Epilepsy Research 116 (2015) 40–52

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Epilepsy Research
journal homepage: www.elsevier.com/locate/epilepsyres

Review article

Pyruvate dehydrogenase complex deficiency and its relationship with

epilepsy frequency – An overview
Suman Bhandary a,b , Kripamoy Aguan a,∗
a
Department of Biotechnology & Bioinformatics, North-Eastern Hill University, Shillong 793 022, India
b
Division of Molecular Medicine, Bose Institute, Kolkata 700 054, India

a r t i c l e i n f o a b s t r a c t

Article history: The pyruvate dehydrogenase complex (PDHc) is a member of a family of multienzyme complexes that
Received 9 March 2015 provides the link between glycolysis and the tricarboxylic acid (TCA) cycle by catalyzing the physio-
Received in revised form 29 June 2015 logically irreversible decarboxylation of various 2-oxoacid substrates to their corresponding acyl-CoA
Accepted 5 July 2015
derivatives, NADH and CO2 . PDHc deficiency is a metabolic disorder commonly associated with lactic
Available online 8 July 2015
acidosis, progressive neurological and neuromuscular degeneration that vary with age and gender. In
this review, we aim to discuss the relationship between occurrence of epilepsy and PDHc deficiency
Keywords:
associated with the pyruvate dehydrogenase complex (E1␣ subunit (PDHA1) and E1␤ subunit (PDHB))
PDH complex deficiency
Epilepsy
and PDH phosphatase (PDP) deficiency. PDHc plays a crucial role in the aerobic carbohydrate metabolism
Metabolic disorder and regulates the use of carbohydrate as the source of oxidative energy. In severe PDHc deficiency, the
Ketogenic diet energy deficit impairs brain development in utero resulting in physiological and structural changes in the
Antiepileptic drugs brain that contributes to the subsequent onset of epileptogenesis. Epileptogenesis in PDHc deficiency is
X-linked linked to energy failure and abnormal neurotransmitter metabolism that progressively alters neuronal
PDHA1 excitability. This metabolic blockage might be restricted via inclusion of ketogenic diet that is broken
up by ␤-oxidation and directly converting it to acetyl-CoA, and thereby improving the patient’s health
condition. Genetic counseling is essential as PDHA1 deficiency is X-linked. The demonstration of the X-
chromosome localization of PDHA1 resolved a number of questions concerning the variable phenotype
displayed by patients with E1 deficiency. Most patients show a broad range of neurological abnormalities,
with the severity showing some dependence on the nature of the mutation in the El␣ gene, while PDHB
and PDH phosphatase (PDP) deficiencies are of autosomal recessive inheritance. However, in females, the
disorder is further complicated by the pattern of X-chromosome inactivation, i.e., unfavorable lyoniza-
tion. Furthermore research should focus on epileptogenic animal models; this might pave a new way
toward identification of the pathophysiology of this challenging disorder.
© 2015 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2. Pyruvate dehydrogenase and its regulatory enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3. Clinical features of congenital PDH complex deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4. Mitochondrial epilepsy and its pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5. Metabolic management and pharmacologic control of PDHc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
6. Molecular targets for antiepileptic drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
7. Genetic etiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
9. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

∗ Corresponding author.
E-mail address: kmaguan@gmail.com (K. Aguan).

