 40-phosphopantetheine
may have therapeutic potential in PKAN to bypass the pantothenate
kinase 2 defect and restore cellular CoA synthesis.
To test this idea, we needed to develop a high-fidelity mammalian
model of PKAN. Published mouse models rely on compound abnor-
malities in order to demonstrate a phenotype, requiring both the dele-
tion of Pank2 and a superimposed second “hit”, either genetic or
metabolic. They include (i) a neuron-specific Pank1+Pank2 double
knock-out model (Sharma et al,2018);(ii)aPank2 knock-out animal
fed a severe ketogenic diet to induce metabolic stress (Brunetti et al,
2014); and (iii) mice administered hopantenate, a toxic chemical that
competes with pantothenate as a substrate for all pantothenate kinases
and causes global depletion of CoA with lethal metabolic changes
(Zhang et al, 2007; Di Meo et al, 2017). A fundamental limitation of
all three models is the inability to attribute disease features specifically
to defective pantothenate kinase 2. As a result, hypotheses of PKAN
pathogenesis based on these models are uncertain, and therapeutics
developed using these models may or may not have efficacy in PKAN
(Brunetti et al, 2014; Di Meo et al, 2017; Sharma et al, 2018).
We sought to develop a mammalian disease model with features
that could be specifically attributed to loss of pantothenate kinase 2.
Employing knowledge of the human disease, we re-investigated our
previously reported mice, which harbor a germline null mutation in
Pank2 and have no detectable pantothenate kinase 2 protein (Kuo
et al, 2005). Though the animals manifested a mild, late-onset
retinopathy and pupillometric defect, similar to features found in
humans (Hayflick et al, 2003; Egan et al, 2005), these features were
strain-specific and required electroretinographic expertise to track
(Kuo et al, 2005). Those limitations, coupled with the long duration
to a clinical phenotype and lack of overt neurological features,
appeared to restrict the utility of this mutant. We returned to these
animals to determine whether disease changes might be detectable
if we isolated the disease-vulnerable globus pallidus region from the
remaining brain tissue and then looked for CoA-related differences.
This idea was based on knowledge of the exquisitely focal human
neuropathology, which is limited to globus pallidus (Woltjer et al,
2015). Our new approach yielded success, revealing critical insights
into disease pathogenesis by demonstrating defects in CoA metabo-
lism, iron homeostasis, dopamine metabolism, and mitochondrial
function in globus pallidus. Moreover, we used this model to
demonstrate that 40-phosphopantetheine ameliorates the primary
CoA metabolic defect and normalizes all secondary perturbations.
Results
Enriching for disease-vulnerable brain tissue reveals CoA
pathway defects in Pank2KO animals
Since PKAN selectively damages globus pallidus, we sought to
isolate this disease-vulnerable region from other brain tissue in the
Pank2 KO mouse for further investigation. We dissected mouse
brain into three regions: globus pallidus-containing (GP), substantia
nigra-containing (SN), and cerebellum (Fig 1B). GP also includes
thalamus, hypothalamus, and striatum. SN also includes ventral
tegmental area, red nucleus, and oculomotor nucleus. The method
of dissection was confirmed for each region based on gene expres-
sion patterns. We found candidate genes using in situ hybridization
data reported in the Allen Brain Atlas (©2016 Allen Institute for
Brain Science. Allen Mouse Brain Atlas. Available from: mouse.b-
rain-map.org) and confirmed high levels of mRNA for Drd1 in GP
(but not SN or cerebellum), Th in SN (but not GP or cerebellum),
and Gabra6 and Calb1 in cerebellum (but not GP or SN) using qRT–
PCR (Appendix Fig S1B).
With this new approach, we set out to determine whether we
could identify perturbations in the CoA pathway and in disease-rele-
vant biomarkers. Using the three brain regions from WT and KO
animals, we measured mRNA expression for the three genes encod-
ing CoA synthetic enzymes that are downstream of pantothenate
kinase (Fig 1A), including Ppcs (phosphopantothenoylcysteine
synthetase), Ppcdc (phosphopantothenoylcysteine decarboxylase),
and Coasy (CoA synthase). The expression of two, Ppcs and Coasy,
was significantly down-regulated in KO animals but only in the GP
region (Fig 1C). Ppcdc mRNA expression did not differ by genotype
(Fig 1C) and was not studied further. Levels of Coasy protein were
also found to be decreased in KO GP only (Fig 1D). For this reason
and because it is the terminal enzyme required for CoA synthesis,
we considered Coasy expression as a candidate biomarker for
further development.
Defective Pank2perturbs iron homeostasis, mitochondrial
function, and dopamine metabolism
A common feature among the NBIA disorders is iron accumulation
in globus pallidus. To assess iron homeostasis in our model, we
measured the expression of iron homeostasis genes, levels of subcel-
lular compartmental iron, and activity of an iron-dependent enzyme.
The expression of Tfrc (transferrin receptor 1), Ireb2 (iron regulatory
protein 2), and Hamp (hepcidin) was significantly decreased in KO
animals in GP only (Fig 2A), and Tfr1 protein levels were also mark-
edly decreased (Fig 2F). These findings suggested that cells in this
region were sensing and responding to increased cytosolic iron. We
confirmed the presence of significantly increased iron levels in cells
isolated from GP in the KO animals in both the cytosolic and mito-
chondrial fractions using subcellular fractionation and inductively
coupled plasma mass spectroscopy (Fig 2B). In contrast, iron levels
in cortex and SN subcellular fractions did not differ by genotype
(Appendix Fig S2A). We confirmed that there were equivalent quan-
tities of mitochondria in tissue samples from KO and WT GP using
mitochondrial DNA quantification (data not shown).
We then sought to determine whether the changes in CoA meta-
bolism and iron homeostasis alter the function of enzymes that
depend on these factors. Mitochondrial aconitase requires iron–
sulfur cluster biogenesis for its activity and also depends on CoA for
production of its substrate, citrate. Mitochondrial aconitase cata-
lyzes the isomerization of citrate to isocitrate in the tricarboxylic
acid cycle, and citrate is formed by the condensation of acetate,
derived from acetyl-CoA, and oxaloacetate. We found loss of activity
of mitochondrial aconitase in KO brain but not in WT brain or liver
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EMBO Molecular Medicine Suh Young Jeong et al
from either genotype (Fig 2C, Appendix Fig S2B). Previous studies
in human PKAN iPSC-derived neurons also reported perturbations
in aconitase and iron homeostasis, but they differ from what we
observed. Specifically, Orellana et al (2016) reported decreased
activities of both mitochondrial aconitase and cytosolic aconitase as
well as TfR1 up-regulation and FtH (ferritin) down-regulation,
suggesting that the iPSC-derived neurons were sensing iron insuffi-
ciency. Reasons for these differences in the different systems are
uncertain.
We sought further evidence for functional defects that could be
attributed to CoA and iron dyshomeostasis. The synthesis of acetyl-
CoA requires pyruvate dehydrogenase (PDH) and depends on
A
C
D
B
Figure 1. Isolating disease-vulnerable brain tissue from disease-protected reveals CoA pathway defects in Pank2KO animals.
A The CoA synthesis pathway, including intermediates and enzymes (boxed).