http://dx.doi.org/10.1016/j.eplepsyres.2015.07.002
0920-1211/© 2015 Elsevier B.V. All rights reserved.
 S. Bhandary, K. Aguan / Epilepsy Research 116 (2015) 40–52
1. Introduction
In each and every cell the pyruvate dehydrogenase complex
catalyzes the irreversible oxidative decarboxylation of pyruvate
to form acetyl-CoA and functions as a gateway to the oxidative
metabolism of carbohydrate within mitochondria. This reaction
interlinks glycolysis, citric acid cycle and the respiratory chain.
Thereby enabling the stepwise transfer of electrons from reducing
equivalents (NADH and FADH2 ) to molecular oxygen and subse-
quent synthesis of ATP (Fig. 1A) (Margineantu et al., 2002). A defect
in any of these pathways of pyruvate utilization may lead to the
accumulation of pyruvate and lactate resulting in lactic acidemia
(Robinson and Sherwood, 1984; Robinson, 1989, 2000; Vary, 1991).
The most common factor that causes lactic acidemia is the defi-
ciency of the pyruvate dehydrogenase complex. Genetic defects in
the pyruvate dehydrogenase complex are associated with a range
of clinical abnormalities in the nervous system, although impaired
development and poor functioning of other tissues including mus-
cle and liver also occur. The brain, unlike other organs, normally
produces ATP almost exclusively from glucose oxidation via gly-
colysis and tricarboxylic acid cycle. Blockage in this process, at the
step of pyruvate oxidation, is catalyzed by PDHc resulting in ATP
deprivation and damage to the brain and other tissues in patients
with moderate and severe deficiencies of PDHc activity. Currently,
there is no effective treatment available and as a result infants
with severe PDHc deficiency survive for only two to three years
at most. Less severe deficiencies in PDHc activity can result in
decreased production of acetyl-CoA in brain. This, in turn, leads to
decreased synthesis of the neurotransmitter, acetylcholine, which
may be a factor contributing to the poorly coordinated move-
ments seen in patients with mild PDHc deficiency. Symptoms may
include neurological manifestations which include developmen-
tal delay/intellectual disability, hypotonia, hypertonia, dystonia,
epileptic seizures, ataxia, and axonal neuropathy (Barnerias et al.,
2010). Thus, control of this enzyme complex plays a decisive role
Fig. 1. (A) A scheme of the metabolic process that links PDHc deficiency to ATP generation and drugs that block energy metabolism in both neurons and astrocytes. (B) The
overall reaction mechanism of the pyruvate dehydrogenase complex.
41
in regulating the fuel used by various tissues of the body. A number
of comprehensive reviews on various aspects of pyruvate dehydro-
genase multienzyme complex have caught attention in the recent
past. However, relatively little attention has been accorded over
the relationship between pyruvate dehydrogenase complex (PDHc)
and epilepsy. The rationale behind this present review is to abridge
the reaction mechanism, the PDH complex regulation, the physio-
logical chemistry of the complex and to define its place in normal
cellular bioenergetics. Moreover we focus on the clinical features
of congenital PDHc deficiency, especially age-associated dysfunc-
tions of the complex, and enumerate recent findings that might
shed new light on the significance of PDHc on epilepsy. Emphasis
is put on the recent literature regarding the evidence of genetic
involvement, key neurological features in human epilepsies and
challenges associated with this mitochondrial disorder.
PDHc is the largest and one of the most complex multienzyme
systems known and the elucidation of its structural organization
and functional mechanisms remains one of the most challenging
problems. Almost 50 years ago, the structure and components of
the mammalian PDH complex were identified (Koike et al., 1963)
and the components constituting the enzyme complex are encoded
by nuclear genes and the disorder is inherited through both X-
linked and autosomal recessive modes of inheritance. The human
pyruvate dehydrogenase multienzyme complex (belonging to the
family of 2-oxoacid dehydrogenase complexes) exists as a sta-
ble, highly organized assembly of 9–10 × 106 Da, each assembly
consisting of three distinct component enzymes termed pyruvate
dehydrogenase (E1, EC 1.2.4.1, a heterotetramer of two ␣ and two
␤ subunits with molecular weights of 41 and 36 kDa, respectively),
dihydrolipoamide acetyltransferase (E2, EC 2.3.1.12, 74-kDa) and
dihydrolipoamide dehydrogenase (E3, EC 1.8.1.4, 55-kDa) (Smolle
et al., 2006; Zhou et al., 2001). Human PDHc also contains an acces-
sory subunit, E3 binding protein (E3BP) that mediates stable E3
integration into the E2 core of the complex. In addition, there are
two regulatory enzymes, pyruvate dehydrogenase kinase (PDK, EC
42 S. Bhandary, K. Aguan / Epilepsy Research 116 (2015) 40–52
2.7.1.99) and phosphatase (PDP, EC 3.1.3.43) interacting with the
complex. The three catalytic enzymes are present in multiple copies
within PDHc and associate into a dodecahedron with an E2 core,
whereby they can efficiently act in concert (Maj et al., 2006; Yu
et al., 2008; Zhou et al., 2001). The overall reaction of PDHc, cat-
alyzed by its three main enzymes, results in the decarboxylation of
pyruvate with the concomitant production of acetyl-CoA, CO2 and
NADH in a series of coordinated reactions involving five coenzymes
TPP, lipoic acid, CoA, FAD and NAD+ (Fig. 1B) (Behal et al., 1993; Frey
et al., 1989; Patel and Korotchkin, 2006; Patel and Roche, 1990;
Perham and Palkman, 1989; Roche and Hiromasa, 2007; Wieland,
1983).
PDHc-E1 is the initial member of the pyruvate dehydroge-
nase complex, an assemblage that plays a pivotal role in cellular
metabolism catalyzing the oxidative decarboxylation of pyruvate
and subsequent acetylation of coenzyme A to acetyl-CoA (Arjunan
et al., 2002; Chandrasekhar et al., 2006). PDHc-E2 plays a key
role in the structure and function of the multienzyme complex. It
has multi-domain structure: one or more lipoyl domains of ∼100
residues, each carrying a lipoamide molecule covalently attached
to a lysine, a so-called peripheral subunit binding domain of ∼50
residues which interacts with both the PDHc-E1 and PDHc-E3
components and a catalytic core domain of 250 residues that con-
tains the acetyltransferase active site and aggregates to form the
inner core of the complex (Packman et al., 1991). PDHc-E3 is
a member of the pyridine nucleotide, FAD-dependent-disulphide
oxidoreductase family that includes glutathione reductase, thiore-
doxin reductase, trypanothione reductase and mercuric reductase
(Mande et al., 1996). These enzymes catalyze electron transfer reac-
tions between pyridine nucleotides (NAD+ or NADP+ ) and their
specific substrates. The electron transfer reactions are mediated by
an active-site cysteine pair and the FAD cofactor. Further details
of the molecular biology and structure of PDH complexes may
be obtained from other reviews (Patel and Roche, 1990; Yeaman,
1989). The PDHc has an important bioenergetic function, gen-
erating acetyl-CoA from pyruvate for further oxidation via the
tricarboxylic acid cycle. In lipogenic tissues it has an additional
biosynthetic role, the provision of acetyl-CoA for the synthesis of
fatty acids and cholesterol. Interconversion between active dephos-
phorylated (PDHA) and inactive phosphorylated (PDHB) PDHc
leads to the rapid establishment of steady states with differing
capacities for flux from pyruvate to acetyl-CoA. Flux through the
complex is subject to extensive regulation as expected from its cen-
tral role in governing carbon flow in the face of variable supply and
demand. The end products of PDH activity, NADH and acyl-CoA,
inhibit complex activity directly, apparently by binding to the E2
and E3 active sites respectively (Fouque et al., 2003). Deficiencies
of the enzyme complex are well documented and mutations are
in PDHA1, which encodes the alpha subunit of E1 (Kanzaki et al.,
1969) but rare mutations have been identified in the genes encod-
ing subunits E1␤ (PDHB) (Lissens et al., 2000), E2 (DLAT) (Brown
et al., 2004), E3 (DLD) (Head et al., 2005) and also the E3-binding
protein (PDHX) (Liu et al., 1993). The complexity of the multitude
of subunits, strict cofactor requirements, and stringent regulation
of the PDHc make it a possible target for damage and subsequent
inactivation during pathologic conditions including ischemia and
neurodegenerative disorders.
Under normal physiological conditions, cerebral function
requires a continuous supply of glucose for its energetic and biosyn-
thetic needs. Pyruvate oxidation via the pyruvate dehydrogenase
enzyme complex (PDHc) represents a step of key importance in
cerebral glucose utilization. Since currently available evidences
suggest that PDHc may be a rate-limiting factor for cerebral
utilization, inherited or acquired deficiencies of PDHc, if suffi-
ciently severe, are likely to have an adverse effect on cerebral
energy metabolism. The complex is entirely nuclear encoded and
comprises three structurally distinct but functionally interdepen-
dent enzymes (Linn et al., 1969a; Popov et al., 1997; Robinson
et al., 1990). PDHc meets all the requirements of a flux generating
enzyme. It catalyzes a non-equilibrium reaction that is irreversible
and regulates the flux of pyruvate-derived acetyl CoA into the TCA
cycle and determines the maximal activity of this pathway during
the oxidation of glucose. As a consequence of its flux generating
characteristics, it is thought to have two important physiological
functions. First, PDHc might play a pivotal role in the selection of
intramuscular fuels, as summarized in the theory of metabolic reg-
ulation termed the “glucose-fatty acid cycle” (Robinson, 2001). This
theory describes the reciprocal relationship between the use of glu-
cose and fat fuel in skeletal muscles. PDHc is thought to act as a
reversible barrier for reducing the flow of pyruvate-derived acetyl
CoA into the TCA cycle when the availability of fat fuel increases and
removing it when the availability of glucose increases. The second
important aspect of PDHc lies in the role of this enzyme in the con-
trol of muscle lactate concentration during exercise and recovery
after exercise.
A deficiency in this enzymatic complex limits the production of
citrate. Because citrate is the first substrate in the citric acid cycle,
the cycle cannot proceed. Alternate metabolic pathways are stim-
ulated in an attempt to produce acetyl-CoA; however, an energy
deficit remains, especially in the central nervous system (CNS). The
magnitude of the energy deficit depends on the residual activity
of the enzyme. Severe enzyme deficiencies may lead to congeni-
tal brain malformation because of a lack of energy during neural
development. Morphological abnormalities occur before a gesta-
tion period of 10 weeks. Maldevelopment of the corpus callosum
is commonly observed in those with prenatal-onset types of pyru-
vate dehydrogenase complex deficiency. Progressive neurological
deterioration varies in neonates with an apparently healthy brain.
Hypomyelination, cystic lesions, and gliosis of the cortex or cerebel-
lum, with gray matter degeneration or necrotizing encephalopathy,
may occur in some individuals with pyruvate dehydrogenase com-
plex deficiency, whereas a gliosis of the brainstem and basal ganglia
with capillary proliferation occurs in those with Leigh syndrome
(Randle et al., 1963). Underlying neuropathology is not usually
observed in individuals whose onset of pyruvate dehydrogenase
complex deficiency occurs during childhood. The most common
form of pyruvate dehydrogenase complex deficiency is caused by
mutations in the X-linked E1 alpha gene; all other causes are due
to alterations in recessive genes (Brown et al., 1994; De Meirleir,
2002; Hansen et al., 1991).
In the last two decades, inherited neurological disorders have
been identified in which there is convincing evidence for genetic
abnormalities of PDHc or one of its constituent enzymes (De
Meirleir et al., 1992). Generally, the more severe the PDHc defect,
the more severe is the accompanying clinical illness whereas the
earlier its onset, the more rapid its progression resulting in more
widespread CNS damage. Thus, patients with less than 15–20%
of normal PDHc activity generally present with “lactic acidosis of
infancy” and severe neurological deficits, whereas patients with
40–50% residual PDHc activity have a milder illness in which
intermittent ataxia is the most prominent neurological symptom.
Specific biochemically proven abnormalities of the El, E2, or E3
components of PDHc have been identified (Morten et al., 1999).
Inherited defects of PDHc regulation have been reported in associ-
ation with Leigh disease (Blass, 1981; Butterworth et al., 1985). In
addition to ischemic brain injury, PDHc is affected also in other
neurologic disorders, viz., Wernicke–Korsakoff syndrome (WKS)
which is characterized by a triad of mental confusion, ataxia, and
ophthalmoplegia. The main etiologic factor is known to be lack
of thiamine, but the biochemical mechanisms involved remain
unclear. A thiamine-dependent enzyme such as PDHc is thought
to play a role in the pathogenesis of WKS. PDHc activity is also
 S. Bhandary, K. Aguan / Epilepsy Research 116 (2015) 40–52
affected in Alzheimer’s disease (AD). Brain lipid peroxidation and
decreased brain glucose utilization are characteristic of this neu-
rodegenerative disease (Butterworth, 1982).
Furthermore, synthetic pathways involving synthesis of
glucose-derived neurotransmitters may be particularly susceptible
to mild impairments of PDHc. The acetyl moiety of acetylcholine
is normally derived from pyruvate, the end product of the gly-
colytic pathway. Thus, any impairment of the pyruvate oxidation
pathway might be expected to cause a reduction in acetylcholine
synthesis. Indeed, studies have shown that a number of substances
that inhibited conversion of 14 C-pyruvate to 14 CO2 in rat brain
preparations resulted in subsequent decrease in conversion of 14 C-
pyruvate to acetylcholine (Sorbi and Blass, 1982). The substrate
analog and alkylating agent 3-bromopyruvate inhibit completely
both cell motility and pyruvate release. The inhibitory substances
tested included the irreversible PDHc inhibitor 3-bromopyruvate
and the competitive inhibitor 2-oxobutyrate (Barnard et al., 1993).
Evidences suggest that the neurotransmitter pools of GABA as well
as glutamate and aspartate are synthesized from glucose via pyru-
vate. Incorporation of glucose into these amino acids is sensitive
to changes in physiological function, whereas incorporation from
other precursors is not. For example, anesthetic doses of sodium
pentobarbitone lead to decreased incorporation of 14 C-glucose into
amino acids, but have no effect on incorporation of 14 C-acetate or
14 C-butyrate (Toshima et al., 1982). Electrical stimulation of guinea
pig cortical slices evokes a selective tetrodotoxin-sensitive release
of amino acids, newly synthesized from 14 C-glucose (Sheu et al.,
1985). Impairment of cerebral glucose metabolism results in reduc-
tion of GABA, glutamate, and aspartate in brain (Gibson et al., 1975).
Another mechanism by which PDHc deficiency may affect cere-
bral function is by alteration of calcium homeostasis. There are
considerable evidences available to suggest that mitochondrial
metabolism can influence neurotransmitter release by regulation
of calcium accumulation. Brain mitochondria appear to be tightly
linked to PDHc activity by the phosphorylation state of the enzyme
complex. Dichloroacetate, the PDHc kinase inhibitor, stimulates
pyruvate-supported calcium accumulation at concentrations at
which it stimulates cerebral PDHc activity (Cremer and Lucas,
1971). Yet another report suggests that PDHc regulation may play
an important role in the modulation of synaptic plasticity involved
in learning and memory function (Potashner, 1978).
2. Pyruvate dehydrogenase and its regulatory enzymes
Regulation of the activity of the pyruvate dehydrogenase com-
plex is of critical importance in all mitochondria-containing cells of
higher eukaryotes. The complex has to be active when the complete
oxidation of glucose is needed for the generation of energy, whereas
its activity has to be severely suppressed when glucose is in short
supply. Regulation of the activity of the complex is accomplished
by two different mechanisms: end-product inhibition by acetyl
CoA and NADH (Butterworth, 1982) and by interconversion of its
pyruvate dehydrogenase (E1) component between active (non-
phosphorylated) form and inactive (phosphorylated) form (Fig. 2)
(Browning et al., 1981). However, PDHc is much more extensively
regulated through its associated kinases and phosphatases (Morgan
and Rottenberg, 1981). Both control mechanisms may be inter-
dependent in vivo since they often rely on the same metabolic
effectors. End-product inhibition is a result of reductive acetylation
of lipoate moieties with concomitant accumulation of El-bound
hydroxyethyl-TPP carbanion, creating an impediment to further
pyruvate decarboxylation (Garland et al., 1964). More significant
physiological regulation occurs by phosphorylation of three ser-
ine residues located on the ␣-subunit (E1˛) catalyzed by pyruvate
dehydrogenase kinase (PDK). Dephosphorylation is catalyzed by
43
Fig. 2. Regulation of PDHc by phosphatase and kinase activities.
pyruvate dehydrogenase phosphatase (PDP). The physiological role
of multi-site phosphorylation remains unclear but it may be a
determinant of differentiation between short- and long-term PDH