B Brain dissection yielding three study regions (red dotted lines) including GP, SN, and cerebellum. Captured and modified from Brain Atlas 2(Allen Institute for Brain
Science).
C Relative quantification of mRNA for Ppcs, Ppcdc, and Coasy by genotype from each of the three study regions. n=11,4,4for WT and n=8,4,4for KO (GP, SN,
Cerebellum, respectively).
D Western blot and quantification for Coasy from GP by genotype and treatment status (pPanSH = 40-phosphopantetheine). Ponceau S was used as a loading control.
n=8,4for WT and n=8,6for KO (vehicle and pPanSH groups, respectively).
Data information: Data shown here were evaluated by either one-way ANOVA or two-way ANOVA depending on the number of variables. **P<0.01, ***P<0.001. All
graphs represent mean s.e.m.
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Suh Young Jeong et al EMBO Molecular Medicine
sufficient quantities of mitochondrial matrix CoA. We found signifi-
cantly decreased PDH activity from GP in KO animals compared
with controls, with no accompanying loss of protein (Fig 2D and F,
Appendix Fig S2C). Because iron is essential for electron transport
chain function, we also measured complex I activity and found a
significant decrease in activity in GP from the KO mouse compared
to WT controls (Fig 2E). Our combined data show that defective
Pank2 causes regional alterations in the expression and activity of a
specific set of enzymes related to CoA metabolism and iron home-
ostasis with functional sequelae in energy metabolism.
Pantothenate kinase-associated neurodegeneration manifests
with dystonia and parkinsonism; however, a specific defect in dopa-
mine metabolism or transport has not been shown. In the GP-
enriched region of KO animals, we saw marked up-regulation in
A
BC
DEF
Figure 2.
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dopamine receptor gene expression (Drd1 and Drd2, Fig 3A and B)
and in Drd1 protein levels (Fig 3A, Drd2 protein was not studied).
In contrast, GABA receptor gene expression (Gabra3 and Gabra6)
was not altered in any of the three brain regions tested in KO
animals (Fig 3C, Appendix Fig S3A). Receptor up-regulation
commonly results from loss of ligand. Therefore, we looked for
evidence of diminished dopamine synthesis by measuring levels of
tyrosine hydroxylase, the rate-limiting enzyme in the dopamine
synthesis pathway, and one requiring iron as a cofactor. Decreased
Th protein levels were found in whole brain from Pank2 KO animals
(Fig 3D), but the mice show no signs of parkinsonism. More refined
studies to detect subtle evidence for dystonia are planned. In
summary, we propose that loss of pantothenate kinase 2 disrupts
dopamine homeostasis, causing the movement disorder observed in
humans.
Within mouse brain, we see a regional pattern of disease changes
that mirror those found in human brain. We therefore expected that
non-diseased tissue from other organ systems would not manifest
the biochemical changes we found in globus pallidus. To our
surprise, both cultured human fibroblasts and immortalized and
fresh lymphocytes from people with PKAN show defects in the CoA
pathway. Using primary cells, we corroborated our mouse findings;
COASY expression was decreased in cultured human fibroblasts and
lymphoblasts (Fig 4A), as was PPCS expression (Appendix Fig S4A).
A
BC D
Figure 3. Dopamine metabolism is impaired in Pank2KO GP.
A Relative mRNA and protein expression of the dopamine receptor Drd1compared by genotype, brain region, and treatment status (pPanSH =
40-phosphopantetheine). n=11,4,4for WT and n=8,4,4for KO (GP, SN, cerebellum, respectively) qRT–PCR. n=5for Western blot for all groups.
B, C Relative mRNA expression of Drd2and GABA receptor Gabra3expression by genotype and brain region. n=11,4,4for WT and n=8,4,4for KO (GP, SN,
cerebellum, respectively).
D Western blot quantification from whole brain of tyrosine hydroxylase compared by genotype. n=5for WT and n=3for KO.
Data information: Statistical significance was determined using ANOVA. *P<0.05, ***P<0.001. All graphs represent mean s.e.m.
◀Figure 2. Regional brain differences in iron homeostasis suggest a mechanism for iron overload in PKAN.
A Relative mRNA expression of Tfrc, Ireb2,and Hamp by genotype and brain region. n=11,4,4for WT and n=8,4,4for KO (GP, SN, cerebellum, respectively).
B Total iron quantity in cytosol and mitochondria from GP using ICP-MS. The data distribution is presented using a box plot, including minimum, first quartile,
median, third quartile, and maximum. The magenta-colored line represents mean. n=6for both genotypes.
C Assay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype. Quantification of aconitase activity in two tissues (brain and liver) from
animals of each genotype by band density. n=4for WT, n=5for KO.
D, E Activity of pyruvate dehydrogenase (D) and complex I (E) in GP by genotype and treatment status (veh = vehicle; pPanSH = 40-phosphopantetheine). n=5for
both genotypes and treatment groups.
F Western blot and quantification of TfR1and PDHE1a1from GP by genotype and treatment status (pPanSH = 40-phosphopantetheine). Ponceau S was used as
total protein loading control and VDAC for the mitochondrial protein loading control. n=6,4for WT and n=6,6for KO (vehicle and pPanSH groups, respectively).
Data information: Data were evaluated by one-way ANOVA or two-way ANOVA depending on the number of variables. *P<0.05,**P<0.01, ***P<0.001. All graphs
except (B) represent mean s.e.m.
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Suh Young Jeong et al EMBO Molecular Medicine
Differences by genotype were also found in circulating cells.
Untransformed fresh blood-derived lymphocytes from people with
PKAN showed decreased expression of COASY compared to age-
matched controls, and similar changes were seen in mice (Fig 4B
and Appendix Fig S6B). Furthermore, we demonstrated defects in
mitochondrial function in patient-derived fibroblasts, as well. Using
A
C
DE F
B
Figure 4. Human cells with PANK2mutations show perturbations in the CoA synthesis pathway and diminished mitochondrial respiration.
A Relative quantification of COASY mRNA in human PKAN primary cell lines. n=5for both genotypes and cell types.
B Relative quantification of COASY/Coasy in fresh, blood-derived lymphocytes from human and mouse, respectively compared by genotype. n=51,35 for human
(control, PKAN, respectively) and n=5for both mouse genotypes.
C Extracellular flux analysis in human fibroblasts showing differences in OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) by genotype.
Oligom; oligomycin, FCCP; carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, 2-DG; 2-deoxy glucose. Quantification of baseline OCR and ECAR is shown, as well.
n=3per genotype with four technical replicates.
D Complex I activity assay using fibroblast lysates. Cells were either treated with vehicle or 50 lM pPanSH for 3days, and total protein extract was used for the
Complex I Dipstick assay n=4for both genotypes and treatment groups.
E Relative mRNA expression of mitochondrially encoded genes important for oxidative phosphorylation from complex I (MT-ND1), complex III (MT-CYB), cytochrome C
(MT-CO1), and ATP synthase (MT-ATP6) in human mutant fibroblasts versus control cells. n=3for both genotypes.
F Calculation of active mitochondria using the JC-1assay by genotype in control and PKAN lymphoblasts. n=2.
Data information: Data were analyzed using one-way or two-way ANOVA for statistical significance. *P<0.05;**P<0.01, ***P<0.001. All graphs represent
mean s.e.m.