complex inhibitions or, alternatively, a reflection of substrate speci-
ficity exhibited by different isoenzymes of the PDH kinase.
Using site-directed mutagenesis of the three phosphorylation
sites of the human PDH-E1 expressed in Escherichia coli, Korotchk-
ina and Patel were able to demonstrate that mutation at site 1
(S263) but not at sites 2 and/or 3 (S271 and S203 respectively)
decreased specific activity of E1 and also increased Km values for
TPP and pyruvate (Pettit et al., 1975). They were also able to demon-
strate that phosphorylation of each site resulted in a complete
inactivation of the E1; however the rates of phosphorylation and
inactivation were site-specific. A number of studies on pig heart
(Linn et al., 1969b; Randle, 1983) and bovine kidney (Korotchkina
and Patel, 2001) indicated that phosphorylation at sites 2 and 3, in
addition to site 1, markedly inhibited the rate of PDH reactivation
by phosphatase. These results are not in agreement with studies
by Teague et al. (1979) who found that the phosphoryl group on
sites 2 and 3 did not significantly affect the rate of dephosphory-
lation at site 1 or the rate of reactivation of the enzyme in bovine
kidney. The physiological relevance of these findings is presently
unknown. This is complicated by the fact that at the time of the
studies the existence of PDH kinase and PDH phophatase isoen-
zymes was not established. Furthermore, in all cases crude PDH
kinase and PDH phosphatase preparations obtained from differ-
ent animal tissues were used. These preparations could potentially
contain more than one isoform of the kinase or phosphatase.
Interestingly, only sites 1 and 3 are conserved in yeast, Caenorhab-
ditis elegans and Drosophila melanogaster with significantly higher
residue conservation around site 1. Site 2 does not seem to be
conserved and is absent in yeast and worms. Mammalian tissues
express four PDK isoenzymes (Sugden et al., 1978) and two pyru-
vate dehydrogenase phosphatase isoenzymes (Sugden et al., 1979).
The four PDKs that regulate the mammalian PDH complex are a
novel class of kinases apparently unrelated to the large families of
serine–threonine and tyrosine kinases as prominent in other mam-
malian regulatory processes (Kerbey and Randle, 1979; Sugden
and Simister, 1980). Though the PDKs represent a related family
44 S. Bhandary, K. Aguan / Epilepsy Research 116 (2015) 40–52
of kinases, the sequence divergence between the four different
isoforms (61–69%) is consistent with their significantly divergent
regulatory behavior (Bowker-Kinley et al., 1998; Huang et al., 1998).
Moreover, PDK isoform function is apparently ancient and essen-
tial as the corresponding isoforms in rodents and humans are at
least 94% conserved as reviewed in (Huang et al., 1998). The PDKs
regulate PDH complex activity by responding to diverse allosteric
modulators. High ratios of acetyl-CoA to CoASH and NADH to NAD+
represent signals of saturation of mitochondrial demand. Eleva-
tions of these ratios are strong allosteric activators of PDKs, shutting
down PDH activity in response to demand saturation (Baker et al.,
2000; Patel and Korotchkin, 2006; Patel and Roche, 1990; Randle,
1986; Roche and Hiromasa, 2007; Roche et al., 2001). Though satu-
ration of mitochondrial demand is most often produced in response
to high fat or carbohydrate intake in healthy animals (Randle et al.,
1988), the altered metabolism of tumor cells also creates a very
new metabolic environment wherein these regulatory processes
may be entrained for different purposes. PDKs are also subject
to allosteric regulation by pyruvate (Pettit et al., 1975). Elevated
pyruvate levels interact synergistically with ADP to inhibit PDK
activity (activating PDH). This PDK inhibition apparently results
from the binding of pyruvate to PDK-ADP inhibiting exchange for
ATP and, thus, phosphorylation. These isoenzymes have unique
regulatory properties and differ in their level of expression in dif-
ferent cell types, thereby establishing a means for tissue-specific
control of the pyruvate dehydrogenase complex (Batenburg and
Olson, 1976). In general, the short-term kinase activity responds to
the relative concentrations of the PDHc substrates NAD+ , CoA-SH
and the kinase product ADP (inhibition) and PDHc products NADH,
acetyl CoA and kinase substrate ATP (activation). This adjusts flux
through PDHc on a moment-to-moment basis together with more
global control through product inhibition and pyruvate concen-
tration. Some determinants of effector control of PDK have been
defined. In addition, the catalytic activity of PDP is Mg2+ and Ca2+
dependent. It is inactive in the absence of Mg2+ and Ca2+ , and its
catalytic activity is stimulated about 10-fold by Ca2+ (Hansford,
1976; Roche and Hiromasa, 2007). Currently, further progress in
studies on PDH kinase is tempered by the lack of understanding as
to how this important enzyme functions. Experiments performed
by Bowker-Kinley et al. (1998) suggest that the catalytic domain
of PDK is likely to be folded similarly to the catalytic domains of
the members of ATPase/kinase superfamily that include molecular
chaperone Hsp90, DNA gyrase B and histidine protein kinases. The
reverse of PDH complex phosphorylation and subsequent activa-
tion is carried out by pyruvate dehydrogenase phosphatase (PDP).
The enzymic activity of phosphatase depends on tissue type and the
intramitochondrial concentrations of Mg2+ , Ca2+ , NADH and insulin
(Pratt and Roche, 1979).
3. Clinical features of congenital PDH complex deficiency
PDHc deficiency is a nuclear-encoded mitochondrial disorder
and a major recognized cause of neonatal encephalopathies asso-
ciated with primary lactic acidosis (Huang et al., 1998). Elevated
lactate accompanied by elevated pyruvate with a normal lactate-
to-pyruvate ratio is a hallmark of PDHc deficiency, shared by a few
other inborn errors. Several prior clinical reviews of PDHc defi-
ciency have been published (Denton et al., 1972; Pettit et al., 1972;
Robinson, 1995). PDHc deficiency can be caused by defects in the
E1␣, E1␤, E2, or E3 subunits. The most common cause of PDHc defi-
ciency is a defect in the E1˛ gene, located on the X chromosome
(Xp22.1–22.2). It is considered an X-linked dominant condition in
that both females and males with an E1˛ gene mutation are affected
with PDHc deficiency. Mutations in the E1˛ gene are typically de
novo (De Meirleir et al., 2006; Kerr and Zinn, 2009; Robinson, 2001).
E1␤ subunit of PDH has been mapped to 3p13-q23. PDHB encodes
E1␤ subunit of PDHc and accounts for only a minority of cases
of PDHc deficiency. Though the severity of enzyme deficiency is
similar to PDHA1, PDHB mutations also present with considerable
phenotypic variability and severity (Marsac et al., 1999). Other rare
subtypes include PDP deficiency which can cause severe metabolic
acidosis in the neonatal period (Brown et al., 1989; Lissens et al.,
2000).
In PDHc deficiency, glucose carbon is diverted from acetyl
CoA synthesis to oxaloacetate formation via pyruvate carboxy-
lase, although overall flux through the TCA cycle is reduced.
Dichloroacetate (DCA) is a structural analog of pyruvate that has
been recommended for the treatment of primary lactic acidemia,
particularly in patients with respiratory chain disorders or PDHc
deficiency (Cameron et al., 2009; Okajima et al., 2008). The benefi-
cial effects of DCA treatment are thought to arise from an increase
in the flux of pyruvate to mitochondria and a decrease in serum
lactate levels mediated by the stimulation of PDHc, as demon-
strated by experiments involving chronic daily supplementation
in rats (Maj et al., 2005). DCA is known to be a potent inhibitor
of E1 kinase, locking PDHc in its unphosphorylated, catalytically
active form (Stacpoole et al., 1997). However, DCA treatment has
remained controversial because of significant side effects such as
reversible peripheral neuropathy, which may limit the long-term
use of this drug. Furthermore, in one study, most of the patients
treated with DCA presented neurologic deterioration advanced to
such a point that no improvement was achieved despite the rever-
sal of lactic acidemia (Pratt and Roche, 1979). Recent reports have
refocused attention on the potential benefits of chronic DCA treat-
ment in PDHc-deficient patients by demonstrating in vitro that DCA
responsiveness may be selective and depend on the type of molec-
ular abnormality. It has been shown that chronic DCA treatment
increases maximum extractable PDHc activity in cultured human
fibroblasts by reducing degradation of the E1 ␣-subunit (Evans and
Stacpoole, 1982) and that a similar effect is observed in the fibro-
blasts of patients harboring mutations affecting the stability of the
E1 ␣-subunit (Whitehouse et al., 1974). Considerable phenotypic
and allelic heterogeneity has been observed for this defect. Pre-
vious studies have reported that a decrease in the stability of the
E1 ␣-immunoreactive subunit may contribute to the expression of
many E1 ␣-mutations (Morten et al., 1998; Stacpoole, 1989); very
few studies to distinguish mutations that impair polypeptide sta-
bility from those impairing catalytic efficiency have been carried
out to date (Morten et al., 1999).
Human PDH complex deficiency is an extremely heterogeneous
disease in its presentation. Very little correlation between clini-
cal course and molecular characteristics has been established so
far. However, the natural history of congenital PDHc deficiency
and the biochemical concomitants of this disease provide insight
into the clinical presentation and underlying mechanisms of many
age-related disorders (Mahbubul Huq et al., 1991). Several car-
dinal clinical features of PDHc deficiency are remarkably similar
to those associated with aging. For example, mental retardation,
hypotonia, ataxia, peripheral neuropathy and exercise intolerance,
are common findings in PDHc-deficient children. Neuroimaging
frequently reveals structural brain abnormalities, including cere-
bral atrophy and ventriculomegaly. No proven therapies exist
for congenital PDHc deficiency, and most affected patients die
within the first two decades of their lifetime. Studies using cul-
tures of skin fibroblasts from patients harboring various mutations
in the PDHc-E1␣ subunit have shown that these cells exhibit
high rates of glycolysis and lactate production compared to sim-
ilarly treated fibroblasts from healthy donors (Fujii et al., 1996). A
particularly intriguing and unexpected finding in PDHc-deficient
fibroblasts was the overexpression of hypoxia inhibitory factor
1␣ (HIF1␣). HIF1␣ transactivates numerous genes involved in
 S. Bhandary, K. Aguan / Epilepsy Research 116 (2015) 40–52
critical pathways of cell metabolism, growth and survival (Otero
et al., 1998), including those encoding glucose transporters and
most glycolytic enzymes. It also transactivates PDK by down-
regulating the PDHc and oxidative phosphorylation (OXPHOS)
(Patel et al., 2011). The mechanism for HIF1␣ overexpression in
PDHc deficiency is unknown, although reactive oxygen species
(ROS) generated by complex III are thought to be required for
activation of HIF1␣ under conditions of hypoxia (Simpson et al.,
2006). In addition, glycolytic metabolites, such as pyruvate, sta-
bilize HIF1␣ by inhibiting its degradation by cytoplasmic prolyl
hydroxylases (Gordan et al., 2007); creating a positive feedback
loop whereby HIF1␣ activity in PDHc deficiency is maintained by
increased glycolytic flux. Regardless of the precise mechanism,
HIF1␣ overexpression may contribute to the Warburg effect oper-
ative in PDHc-deficient cells and to further inhibition of residual
PDHc activity. Congenital defects in the PDHc and respiratory chain
enzymes share many common clinical features (Kim et al., 2006;
Semenza, 2011).
An etiologic link between PDHc deficiency and clinically
expressed disease is best demonstrated in children born with spo-
radic or inherited mutations in the E1␣ subunit or in the E2 or E3
enzymes of the complex, or in PDHc phosphatase (Klimova and
Chandel, 2008; Lu et al., 2005). To date, there have been no reports
of congenital defects involving the E1␤ subunit or any PDK isoform.
PDHc deficiency accounts for about 15% of known causes of congen-
ital lactic acidosis and is most often due to various point mutations
in the E1␣ subunit gene that reduce the catalytic activity or the
stability of the protein complex. The phenotype typically presents
between birth and infancy. Newborns with severe defects in PDHc
generally develop fulminant lactic acidemia and rarely survive the
neonatal period.
4. Mitochondrial epilepsy and its pathophysiology
The most commonly form of PDHc deficiency is caused by
mutations in the X-linked E1˛ gene and is approximately equally
prevalent in both males and females. However, a greater severity
of symptoms tends to affect males more often than heterozygous
females. This can be explained by x-activation, as females carry
one normal and one mutant gene. The pathophysiology leading
to epilepsy in mitochondrial disorders is not clear. Evidence from
mouse models has shown that specific inhibitors of the respiratory
chain may induce seizures. Subcutaneous injection of potassium
cyanide (KCN, inhibits COX) resulted in dose-dependent tonic
seizures in treated mice, whilst 3-nitropopionate (inhibitor of com-
plex II) induced clonic seizures again in a dose-dependent manner
(Patel et al., 2011). Energy failure undoubtedly plays a role but nei-
ther explains the phenotypic variability of mitochondrial epilepsy,
nor the reason why epilepsy is not a feature of all mitochondrial
disorders. Other aspects of mitochondrial dysfunction, such as ROS
production, abnormal calcium handling, and increased apoptosis,
are also likely to contribute to seizure generation. Brain lipid perox-
idation levels were increased in the KCN mouse model, supporting
a role for ROS formation in the pathogenesis of seizures in these
animals (Scaglia et al., 2004). There is some evidence that tonic
seizures themselves can trigger mitochondrial dysfunction (Lissens
et al., 2000), implicating a vicious spiral in the etiology of mito-
chondrial epilepsy. For example, a male who presented with acute
encephalopathy and pathogenic POLG mutations had evidence of
acute disseminated encephalomyelitis on histological examination
of a brain biopsy (Yamamoto and Tang, 1996). Finally, in individuals
with occasional mitochondrial disease, seizures may be secondary
to electrolyte disturbances arising from severe renal tubulopa-
thy. The pathogenesis of mitochondrial epilepsy remains poorly
understood, contributing to the immense difficulties in treating
45
this condition. The exact prevalence of mitochondrial epilepsy is
not known, but seizures have been reported to occur in ∼35–60%
of individuals with biochemically confirmed mitochondrial dis-
ease (Urbanska et al., 1998; Yamamoto and Tang, 1996). In another
study, one-third of individuals with refractory seizures were found
to have biochemical evidence of mitochondrial dysfunction (Kunz,
2002). Few reports have systematically examined epilepsy pheno-
types in the context of mitochondrial disease (Debray et al., 2007;
Harris et al., 2010; Khurana et al., 2008). Epilepsy is a poor progno-
stic sign in mitochondrial disease, and there is an urgent need for
formal clinical trials of candidate treatments, including the keto-
genic diet and novel therapeutic agents.
5. Metabolic management and pharmacologic control of
PDHc
Mitochondrial epilepsy can be very difficult to manage. Unfor-
tunately, no generalized, effective treatment has been described
for PDHc deficiency. Several strategies have been employed, with
variable success. These include the use of a ketogenic diet (KD),
supplementation with thiamine, and administration of dichloroac-
etate. The role of other vitamins and nutritional supplements is not
clear, and so far has not been supported by clinical trials (Parikh
et al., 2008). However, the prognosis for mitochondrial epilepsy
is extremely poor, and there is clearly an urgent need for novel
treatments for the individuals. A fatal outcome was observed in
45% (22/56) of a French cohort, and 50% of these individuals died
within 9 months of epileptic onset (El Sabbagh et al., 2010). The
KD includes 80% fat, 15% protein, and 5% carbohydrate; the ratio
of fat to carbohydrate plus protein ranges from 2:1 to 4:1, with
higher ratios seen as more restrictive but more effective (Lee et al.,
2008). Most of the fat in the classic, most commonly used KD
is provided as long-chain saturated triglycerides. The restricted
carbohydrate and high-fat intake will result in reduced glucose uti-
lization and increased ketone body production by mitochondrial
beta-oxidation, with a shift away from glycolysis toward increased
TCA cycle activity and direct mitochondrial metabolism of ketone
bodies. Ketone bodies are metabolized to acetyl-CoA, which feeds
into the TCA cycle and then to the respiratory chain/OXPHOS
system to generate adenosine triphosphate (ATP), may at least par-
tially bypass complex I.
Despite nearly a century of clinical use, the mechanisms under-
lying the efficacy of the KD have not fully understood. Several
mechanistic theories by which KD acts to reduce seizure activ-
ity in the brain have been proposed (Bough and Rho, 2007;
Schwartzkroin, 1999). The increased mitochondrial ATP produc-
tion and reduced glycolytic ATP production will selectively activate
ATP-sensitive potassium channels that will, in turn, have a stabi-
lizing effect on the neuronal cell membrane (Danial et al., 2013;