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extracellular flux analysis of cultured primary fibroblasts from
PKAN patients, we measured oxygen consumption rate (OCR) as a
proxy for respiration and extracellular acidification rate (ECAR) as a
surrogate for glycolysis. We found decreased OCR and increased
ECAR in PKAN cells compared with control cells (Fig 4C), adding to
previously published work done on iPSC-derived neurons (Santam-
brogio et al, 2015). Complex I activity in PKAN fibroblasts was
decreased significantly, as well (Fig 4D). Mitochondrial aconitase is
known to serve a distinct function in coordinating nuclear and mito-
chondrial gene expression (Chen et al, 2005). Thus, we asked if
expression of mitochondrially encoded genes might be affected in
these cells. Indeed, we found decreased expression of genes encod-
ing subunits for complexes I, II, IV, and V (Fig 4E), suggesting a
further basis for reduced respiration. Impaired oxidative phosphory-
lation would lead to loss of the mitochondrial membrane potential,
which we confirmed in patient-derived cells using the JC-1 assay
(Fig 4F). The rise in ECAR suggests that patient cells had undergone
a glycolytic shift, which may also explain the metabolic perturba-
tions that we observed in mouse brain. Loss of respiratory capacity
can lead to a shift toward glycolysis for energy production. Thus,
the human PKAN cell data replicated and expanded results from the
KO mouse, corroborating defects in CoA metabolism and dysfunc-
tion of mitochondria.
We have identified a set of biomarkers that reflect defects in CoA
synthesis, iron homeostasis, mitochondrial function, and dopamine
metabolism in experimental mammalian models of PKAN. These
perturbations mirror key features of the human disease and suggest
that our models can be used to understand PKAN pathogenesis and
to investigate candidate therapeutics.
PPARaagonists fail to rescue brain CoA pathway defects
Why is the globus pallidus selectively vulnerable to loss of
pantothenate kinase 2 function? We considered whether differences
in compensation by other pantothenate kinase proteins might
explain regional CoA pathway defects in brain. We analyzed Pank1
and Pank3 expression as a function of brain region and Pank2 geno-
type. Pank1 produces two protein isoforms, Pank1aand Pank1b,
which differ in their transcriptional and enzymatic regulation (Rock
et al, 2000, 2002) (Ramaswamy et al, 2004). We identified a para-
doxical decrease in Pank1aexpression only in KO GP compared to
WT, with no change in Pank1bexpression (Fig 5A). We found
no differences in other brain regions or Pank3 expression (Fig 5A
and B).
This observation suggested a possible therapeutic approach to
compensate for Pank2 loss via Pank1aup-regulation. Previously,
selective up-regulation of human PANK1atranscription by a PPARa
agonist, bezafibrate, was reported in cultured hepatoblastoma cells
(Ramaswamy et al, 2004). We asked whether the PPARaagonists
bezafibrate and gemfibrozil might normalize Pank1aexpression in
GP in the KO animals. Oral dosing for 14 days with bezafibrate
(0.8 mg/g body weight) and gemfibrozil (1.2 mg/g body weight)
failed to increase expression of either Pank1aor Coasy in GP in KO
animals (Fig 5C). To confirm that each compound was consumed
and reached brain, we analyzed expression of a gene known to be
up-regulated by PPARaagonists (Chen et al, 2017). We quantified
expression in GP of Cpt1c, encoding a neuronal isoform of carnitine
palmitoyltransferase (Chen et al, 2017), and demonstrated that both
fibrate compounds up-regulated expression of Cpt1c in GP from
animals of both genotypes, confirming that the compound altered
the expression of other genes in brain as expected (Appendix Fig
S5B). These results suggested that Pank1 expression may be regu-
lated differently by tissue type or species or both. Alternatively, a
primary defect in Pank2 or its metabolic sequelae might block
PPARaup-regulation of Pank1aexpression. Regardless, this thera-
peutic approach proved unlikely to yield benefit in PKAN.
A
B
C
Figure 5.Pank1expression differences and recovery attempt using
PPARaagonists.
A, B Relative expression of Pank1a, Pank1b,total Pank1,and Pank3in mouse
brain regions. n=8,8,4for both genotypes (GP, SN, cerebellum,
respectively).
C Relative expression of Pank1aand Coasy in GP from WT and KO mice
treated for 14 days with bezafibrate (0.8mg/g body weight) or
gemfibrozil (1.2mg/g body weight). n=8for both genotypes treated
with vehicle, n=5for both KO groups treated with either bezafibrate or
gemfibrozil.
Data information: Data were evaluated by one-way or two-way ANOVA
depending on the number of variables. *P<0.05,**P<0.01, ***P<0.001. All
graphs represent mean s.e.m.
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AB
C
D
Figure 6.
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40-phosphopantetheine normalizes CoA, iron, and dopamine
biomarkers as well as complex I and PDH activities
Using our mouse model, we asked whether the CoA metabolic defect
could be corrected by providing alternate substrates, thereby bypass-
ing the pantothenate kinase defect. We administered equimolar
doses of pantetheine, 40-phosphopantetheine, or CoA orally to mice
for 14 days and then analyzed PKAN-related biomarkers. Pantothen-
ate (vitamin B
5
) was administered as a control. Only 40-phosphopan-
tetheine normalized Coasy expression, suggesting that Coasy
expression is inducible by substrate availability (Fig 6A). Ppcs,
which encodes an enzyme upstream of 40-phosphopantetheine, was
predictably unchanged by the administration of 40-phosphopan-
tetheine or any of the other compounds tested (data not shown). We
observed a dose–responsive normalization in expression of Coasy,
Tfrc, Ireb2, and Drd1 (Fig 6B) and correction of protein levels of
Coasy, Tfr1, and Drd1 in KO GP (Figs 1D and 2F, and 3A). Even the
lowest dose of 40-phosphopantetheine (0.82 lg/g) was effective in
fully correcting the CoA- and iron-related gene abnormalities. In
addition, PDH and complex I activities in KO GP were fully recov-
ered (Fig 2D and E). These data suggest that 40-phosphopantetheine
is orally bioavailable and crosses the blood–brain barrier. With
higher doses of 40-phosphopantetheine, we observed progressively
lower levels of Coasy expression in GP from both WT and KO
animals (Fig 6B), suggesting that Coasy expression is repressible by
product inhibition. Coasy expression in KO mouse circulating
lymphocytes was also normalized by 14 days of administration of
40-phosphopantetheine dosed at 5 lg/g, and we observed the same
inverse dose response seen at higher doses in both WT and KO
animals (Appendix Fig S6B). While an alternate hypothesis to be
considered is that lower levels of 40-phosphopantetheine alleviate
basal repression in the KO animal, this idea does not explain similar
observations in WT animals. Therefore, we favor the more parsimo-
nious interpretation of product inhibition to explain all results. Our
data indicate that 40-phosphopantetheine corrects the primary CoA
pathway-related biomarkers and normalizes secondary defects in
iron, dopamine, complex I, and PDH, a striking finding.