Hartman and Rho, 2012). The KD may have a role in altering neu-
rotransmitter function by disrupting excitatory glutamate synaptic
transmission and facilitating an increased production of inhibitory
␥-aminobutyric acid (GABA) in the brain tissue. As a result, hyper-
polarization of neurons occurs, stabilizing synaptic function and
increasing resistance to seizures throughout the brain. Based on
the tricarboxylic acid mechanism, the ketogenic diet is an appro-
priate first-line therapy for patients with seizures associated with
metabolic disorders and pyruvate dehydrogenase complex defi-
ciency (Bough and Rho, 2007; Danial et al., 2013; Hartman and Rho,
2012). The KD alters the energy metabolism in the brain, there-
fore altering brain excitability and enhances alterations in synaptic
transmission. It leads to changes in neuronal and perhaps glial cell
properties thereby reducing excitability. It is also associated with
changes in a variety of circulating factors which act as neuromod-
ulators that can regulate central nervous system excitability and
46 S. Bhandary, K. Aguan / Epilepsy Research 116 (2015) 40–52
Table 1
Nutritional and potential therapeutic interventions that increase PDHc activity.
Intervention Probable mechanism
Dichloroacetate Inhibits PDKs
l-Carnitine Decreases acetyl CoA/CoA
Omega-3 fatty acids Inhibit PDKs
Etomoxir Inhibits Long chain fatty acids (LCFA) oxidation
Insulin Inhibits lipolysis, LCFA oxidation
Pyruvate Inhibits PDKs; scavenges oxygen free radicals
Thiamine Inhibits PDKs; cofactor
alters the extracellular environment of the brain, which helps to
decrease the neuronal excitability. The main stream of those pro-
posed mechanisms of KD is that it changes the normal metabolism
of neurons, resulting in varying neuronal excitability, as well as
modifying neural circuits and cellular properties to enhance and
normalize neuronal function. Other hypothesized mechanisms of
KD action include increased polyunsaturated acid levels which may
accord anticonvulsant benefit via modifications in cell membrane
composition, stimulation of nuclear receptor proteins (e.g., peroxi-
some proliferator-activated receptor ␣) and decreased production
of ROS by upregulation of mitochondrial uncoupling protein activ-
ity and expression (Danial et al., 2013). Preclinical evidence has
suggested that ketones may directly contribute to the anticonvul-
sant mechanism of the KD. Although it does not appear that ketones
antagonize excitatory receptors (i.e., AMPA or NMDA) or activate
inhibitory GABAA receptors directly (Donevan et al., 2003; Thio
et al., 2000). Recent evidence suggests that energy source for the
brain is switched from glucose to ketone bodies (acetoacetate and
␤-hydroxybutyrate). Ketone bodies directly regulate neural exci-
tation and seizures via ATP-sensitive K+ channels (KATP channels)
(Ma et al., 2007). These data show that ketones directly dimin-
ish the spontaneous firing rate of neurons in the substantia nigra
pars reticulata, a region of the brain known to be critical to seizure
propagation (Iadarola and Gale, 1982; Lutas and Yellen, 2013).
A number of case reports have described ketogenic diet in
the treatment of children with mitochondrial disease (Chinnery
et al., 2006), but few studies have examined the effects of keto-
genic diet in a more systematic way. Overall, there is a suggestion
that, although seizure frequency may be reduced on a ketogenic
diet, the diet does not appear to influence the relentlessly pro-
gressive course of mitochondrial disease in many individuals. It is
not clear whether some subgroups of mitochondrial disease may
respond better than others to a ketogenic diet. Formal clinical tri-
als are needed, with the aim of determining which individuals
are likely to benefit from ketogenic diet. DCA is the most potent
lactate-lowering agent available because it stimulates the rate-
determining step in aerobic glucose oxidation. Consequently, it
has been employed to improve acid-base status and mitochondrial
energetics in a number of acquired and congenital disorders (El
Sabbagh et al., 2010). This drug inhibits all PDK isoforms except the
testes-specific PDK3 and converts phosphorylated E1␣-subunits
into their unphosphorylated, catalytically active form within min-
utes of its oral or parenteral administration. Agents that inhibit
either the release of long chain fatty acids from adipose tissue
(e.g., insulin, nicotinic acid) or their ␤-oxidation (e.g., insulin, eto-
moxir) could reduce the mitochondrial concentration of acetyl CoA
derived from lipid and could thereby decrease the acetyl CoA/CoA
ratio (Freeman et al., 2000). A similar mechanism may explain the
ability of l-carnitine to activate the PDHc (Seo et al., 2007), since
l-carnitine can be acetylated by acetyl CoA via carnitine acyltrans-
ferase, thereby liberating free CoA (Table 1).
Recent observations of Sada et al. raised a surprising question:
“Should the focal point of antiepileptic drugs (AEDs) development
actually be neurons?” There is now considerable evidence that
References
Joshi et al. (2009), Stacpoole (1997), Bersin and Stacpoole (1997)
and Broderick et al. (1992)
Katayama and Welsh (1989) and Chandy and Ravindra (2000)
Andreassen et al. (2001) and Fouque et al. (2003)
Broderick et al. (1992)
Bresolin et al. (1982)
Xi et al. (2008), Fryer et al. (1995), and Jucker et al. (1999)
Randle et al. (1988) and DeBoer et al. (1993)
inexcitable elements of the central nervous system, such as astro-
cytes, the vasculature, and the immune system, play a greater role
in epileptic seizures (Kaminski et al., 2014; Vezzani et al., 2011).
The report by Sada et al. (2015) pointed out epilepsy as a disease of
energy metabolism rather than neuronal discharge, a strong shift
in the neurocentric view of epilepsy. This group reported that inhi-
bition of lactate dehydrogenase (LDH), an enzyme critical in the
metabolic cross-talk between astrocytes and neurons, blocked neu-
ron excitation in vitro, and also prevented seizures in an animal
model of epilepsy. Although LDH may dehydrogenate several sub-
strates, the one highlighted by the authors is lactate. Generation
of lactate from pyruvate in astrocytes, followed by its transport to
neurons, allows lactate to become an energy substrate for neurons
when energy demand is high (Fig. 1A). Sada et al. also showed that
one of the currently available AEDs, stiripentol, exerts its effect – by
inhibiting LDH. This finding is attractive because many AEDs have
mechanisms of action that are not well explained. The authors also
make the case that KATP channels in neurons are likely to mediate
the effects of LDH inhibition. In this study (Sada et al., 2015), they
found that neural activities and seizures can be suppressed by LDH
inhibition and also found that LDH can be inhibited by stiripentol
and its analog. There are no antiepileptic drugs that act on metabolic
pathways; therefore, LDH inhibitors would be the first antiepileptic
drug to mimic ketogenic diets. This study opens a realistic path to
develop compounds for drug-resistant epilepsy by targeting LDH
enzymes with stiripentol derivatives.
Finally, we sought information relating to the clinical man-
agement of PDHc deficiency. Alkali, usually sodium bicarbonate,
was administered often but sporadically, usually as a temporary
treatment for acute acid–base decompensation. The most com-
mon interventions used chronically were thiamine (vitamin B1),
a ketogenic diet, comprising at least 65% of total calories, and DCA.
Thiamine doses varied, from a few milligrams per day to doses
exceeding 1 g/day. These latter cases typically involved patients in
whom PDHc deficiency was considered due to a mutation affecting
the interaction of thiamine pyrophosphate with the E1␣-subunit.
The frequency with which ketogenic diets were employed may
be underrepresented because many reports failed to specify the
proportion of fat calories. Some patients received intravenous
DCA for acute treatment of lactic acidosis but the majority of
patients received DCA for chronic oral treatment at doses usually of
25 mg/kg/d or more. No intervention for PDHc deficiency has been
evaluated in a randomized controlled trial.
6. Molecular targets for antiepileptic drugs
Anti-epileptic drugs (AEDs) work in various ways to reduce
excitation in the brain and prevent seizures. AEDs make the brain
less likely to have seizures by altering and reducing the excessive
excitability of the neurones that normally cause a seizure. Different
AEDs work in different ways and have different effects on the brain.
The way AEDs work is not fully understood. There are several dif-
ferent ways in which AEDs stop seizures by working on particular
‘targets’ in the brain (Fig. 3). AEDs may affect the neurotransmitters
 S. Bhandary, K. Aguan / Epilepsy Research 116 (2015) 40–52 47