Human cells showed rescue with 40-phosphopantetheine treat-
ment, as well. Primary PKAN fibroblasts treated for 24 h normalized
their expression of COASY and TFRC in a dose-dependent manner
(Fig 6C and Appendix Fig S6C). We again observed an inverse dose
response in COASY expression at higher doses suggesting down-
regulation as a result of product inhibition. 40-phosphopantetheine
decreased the glycolytic shift and normalized complex I activity,
indicating improved mitochondrial respiration in this in vitro human
model (Figs 4D and 6D) and corroborating our findings of func-
tional correction in mouse brain.
We evaluated the duration of effect of 40-phosphopantetheine on
brain biomarkers after its withdrawal. As before, we administered
40-phosphopantetheine orally for 14 days at 5 lg/g and then ceased
administration and sacrificed animals at 0, 1, 2, 3, and 7 days post-
cessation. We found that expression in GP of Coasy, Tfrc, and Drd1
all drifted back to pre-treatment levels in a time-dependent manner
over 7 days (Fig 7A). These results suggest that 40-phosphopan-
tetheine can be titrated to correct the biomarker abnormalities
resulting from loss of pantothenate kinase 2, albeit with the need to
sacrifice animals for the analysis. In this way, the earliest patho-
physiologic changes in PKAN can be investigated in a highly tract-
able true model system.
We found no evidence for toxicity of 40-phosphopantetheine. A
dose of 20 lg/g was administered orally for 14 days to wild-type
mice. Histologic analysis of brain, spinal cord, heart, muscle, liver,
kidney, and spleen by two pathologists blinded to treatment status
revealed no differences (data not shown).
In summary, our data showed perturbations in a set of disease-
relevant biomarkers in mouse and human models of PKAN that
were corrected by 40-phosphopantetheine. Defective pantothenate
kinase 2 resulted in a CoA pathway defect and secondary perturba-
tions in iron homeostasis, dopamine metabolism, complex I and
PDH activities, and mitochondrial respiration. From these, we have
identified candidate pharmacodynamic biomarkers. Our data
showed that 40-phosphopantetheine corrected the CoA-related
defects and resolved all secondary abnormalities in both models.
With these results, we have demonstrated the fidelity of our mouse
model in recapitulating key features of the human disease and
provided evidence in support of 40-phosphopantetheine as a candi-
date therapeutic for PKAN.
Discussion
We present a mouse model of PKAN with CoA, iron, and dopamine
metabolic defects that can be specifically attributed to loss of
pantothenate kinase 2 function. This model recapitulates key
features of the human disease, including iron accumulation and
brain region specificity, and therefore represents a tractable system
with which to investigate disease pathogenesis and evaluate candi-
date therapeutics.
High value has been placed on mouse models with features that
mimic the clinical manifestations of disease in humans. Animals
showing only biochemical and molecular changes have garnered
less favor as models, even when those changes are specifically attri-
butable to the primary cause of disease, as in single-gene disorders.
In fact, such models are likely to be more informative for
◀Figure 6.40-Phosphopantetheine corrects markers of CoA, iron, and dopamine dyshomeostasis and mitochondria l dysfunction in Pank2KO mice.
A Relative quantification of Coasy mRNA from GP compared by treatment status using four compounds: 40-phosphopantetheine (pPanSH), pantetheine, vitamin B
5
, and
CoA. n=8(WT-veh), 5(KO-veh), 5(KO-pPanSH), 4(KO- other three compounds).
B Correction of Coasy, Tfrc, Ireb2,and Drd1expression in GP by 40-phosphopantetheine over a range of doses. n=8(WT-veh), 5(KO-veh), 5(KO-0.82), 4(WT-5), 5(KO-5),
5(KO-8.2), 5(KO-10). 5(WT-20), 5(KO-20).
C Relative quantification of COASY mRNA from human fibroblasts treated with pPanSH. n=3for both genotypes.
D ECAR measured in cultured human fibroblasts by genotype and treatment status. Cells were treated for 3days with 40-phosphopantetheine (pPanSH) or vehicle (veh).
n=3for both genotypes.
Data information: Data were evaluated by one-way or two-way ANOVA depending on the number of variables. Asterisks shown in (B–D) represent statistical significance
compared to the vehicle-treated control groups. *P<0.05,**P<0.01, ***P<0.001. All graphs represent mean s.e.m.
ª2019 The Authors EMBO Molecular Medicine 11:e10489 |2019 9of 17
Suh Young Jeong et al EMBO Molecular Medicine
investigating critical early disease changes before cells and tissues
sustain extensive damage. Molecular perturbations precede clinical
manifestations and are arguably more informative than an overt
neurological sign. We now report Pank2 KO animals with abnormal
levels of CoA-, iron-, and dopamine-related biomarkers, diminished
activities of complex I and pyruvate dehydrogenase, and mitochon-
drial dysfunction, all of which were found to be regionally localized
in brain. These changes represent the molecular cascade of early
PKAN pathogenesis resulting from loss of pantothenate kinase 2.
We favor a hypothesis of PKAN pathogenesis proposed by
Lambrechts et al (2019) that integrates and explains the myriad
cellular changes we report (Fig 8). If there is a deficiency of CoA in
PKAN, processes in which there is net consumption of CoA may be
most sensitive to that loss. CoA is consumed for the phosphopan-
tetheinyl activation of certain proteins, whereas most other path-
ways that utilize CoA result in no net change in levels. These
activated proteins play essential roles in mammals, including as
acyl carrier proteins (Praphanphoj et al, 2001; Donato et al, 2007).
Mitochondrial acyl carrier protein (mtACP), which requires phos-
phopantetheinyl activation, is essential for electron transport, type
II fatty acid synthesis, iron–sulfur cluster biogenesis, and tRNA
processing via RNase P (Chuman & Brody, 1989; Brody et al, 1997;
Autio et al, 2008; Hiltunen et al, 2009; Van Vranken et al, 2016,
2018). Moreover, mtACP is a key factor in coordinating the cellular
response to nutrient availability with intermediary metabolism via
its requirement for acetyl-CoA (Hiltunen et al, 2010; Kursu et al,
2013; Van Vranken et al, 2018). If phosphopantetheinylation of
mtACP was impeded by a defect in pantothenate kinase 2, then we
would expect to see perturbations in these pathways. Specifically,
failure to phosphopantetheinylate mtACP would be predicted to
lead to impaired complex I activity resulting in decreased oxidative
phosphorylation and loss of mitochondrial membrane potential,
impaired lipoic acid production with loss of activity of lipoylated
enzymes, and impaired iron–sulfur cluster biogenesis with loss of
activity of Fe-S-dependent enzymes and processes ultimately lead-
ing to iron dyshomeostasis.
Indeed, we observe precisely these changes. mtACP is the
NDUFAB1 subunit of complex I, and complex I activity is dimin-
ished in our PKAN models (Figs 2E and 4D). Mitochondrial fatty
acid synthesis requires the phosphopantetheinyl arm of mtACP to
transport reaction intermediates between catalytic components in
order to generate acyl-ACPs including octanoyl-ACP, which is the
precursor of lipoic acid. Lipoic acid is consumed in the post-transla-
tional lipoylation of proteins, including pyruvate dehydrogenase,
which requires this modification for its activity. Thus, the decrease
in PDH activity that we report (Fig 2D and F) is hypothesized to
arise from a decrease in lipoylation resulting from inactive mtACP.