Table 2
Summary of the principal mechanism of actions of clinically approved antiepileptic drugs.

AED Year of approval in Presumed mode of action

the US and Europe

Phenobarbitol 1912 GABA potentiation

Primidone 1954 GABA potentiation
Diazepam 1963 GABA potentiation
Clonazepam 1968 GABA potentiation
Clobazam 1975 GABA potentiation
Progabide 1985 GABA potentiation
Vigabatrin 1989 GABA potentiation
Tiagabine 1996 GABA potentiation
Phenytoin 1938 Na+ channel blocker
Carbamazepine 1964 Na+ channel blocker
Lamotrigine 1990 Na+ channel blocker
Oxcarbazepine 1990 Na+ channel blocker
Zonisamide 2000 Na+ channel blocker
Rufinamide 2004 Na+ channel blocker
Lacosamide 2008 Na+ channel blocker
Eslicarbazepine 2009 Na+ channel blocker
Trimethadione 1946 T-type Ca2+ channel blocker
Ethosuximide 1958 T-type Ca2+ channel blocker
Gabapentin 1993 ␣2 ␦-subunit of Ca2+ channel blocker
Pregabalin 2004 ␣2 ␦-subunit of Ca2+ channel blocker
Levetiracetam 2000 SV2A modulation
Retigabine 2011 K+ channel activator
Perampanel 2012 Glutamate (AMPA) receptor antagonist
Valproate 1967 Multiple (GABA potentiation, Glutamate (NMDA) inhibition, Na+ and T-type Ca2+ channel blocker)
Felbamate 1993 Multiple (GABA potentiation, Glutamate (NMDA) inhibition, Na+ and T-type Ca2+ channel blocker)
Topiramate 1995 Multiple (GABA potentiation, glutamate (AMPA) inhibition, Na+ and Ca2+ channel blocker)
Stiripentol 2002 GABA potentiation and Na+ channel blocker

responsible for sending messages, and/or altering the activity of
the cell by changing how ions flow into and out of the neurones
(Benarroch, 2007, 2009; Bialer and White, 2010; Brodie et al., 2011;
Kohling, 2002; Kwan et al., 2001; Meldrum and Rogawski, 2007).
Some AEDs (such as phenytoin, lamotrigine and carbamazepine)
work by affecting the sodium channels of neurones. AEDs that
target calcium channels (such as lamotrigine and topiramate) work
by blocking the calcium channels. This prevents messages being
sent across the synapse from one neurone to another either by
stopping the release of neurotransmitters or by preventing cal-
cium entering the second neurone. GABA is a type of inhibitory
neurotransmitter in the brain, which effectively stops brain mes-
sages from continuing to be sent. GABA helps chloride ions pass into
neurones, which affects the resting membrane potential of the cell
and makes it difficult for the neurone to send messages. AEDs that
work on the GABA system and its receptors are agonists, and effec-
tively increase the movement of chloride into cells, and increase
the switching off of messages. AEDs such as gabapentin work by
increasing the production of GABA, and sodium valproate and viga-
batrin work by decreasing the breakdown of GABA, both of which
result in an increased amount of GABA. There is, however, credible
evidence to support selective binding of levetiracetam and its struc-
tural analog brivaracetam bind to synaptic vesicle glycoprotein 2A
(SV2A), with little or no affinity for other members of the same
protein family, and an impressive correlation between SV2A bind-
ing affinity and the anticonvulsant efficacy in audiogenic seizure
sensitive mice. Drugs that effect and prevent glutamate uptake pre-
vent glutamate from helping the movement of ions through the
cell membrane and so prevent the spread of the messages from
one neurone to another. The AED perampanel works specifically on
glutamate receptors, while some other AEDs (such as topiramate)
work on glutamate receptors as well as other targets. Different AEDs
use different targets, or a combination of targets. For some it is
known which targets they use, but for others it is not yet known.
Proposed mechanisms of action of some of the currently available
AEDs have been summarized in Table 2.

7. Genetic etiology

PDHc deficiency has been considered one of the most common

Fig. 3. Schematic representation of the commonly available AEDs and their possible
mechanisms of action.
biochemically proven causes of congenital lactic acidosis. Regard-
less of which subunit or component of the complex is defective,
most patients present within the first few months of life with both
clinical and biochemical evidence of disease. Any congenital or
48 S. Bhandary, K. Aguan / Epilepsy Research 116 (2015) 40–52
acquired defect in any PDHc component may give rise to lactic
acidosis and to cellular energy failure; the latter most commonly
expressed as progressive neurological and neuromuscular deteri-
oration. The vast majority of proven mutations causing decreased
activity of PDHc have been found within the coding region gene
for the E1␣ subunit. To date, no mutation of E1␤ subunit has been
reported in human PDHc deficiency. Mutations of E1␣ resulting in
instability and loss of immunoreactive protein are commonly asso-
ciated with concomitant loss of both E1␣ and ␤ immunoreactivity,
presumably reflecting the critical role of binding between ␣ and ␤
subunits for their mutual stability. Mutations have been described
in the less common defects affecting E2, E3 and E3-binding pro-
tein (Henneberry et al., 1989). E3 deficiency results in multiple
␣-ketoacid dehydrogenase deficiencies. In general, those who died
were younger, had earlier clinical onset and had lower PDHc activ-
ity.
The concept of a gene therapy approach for mitochondrial
diseases is not new, but theoretical and experimental contrib-
utions have so far been focused mainly on overcoming defects in
mitochondrial DNA (Butterworth et al., 1993; Butterworth, 2003;
Rigobello and Bindoli, 1993). A more general and potentially effec-
tive gene therapy approach for diseases involving mitochondrial
energy failure may be to target the PDHc, specifically the E1␣ sub-
unit. This postulate is based on the following considerations. First,
the integrity of the complex is critical for oxidative phosphory-
lation and ATP synthesis to occur normally. Second, the activity
(phosphorylation state) of the E1␣ subunit primarily determines
the overall catalytic activity of the PDHc and most acquired or
congenital deficiencies of the complex appear to be due to pertur-
bation of this subunit (Chrzanowska-Lightowlers et al., 1995; Kerr
et al., 1996). Third, the E1␣ subunit is relatively hydrophilic and,
because it is nuclear encoded in mammals, it can be imported into
mitochondria. As summarized here and as reviewed more exten-
sively in other reports (Bigger et al., 1999; Lissens et al., 2000; Zullo,
2001), a new scientific paradigm that has evolved over the last two
decades emphasizes mitochondrial energy failure as an important
concomitant factor in the etiology or progression of many different
acquired and hereditary diseases. Recently, a vector using recombi-
nant adeno-associated virus (rAAV) that contained a fusion protein
of full-length E1␣ and the reporter gene green fluorescent protein
was used to deliver wild type E1␣ into mitochondria after injec-
tion of the construct in vivo into the central nervous system of rats
and in vitro into human cells. In vitro studies demonstrated a cor-
rection of approximately 30% of wild-type PDHc activity in PDHc
deficient patient fibroblasts (Owen et al., 2002; Peden et al., 2004).
To confirm gene expression from the rAAV-E1␣ vector, RNA was
extracted from cultured fibroblasts from a healthy adult and from
a patient with PDHc-E1␣ deficiency. These findings require replica-
tion in cells from other E1␣ deficient subjects and, ultimately, must
be confirmed in vivo, preferably in an animal model of the disease, if
one becomes available. Therapeutic strategies have focused on the
modulation of signaling, mediated by the main classic excitatory
and inhibitory neurotransmitters, glutamate and GABA. However,
over the past 30-years, increasing attention has been focused on
a group of bioactive peptides, including galanin and neuropeptide
Y, which are abundantly expressed in the brain. The preferential
release of neuropeptides under conditions of increased neuronal
activity, and in particular during seizures, has encouraged inves-
tigation of their role in seizure modulation (Hokfelt, 1991). Both
galanin (a 29-amino-acid neuropeptide released during seizures
that inhibits glutamate release in the hippocampus) and neu-
ropeptide Y (a 36-amino acid polypeptide) have been shown to
antagonize excitatory glutamatergic neurotransmission in the hip-
pocampus (Zini et al., 1993). Antiseizure effects have been obtained
by increasing the strength of inhibitory signals (by supplementing
specific GABAA receptor subunits or inhibitory neuropeptides like
galanin or neuropeptide Y) or by reducing the strength of excitatory
signals (by knocking down NMDA receptor subunits) (Simonato,
2014). Gene therapy aimed at producing antiepileptogenic effects
is a combination therapy based on the supplementation of the
neurotrophic factors brain-derived neurotrophic factor (BDNF) and
fibroblast growth factor 2 (FGF-2). The goal of this approach was
to increase the extracellular levels of FGF-2 and BDNF by gen-
erating cells capable of constitutively but transiently secreting
these factors; achievement of this goal was verified by performing
in vitro and in vivo analysis of both NTFs processing and release
(Paradiso et al., 2009). Richichi et al. (2004) studied the effect
on acute kainite-induced seizures and kindling epiletogenesis of
long-lasting neuropeptide Y overexpression by local application
of recombinant AAV vectors in the rat hippocampus. The authors
used vectors with different serotypes and clearly showed that tis-
sue can be more efficiently targeted by varying capsid genes. rAAV
serotype 2 (rAAV2) vector increased neuropeptide Y expression in
hilar interneurons only, whereas the chimeric serotypes 1 and 2
vector caused far more widespread expression including mossy
fibers, pyramidal cells and subiculum (Davidson et al., 2000).
Researchers are attempting to develop strategies for globally
delivering genes to the brain by crossing the blood–brain bar-
rier (BBB) after administering vectors in the peripheral blood. For
gene therapy, a vector can be conjugated to a ligand that mimics
the natural ligand for the receptor, e.g., transferrin or insulin. The
vector–ligand conjugate remains intact and unmodified while in
transit and is therefore released intact into the interstitial space.
Recently, AAV vectors have been shown to undergo transcytosis
of the rodent BBB (Di Pasquale and Chiorini, 2006; Foust et al.,
2009). However, much work remains to be done to prove that this
approach can be applied to the treatment of genetic epilepsies.
The researchers from the Imperial College London studied sam-
ples of brain tissue removed from patients during neurosurgery
for their epilepsy and identified a gene network that was highly
active in the brain of these patients, and then discovered that an
unconnected gene, Sestrin 3 (SESN3), acts as a major regulator
of this epileptic gene network. This is the first time SESN3 has
been implicated in epilepsy and its co-ordinating role was con-
firmed in studies with mice and zebrafish. Instead of studying
individual genes, which has been the usual approach in epilepsy to
date, this research group has developed novel computational and
genetics techniques to systematically analyze the activity of genes
(‘systems genetics’ approach) in epilepsy (Johnson et al., 2015). Fur-
ther research is required to enumerate the role of gene therapy in
humans (Chrzanowska-Lightowlers et al., 1995).
8. Conclusion
Pyruvate dehydrogenase complex deficiency is one of the
numbers of mitochondrial dysfunctions resulting in impaired pro-
duction of ATP, accumulation of lactic acid and typically severe
systemic dysfunction. Defects of the human pyruvate dehydroge-
nase complex may cause potentially severe neurological disease or
death, but they are present with great variability due to the tissue
specificity of metabolic fuel requirements. Although over 40 years
have elapsed, since the first description of a congenital deficiency
of the PDHc (Bigger et al., 1999), the incidence and prevalence of
this life-threatening condition is yet to be unfurled. Several earlier
reviews exist of the clinical and biochemical characteristics or of the
molecular genetic etiologies of PDHc deficiency (Blass et al., 1970;
Kerr and Schmotzer, 2004; Owen et al., 2000; Robinson, 1995;
Stacpoole and Gilbert, 2006; Zullo, 2001). However, there has been
no recent comprehensive analysis of the natural history of the dis-
ease, nor attempts to discern phenotypic differences or predictable
outcomes based on biochemical defects or mutations in specific
 S. Bhandary, K. Aguan / Epilepsy Research 116 (2015) 40–52
components of the complex. In the present review, we have sum-
marized the clinical, biochemical and genetic findings contained
in published reports of PDHc deficiency. We sought associations
among various pathological indices that could provide new insight
into the pathobiology and clinical course of this disease. In recent
years, much has been learned about the molecular basis of these
disorders, which predominantly affect the E1␣ subunit of pyru-
vate dehydrogenase, encoded by chromosome X. Other subtypes
are inherited in an autosomal recessive manner. Diagnosis is avail-
able both by biochemical and molecular means. Management is still
far from ideal although early institution of a ketogenic diet may
be helpful in some cases. Dichloroacetate has not found universal
acceptance due to its side effects of peripheral neuropathy and that
it does not reverse the central nervous system problems. However,
factors that account for the great variability observed in affected
individuals have been only partially explained. More research is
needed for the optimal management of epilepsy and pathogenesis
of epilepsy in the setting of PDHc deficiency. A better understand-
ing of these variables may provide future guidance to find more
effective therapy, which at present is of limited benefit.
9. Future directions
The association of mitochondrial abnormalities and disease in
human has only been recognized since 1962. Numerous clinical
descriptions were reported, unfortunately, treatment is unsatisfac-
tory and most patients fail to respond to medications. Every day
more and more information is available about the structure and
function of the human PDHc. The challenge now lies in trying to
integrate this information and achieve a full appreciation of this
metabolically important complex and its role in the context of cell
as well as specific tissues. When it comes to specific subcomplexes,
El, E2, and E3, research studies by various scientific groups in the
past seem to have been concentrated especially on the E1˛ subunit
and to a lesser extent on E2 and E3. This leaves subcomplexes E1␤
and E2 somewhat neglected. Therefore good functional studies uti-
lizing a variety of reliable systems are required for the PDH complex
components. There are some clinical findings that may be helpful
in detecting those infants, children and adolescents with possible
PDHc mutations that may help the clinician with improved medica-
tion management. The ketogenic diet may be an option for selected
children. Clearly the study of epilepsy and mitochondrial disease
or more precisely PDHc deficiency, is in its infancy. Much work
remains to be done and better treatment options are needed. How-
ever probably the most challenging and interesting work would
be the determination of the molecular basis underlying the mul-
tiple mitochondrial enzyme deficiency syndrome, which includes
severe deficiency of the pyruvate dehydrogenase complex. If we can
develop medication to target this gene in the brain, then the hope
is that we could influence the whole epileptic gene network rather
than individual parts and in turn achieve more effective treatments.
There is a significant unmet medical need in epilepsy.
Conflict of interest statement
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Acknowledgements
The authors would like to acknowledge financial support by
the Department of Biotechnology, Govt. of India. Helpful discus-
sion with Dr. Aparajita Ghosh, Bose Institute, Kolkata, India is
acknowledged.
49
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