Moreover, Lambrechts et al (2019) show that under conditions of
PANK2 depletion, levels of holo-mtACP were decreased in mamma-
lian cells and in Drosophila cells, and lipoylation of PDH was also
decreased. Acyl-ACPs are also required for electron transport chain
complex assembly and for iron-sulfur cluster biogenesis through
their interactions with LYR proteins (Maio et al, 2014; Maio &
Rouault, 2015). Therefore, loss of acyl-ACPs is the proposed mecha-
nism leading to impaired iron–sulfur cluster formation, which
would cause loss of activities of iron–sulfur cluster-dependent
complexes and enzymes, including complex I and mitochondrial
aconitase, and iron dyshomeostasis. Finally, mtACP is required for
processing of mitochondrial tRNAs by RNase P (Autio et al, 2008),
and a defect in mtACP might further impair respiration by limiting
the synthesis of mitochondrially encoded subunits to form other
complexes required for electron transport. Though alternative expla-
nations could be proposed for each result that we observe, a defect
in mtACP activation explains all of the observed perturbations via a
single direct link to CoA, thereby reflecting the most parsimonious
interpretation of our data and providing a compelling hypothesis of
PKAN pathogenesis.
A
Figure 7. Withdrawal of 40-phosphopantetheine causes brain
biomarkers to return to pre-treatment levels.
Relative quantification of Coasy, Tfrc, and Drd1 mRNA from GP by number of
days post-cessation of treatment with 5 lgof4
0-phosphopantetheine per g of
body weight. n=11 (WT-veh-D0), 5 (KO-veh-D0), 4 (WT-pPanSH-D0), 5 (KO-
pPanSH-D0), 5 (KO-pPanSH-D1), 5 (KO-pPanSH-D2), 5 (KO-pPanSH-D3), 5 (WT-
pPanSH-D7), 4 (KO-pPanSH-D7). Data shown were evaluated by one-way
ANOVA. Asterisks represent statistical analyses compared to the WT vehicle
group. *P<0.05, **P<0.01, ***P<0.001. All graphs represent mean s.e.m.
10 of 17 EMBO Molecular Medicine 11:e10489 |2019 ª2019 The Authors
EMBO Molecular Medicine Suh Young Jeong et al
Why might mtACP function be impaired but not that of other
CoA-dependent processes? Lambrechts et al (2019) have indica-
tions that levels of CoA are not decreased but that levels of holo-
mtACP are decreased. Perhaps differences in the synthesis and
regulation of subcellular compartmental CoA pools explain this.
We hypothesize that a specific pool of CoA is needed to phospho-
pantetheinylate mtACP, that its genesis requires pantothenate
kinase 2, that this pool is depleted in PKAN and underlies its
molecular pathogenesis, and that exogenous 40-phosphopantetheine
can replenish that pool. However, within the whole cell pools of
CoA, this deficiency cannot be detected. The localization of several
CoA synthesis enzymes to mitochondria, including PANK2, PPCS,
and COASY but not PPCDC (Hortnagel et al, 2003; Johnson et al,
2004; Kotzbauer et al, 2005; Uhlen et al, 2010; Rhee et al, 2013;
Dusi et al, 2014, and Appendix Fig S2F), raises the intriguing possi-
bility that CoA pathway intermediates such as 40-phosphopan-
tetheine might traverse organellar membranes and serve to
replenish compartmental pools of CoA (Srinivasan et al, 2015;
Sibon & Strauss, 2016). Alternatively, since pantothenate kinases
can directly phosphorylate pantetheine to yield 40-phosphopan-
tetheine (Levintow & Novelli, 1954; Strauss, 2010), mitochondria
may depend on pantothenate kinase 2 and this alternate synthesis
pathway in order to directly produce CoA destined specifically for
the phosphopantetheinylation of mtACP. Although the only known
phosphopantetheinyl transferase in mammals localizes to cyto-
plasm (Beld et al, 2014), a separate mitochondrial phosphopanteth-
einyl transferase similar to one found in yeast may exist (Stuible
et al, 1998). CoA is the required substrate for all phosphopanteth-
einyl transferases characterized to date; none can use 40-phospho-
pantetheine (Beld et al, 2014). Recent reports that cellular CoA can
be synthesized directly from exogenous sources of 40-phosphopan-
tetheine would predict the potential for this key intermediate to
rescue cellular changes in our disease models (Srinivasan et al,
2015; Lambrechts et al, 2019). Here, we suggest that exogenous 40-
phosphopantetheine can serve to replenish the CoA pool that is
specifically depleted in PKAN.
We show that 40-phosphopantetheine can correct the primary
CoA metabolic defect as well as the secondary changes observed.
The brain abnormalities are fully corrected at the level of gene
expression, protein quantity, and enzyme activity following the oral
administration of 40-phosphopantetheine. These changes in gene
expression represent a pharmacodynamic biomarker set that can
serve for the preclinical development of PKAN therapeutics. CoA
synthase gene expression was also found to be decreased in circulat-
ing cells, representing a marker in accessible tissue that could be
tracked during an interventional study. Moreover, circulating cell
biomarker levels appear to change in parallel with those in brain in
our mouse model, supporting their use as a possible pharmacody-
namic surrogate marker in clinical trials of rational therapeutics for
PKAN.
Only 40-phosphopantetheine corrected Coasy expression in our
model; vitamin B
5
, pantetheine, and CoA failed to normalize this
biomarker. Neither pantetheine nor CoA is stable in serum. CoA is
probably dephosphorylated by intestinal phosphatases and subse-
quently catabolized to pantetheine, and pantetheine is degraded by
serum pantetheinases to pantothenate and cysteamine (Shibata
et al, 1983; Wittwer et al, 1985). Thus, neither of these compounds
when fed orally would be expected to reach the target tissue intact.
In contrast, 40-phosphopantetheine seems to escape these degrada-
tions. The fact that oral administration of 40-phosphopantetheine
normalizes brain biomarkers in our in vivo model strongly suggests
that this molecule is not degraded by intestinal phosphatases, read-
ily crosses membranes, and reaches brain intact. This is further
supported by published data documenting measurable quantities of
Figure 8. PKAN pathogenesis is hypothesized to center around mitochondrial ACP and its activation by 40-phosphopantetheine.
Our current model predicts that defects in pantothenate kinase 2 impair the phosphopantetheinylation of mtACP and lead to impaired complex I activity (Fig 2E), iron–sulfur
biogenesis (Fig 2C), and mitochondrial fatty acid synthesis. Lipoic acid modification is required at the E2 domain of PDH complex. Decreased production of lipoic acid in
mitochondria would diminish activity of this enzyme (Fig 2D). The clinical features of PKAN, including abnormal iron accumulation and parkinsoni sm, can be explained by
these metabolic defects. Their rescue by exogenous 40-phosphopantetheine suggests that the CoA pool critical for mtACP phosphopantetheinylation is at least partially
dependent on pantothenate kinase 2.
ª2019 The Authors EMBO Molecular Medicine 11:e10489 |2019 11 of 17
Suh Young Jeong et al EMBO Molecular Medicine
endogenous 40-phosphopantetheine in wild-type mouse serum
(Srinivasan et al, 2015).
Our disease model studies have revealed new information about
the CoA pathway and its regulation. Loss of pantothenate kinase 2
function diminishes levels of Ppcs and Coasy expression to ~40–60%
of control levels. This difference is hypothesized to reflect the
portion of CoA synthesis that is specifically attributable to
pantothenate kinase 2. We further suggest that regulation of expres-
sion of the CoA synthase gene can be controlled by levels of
substrate, 40-phosphopantetheine, and product, CoA, either directly
or possibly via the CoAlation of transcription factors or regulatory
proteins (Tsuchiya et al, 2017, 2018; Gout, 2018).
Important questions have been raised by our studies that remain
unanswered. What is the basis for the selective vulnerability of GP
to a loss of function of pantothenate kinase 2? There are myriad
possibilities but no data yet to support any one; they include
regional differences in energy demand, cell membrane composition,
mitochondrial fatty acid synthesis, iron–sulfur cluster biogenesis,
CoA demand, vascular anatomy, and vulnerability to hypoxia.
PKAN biochemical changes are regionally specific in brain; so, why
are similar abnormalities seen in “non-diseased” tissues such as
fresh lymphocytes and cultured fibroblasts? For this observation, we
have no strong hypothesis. Given the apparent capacity for exoge-
nous 40-phosphopantetheine to easily traverse cell membranes, why
are endogenous sources from other subcellular compartments, cells,
or tissues unable to mitigate the disease process in PKAN? Our
incomplete knowledge about the CoA pathway and organellar dif-
ferences in CoA metabolism and regulation precludes our offering a
compelling hypothesis at this time.
Brain iron accumulation is a prominent feature of PKAN, yet the
mechanism underlying iron dyshomeostasis has perplexed the field
since discovery of the pantothenate kinase 2 gene (Zhou et al,
2001). We show that cells in GP are correctly sensing and respond-
ing to high iron in both the cytosolic and mitochondrial fractions.
However, we also show impaired function of an iron–sulfur cluster-
dependent enzyme, mitochondrial aconitase, suggesting that these
cells are starved for bioavailable iron. Impaired mitochondrial func-
tion, insufficient iron-–sulfur cluster biogenesis, or sequestration of
iron could all lead to errant signaling between cytosol and mito-
chondria to acquire more iron. Loss or mis-sensing of bioavailable
iron is a common theme leading to mis-communication between
organelles and signaling for increased uptake, a phenomenon that is
hypothesized to explain iron accumulation in many neurodegenera-
tive disorders (Babcock et al, 1997; LaVaute et al, 2001; Calmels
et al, 2009; Richardson et al, 2010; Rouault, 2012; Matak et al,
2016). Our data suggest that this mechanism is at work in PKAN, as
well.
This concept calls into question the rationale for chelation as a
therapeutic approach in PKAN and other neurodegenerative disor-
ders if bioavailable iron is, in fact, insufficient. Alternatively, chela-
tion may complement a rational therapeutic approach by
accelerating the removal of mis-sequestered iron that might be
contributing to disease (Klopstock et al, 2019). We note that exoge-
nous 40-phosphopantetheine corrects iron dyshomeostasis in both
our in vivo and in vitro models, suggesting that resolution of the
primary CoA defect may be sufficient to correct downstream seque-
lae, including iron accumulation. Though we have not proven that
correction of the iron defect derives directly from correction of the
CoA metabolic pathway defect, this would be the most parsimo-
nious interpretation of our data. Moreover, this interpretation would
fit with the prediction that re-activation of mtACP would correct
iron–sulfur biogenesis. On this basis, we suggest that rescue of the
CoA metabolic defect in PKAN may be sufficient to enable cells to
re-establish iron homeostasis without any other interventions.
While loss of iron–sulfur clusters is a compelling explanation for the
observed decrease in mitochondrial aconitase activity, this defect
may instead result from slowing of the tricarboxylic acid cycle due
to lack of CoA or from lack of acetyl-CoA due to a defect in PDH. To
investigate such mechanistic questions, these tractable disease
model systems now can serve as critical tools.
Our findings of a dopamine metabolic defect are consistent with
the clinical features of dystonia and parkinsonism that are observed
in PKAN. Dopaminergic neurons are known to be vulnerable to
complex I dysfunction resulting from exposure to the selective
toxins rotenone and MPTP (Ferrante et al, 1997; Sriram et al, 1997;
Greenamyre et al, 1999; Matak et al, 2016). While the mechanism
for this vulnerability is unclear (Choi et al, 2008), we hypothesize
that a complex I defect, possibly in combination with oxidative
damage from increased iron, underlies the dopaminergic defect in
PKAN. Furthermore, iron is a cofactor for tyrosine hydroxylase
activity, leaving open the possibility that iron dyshomeostasis might
contribute in multiple ways to the dopamine metabolic perturba-
tions that we see in mouse brain. We note that the dopamine defect
is reversible and propose that this results from correction of the CoA
pathway defect and subsequent rescue of complex I function and
iron homeostasis.
Taken together, our results provide strong evidence for a phar-
macodynamic biomarker set that includes proximal, remote, and
surrogate markers for use in preclinical as well as clinical studies.
These markers of disease are linked to a cascade of molecular
changes that can explain key features of PKAN, including pallidal
iron accumulation. These features are recapitulated in our mouse
disease model, in which all perturbations can be specifically attrib-
uted to a defect in pantothenate kinase 2, an important fact that
distinguishes this from other published models. We have identified
new factors that regulate the CoA pathway. In addition, we provide
a new framework and research resources with which to investigate
the cellular role of pantothenate kinase 2 and the vulnerability of
the globus pallidus to its dysfunction. Finally, we offer strong
evidence in support of a CoA pathway intermediate, 40-phosphopan-
tetheine, as a therapeutic for PKAN.
Materials and Methods
Animals and Cells
A murine model with germline Pank2 null mutation on C57/BL6
background was generated by inserting a stop codon into exon 2
as reported previously (Kuo et al, 2005). This background was
maintained by backcrossing to pure C57/BL6 mice (Jackson Labo-
ratories) every 3–4 years. Mice were housed at Oregon Health &
Science University vivarium under the care of Department of
Comparative Medicine. Animals were housed under 12-h light
cycle and cared with daily monitoring while having free access to
chow and water. All experimental protocols were pre-approved by
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EMBO Molecular Medicine Suh Young Jeong et al
OHSU IACUC (protocol no; IP00000450) and following NIH Office
of Laboratory Animal Welfare and NC3Rs guidelines. All animals
used for experiments were between 3–6 months of age and n=3–
11 per group in each experiment, and both genders were used.
The exact number of animals used in each experiment is listed in
the figure legend.
Human primary fibroblasts and lymphoblasts were generated
using biopsied samples from PKAN patients and age-matched
healthy controls using previously published methods (Rittie &
Fisher, 2005; Darlington, 2006). Briefly for fibroblasts, following
informed consent, a 2-mm skin punch biopsy was obtained with
local anesthesia and plated after digesting with proteinases at 37°C.
After 2–3 weeks, the tissue piece was removed from culture, and
fibroblasts were expanded and stored at the lowest passage number.
For the lymphoblasts, leukocytes were separated from patient blood
using gradient centrifugation (Histopaque, Millipore Sigma) and
transformed using Epstein–Barr virus (ATCC). This transformation
of human blood cells is approved by OHSU Institutional Biosafety
Committee (protocol no. IBC-11-28). All subjects and the sample
collection for the repository are part of OHSU’s IRB-approved proto-
col e7232 with additional work covered by protocol e144.
Human sample collection using PAXgene blood RNA tubes
Informed consent was obtained from all subjects, and the experi-
ments conformed to the principles set out in the WMA Declaration
of Helsinki and the Department of Health and Human Services
Belmont Report. All subjects were consented as part of OHSU’s IRB-
approved protocol e7232 with additional work covered by protocol
e144. Human blood from PKAN patients and age-matched controls
was collected using PAXgene
Blood RNA tubes (BD Biosciences).
After a 24-h incubation to ensure lysis of red blood cells, remaining
cells were pelleted and stored at 80°C. Later, total RNA was
isolated (Qiagen) and analyzed using qRT–PCR method.
Mouse brain trisection
Mouse brain was removed from skull and cut sagittally in the
middle. The olfactory bulb was removed, and the rest of the hemi-
sphere was cut at Bregma 0.25, 2 and 5 mm. Cortex was
removed from the GP-containing piece to generate a GP-enriched
sample (GP). Cortex and superior colliculus were removed from the
SN-containing piece to yield a SN-enriched sample (SN). Brainstem
was removed from the posterior brain piece to isolate cerebellum.
There were no differences in trisected area between genotypes in
terms of tissue atrophy, tissue weight, or total protein per tissue
weight (data not shown). This was a relatively crude dissection
method for each area, and other brain areas were present in the GP-
and SN-enriched samples. These areas are thalamus, hypothalamus,
striatum and some parts of pallidum for GP, and part of the
midbrain including ventral tegmental area, red nucleus, and oculo-
motor nucleus for SN. Enrichment of each area was confirmed by
qRT–PCR of known region-specific genes (Appendix Fig S1B).
qRT–PCR
Total RNA was isolated from various samples, and genomic DNA
was removed using gDNA eliminator column (Qiagen). Total RNA
from both mouse brain and PAXgene Blood RNA tube-isolated
lymphocytes was extracted using phenol/chloroform method with
QIAzol (Qiagen), and primary human cell RNA was extracted using
RNeasy Plus Mini Kit (Qiagen). Total RNA concentration was
measured using Epoch plate reader (BioTek), and 1 lg of RNA was
subject to reverse transcription following manufacturer’s protocol
(SuperScript
TM
III First-Strand Synthesis System, ThermoFisher
Scientific). Real-time analyses of these cDNA were performed using
a Rotor-Gene Q real-time thermal cycler and Rotor-Gene SYBR
Green PCR Kit (Qiagen) following manufacturer’s protocol. All data
were normalized to the expression of housekeeping genes (Gapdh
for mice, 18s for human) and then normalized to the control group
expression (Comparative C
T
Method, DDC
T
). Two factors that are
crucial for the DDC
T
method, the equal expression of housekeeping
gene and primer efficiencies, were listed in Appendix Fig S1A. The
complete list of primers can be found in Appendix Table S1.
Westerns and antibodies
Total protein was isolated and quantified using a modified Bradford
assay (Bio-Rad). Western blot analyses were performed using the
following primary antibodies: mouse anti-COASY (Santa Cruz
sc-393812, 1/1,000), rat anti-D1 dopamine receptor (Sigma D2944,
1/1,000), mouse anti-TFR1 (ThermoFisher 13-6800, 1/3,000), rabbit
anti-pyruvate dehydrogenase (Cell Signaling 3205, 1/3,000), rabbit
anti-VDAC (Cell Signaling 4661, 1/5,000), mouse anti-MSH6 (BD
610918, 1/10,000), and mouse anti-bactin (ThermoFisher AM4302,
1/10,000). Most of the primary antibody binding was visualized
with the traditional method using a HRP-conjugated secondary anti-
body (Jackson ImmunoResearch, 1/10,000) and ECL substrate
(SuperSignal
TM
West Pico PLUS, ThermoFisher). However, anti-
DRD1 and its loading control (anti-MSH6) were visualized using a
biotinylated secondary antibody and Alexa Fluor
TM
488-conjugated
streptavidin (ThermoFisher, 1/4,000). The fluorescent Western blot
signal was captured using iBright
TM
FL1000 (ThermoFisher).
Samples, densitometry, and statistical analysis
For human studies, sample size was determined based on the
number of available human patients and their age-matched controls.
For mouse studies, minimum of three animals per genotype and
treatment group to maximum of 11 were used to ensure statistical
analyses. All samples were assigned with non-descriptive,
anonymizing ID to hide genotype and treatment status. Samples
were equally distributed into various treatment groups. All biochem-
ical analyses that resulted in bands were measured using Image
Studio
TM
Lite by Li-Cor (www.licor.com/bio/image-studio-lite).
Statistical analyses were performed using SigmaStat 4.0 (Systat Soft-
ware) with either one-way ANOVA or two-way ANOVA depending
on the number of variables within experiments. All the dataset
passed the Shapiro–Wilk normality test and Brown–Forsythe equal
variance test (SigmaStat). Statistical significance was determined
when P<0.05 and labeled with * or **P<0.001, or ***P<0.0001
in figures to avoid clutter. Most of the data figures contain only
biologically meaningful statistical analyses, with the full analyses
provided throughout the Appendix figure section and
Appendix Table S2. Unless stated specifically in the figure legend,
all the data were presented as mean s.e.m.
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Suh Young Jeong et al EMBO Molecular Medicine
ICP-MS including mitochondrial isolation
Dissected mouse brain was snap-frozen in liquid nitrogen and
stored in a nitric acid-washed tube at 80°C. Crude mitochondrial
isolation was performed using a Mitochondria Isolation Kit (Abcam)
and checked for cross-contamination in each fraction using Western
blot assays. Both mitochondrial and cytosolic fractions were
analyzed for various elements in collaboration with Dr. Martina
Ralle (Elemental Analysis Core, OHSU) using ICP-MS (Inductively
Coupled Plasma Mass Spectroscopy). A small portion of each
subcellular fraction was used to measure DNA quantity (Qiagen)
and used as the normalization factor.
Activity assays
Activities of two [Fe-S] containing enzymes, mitochondrial and
cytosolic aconitase, were measured as an in-gel assay as previously
published (Tong & Rouault, 2006). Briefly, total protein from mouse
brain and liver was extracted and run in a non-denaturing gel and
incubated with a reaction buffer containing cis-aconitate. The enzy-
matic reaction produces four distinct bands (two for mitochondrial
and two for cytosolic), and the band densities were measured. A
duplicated denaturing gel was produced and stained with Coomassie
Brilliant Blue to serve as a loading control.
Activities of two mitochondrial enzyme complexes (complex I
and PDH) were measured using Dipstick assays following manufac-
turer’s protocols (Ambion). Briefly, total protein from dissected
mouse GP or cultured cells was extracted using the extraction buffer
with mild detergent, and total protein concentration was measured
using Bradford assay. To check potential saturation of capturing
antibodies, only WT GP samples were analyzed using increasing
amounts of protein. Once a saturation curve was established,
approximately ¾point within the linear range was selected as the
loading amount (6 lg for PDH activity assay, and 2 lg for the
complex I activity assay). Then, samples from each group were
wicked through a Dipstick containing a band of capturing antibod-
ies, and wash buffer was applied. The target-specific substrates were
then converted to colored bands and their densities analyzed.
Extracellular flux analysis and JC-1assay
Primary non-transformed fibroblasts were cultured in standard
media and plated in Seahorse XF24 Analyzer plates (Agilent) the
day before each experiment (10,000–20,000 cells per well depending
on the growth speed). On the day of the experiment, cells were
washed and switched to the XF Base Medium one hour before
experiment (2 mM glutamine was added for the ECAR assay per
manufacturer’s recommendation). Both ECAR and OCR were
measured following manufacturer’s protocol. After the experiment,
cells were washed, and live cells were counted as a normalization
factor.
The relative population of cells with active mitochondria was
measured using a JC-1 dye following manufacturer’s protocol (Ther-
moFisher Scientific). Briefly, both control and PKAN lymphoblasts
were washed and incubated with JC-1, and the emission fluorescent
intensities at 595 nm (red) and 535 nm (green) were measured. The
red/green ratio from the control cells was set at 100%, and the
PKAN cells were plotted as a relative percentage.
Treatment protocols
Both PPARaagonists were purchased from Millipore Sigma. Both
WT and KO mice were treated with bezafibrate (0.8 mg/g body
weight) or gemfibrozil (1.2 mg/g body weight) by mixing with
ground standard mouse chow for 14 days. Singly caged mice had
limited access to food to ensure correct dose delivery (170 mg
chow/g body weight). After 14 days, mice were sacrificed and GP
area was dissected and analyzed for expression of a gene considered
to serve as a positive control for treatment (Cpt1c, Appendix Fig
S5B).
Both pantetheine and vitamin B
5
were purchased from Millipore
Sigma. Coenzyme A was purchased from CoALA Biosciences
(Austin, TX), and highly pure (>97%) 40-phosphopantetheine
(pPanSH) was provided by Dr. Ody Sibon (University of Groningen,
the Netherlands). For the efficacy test (Fig 6A), all drugs were deliv-
ered orally in 10% sucrose solution for 14 days at 5 lg/g for
pPanSH and at molar equivalent dosages for all others. After
14 days, mice were sacrificed, and dissected brain was analyzed
for gene expression recovery. After this experiment, a dose determi-
nation experiment was performed using only pPanSH from 0 to
20 lg/g dose range (Fig 6B and C). For the in vitro study, primary
human fibroblasts were treated with pPanSH at various concentra-
tions (0–200 lM) for 24 h. Cells were then washed and pelleted,
and qRT–PCR analyses were performed to analyze expression of
various genes. For the extracellular flux analysis, cells were treated
The paper explained
Problem
PKAN is a rare, high burden movement disorder for which there are
no approved disease modifying agents. The lack of a high-fidelity
mouse model has impeded understanding of disease pathophysiology
and development of rational therapeutics.
Results
Using a mouse null mutant of Pank2, the mitochondrial isoform of
pantothenate kinase, we show a defect in coenzyme A metabolism
that is revealed by careful dissection of brain into disease-vulnerable
(GP) and disease-protected (SN and cerebellum) regions, an idea based
on the exquisitely focal pathology found in the human disorder. In
addition, we see increased cellular iron levels and perturbations in
iron-related proteins and in iron-sulfur cluster-dependent enzyme
activities. Defects in electron transport chain function and pyruvate
dehydrogenase activity were also observed. Mitochondrial acyl carrier
protein activation by coenzyme A is hypothesized to be the central
factor tying together these disparate metabolic changes and under-
lying the molecular cascade of PKAN pathogenesis. All metabolic
perturbations are normalized by oral administration for 2weeks of a
CoA pathway intermediate, 40-phosphopantetheine.
Impact
We describe a mouse model of PKAN in which all features are specifi-
cally attributable to loss of pantothenate kinase 2activity. Recapitula-
tion of key human disease features, including iron accumulation in
globus pallidus and dopamine dyshomeostasis, supports the fidelity of
this model and its utility for delineating disease pathogenesis and
testing candidate therapeutics. Correction of all disease features by
40-phosphopantetheine provides compelling evidence to support a
clinical trial in PKAN.
14 of 17 EMBO Molecular Medicine 11:e10489 |2019 ª2019 The Authors
EMBO Molecular Medicine Suh Young Jeong et al
with 50 lM pPanSH for three days in standard culture media, and
pPanSH was replenished every 24 h based on its half-life in serum
(Srinivasan et al, 2015).
For the withdrawal study, both WT and KO mice were treated
with either vehicle or pPanSH at 5 lg/g for 14 days. Each group
of mice was then sacrificed at day 0, 1, 2, 3, or 7 and their
brains dissected and prepped to analyze the duration of effect of
pPanSH.
Expanded View for this article is available online.
Acknowledgements
We are grateful to PKAN families worldwide, who have partnered with us in
this important project and provided steady support and inspiration. This work
was supported by NIH R21HD088833 (S.J.H.) and R01NS109083 (S.J.H.), the
NBIA Disorders Association (S.Y.J.), Friends of Doernbecher (P.H.), the OCTRI
Biomedical Innovation Program (P.H.), and the Collins Foundation (P.H.). The
OHSU Medicinal Chemistry Core was instrumental in supporting this work
through creativity, perseverance, and drive. We thank the OHSU Elemental
Analysis Core for expert support. Research reported in this publication was
supported by the National Center for Advancing Translational Sciences of the
National Institutes of Health under award number UL1TR0002369. The
content is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of Health.
Author contributions
SYJ, SJH, OCMS, PH, RL, and RW designed the research studies, analyzed data,
and wrote the manuscript. SYJ, RF, JC, TP, NG, MZ, MR, and MD conducted
experiments, and acquired and analyzed data. AMG, RF, JH, DZ, LS, PR, AF, and
KW acquired samples or conducted experiments. AP, HJ, and AN generated
essential reagents.
Conflict of interest
Drs. Jeong, Hayflick, and Hogarth are co-inventors on a patent application for
the PKAN biomarker set. Drs. Hayflick and Sibon are co-inventors on a patent
application for 40-phosphopantetheine for use in disorders exclusive of PKAN.
Dr. Sibon is a co-inventor on a patent application for acetyl-40-phosphopan-
tetheine for use in PKAN and in related disorders. Dr. Hayflick is a non-compen-
sated member of the Scientific Advisory Board of BioPontis Alliance, a non-profit
organization. Drs. Hayflick and Hogarth are non-compensated members of the
Scientific and Medical Advisory Board of the NBIA Disorders Association, a non-
profit lay advocacy organization. Dr. Hayflick is a non-compensated member of
the Scientific and Medical Advisory Board of the NBIA Alliance, a non-profit lay
advocacy organization. Drs. Hayflick and Hogarth serve as non-compensated
executives for the Spoonbill Foundation, a not-for-profit organization that may
benefit from the results of this research and technology. This potential conflict
of interest has been reviewed and managed by OHSU.
For more information
(i) http://nbiacure.org
(ii) https://www.nbiadisorders.org
(iii) http://www.nbiaalliance.org
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