Furthermore, PLA2G6 has also been implicated in a number of other
brain diseases including Alzheimer’s disease and bipolar
disorder. More recently, PLA2G6, at the PARK14 locus,
has also been characterized as the causative gene in a subgroup of
patients with autosomal recessive early-onset
dystonia-parkinsonism. Interestingly these patients do not display
cerebellar atrophy or basal ganglia iron on MRI, and
neuropathological examination reveals widespread Lewy body
pathology and the accumulation of hyperphosphorylated tau. These
clinical and neuropathological features further support a link
between PLA2G6 mutations and parkinsonian disorders, and
demonstrate the clinical heterogeneity in PLA2G6-associated
neurodegeneration (Kinghorn, 2015 and references therein, see
below).
Support for a loss-of-function of PLA2G6 enzyme activity in
causing disease comes from a recent study on recombinant wild-type
and mutant human PLA2G6 proteins. This demonstrated that mutations
in PLA2G6 associated with infantile neuroaxonal
dystrophy or neurodegeneration with brain iron accumulation result
in encoded proteins that exhibit <20% of control levels of
phospholipase and lysophospholipase activities. Conversely,
mutations associated with dystonia-parkinsonism do not impair
catalytic activity. The differential enzymatic activities
associated with PLA2G6 mutations in infantile
neuroaxonal dystrophy/neurodegeneration with brain iron
accumulation and dystonia-parkinsonism may explain the relatively
later onset and milder phenotype seen in the latter. Furthermore,
the preserved enzymatic function of dystonia-parkinsonism causing
mutations must be interpreted with caution as there may be
differences in PLA2G6 activity in vivo that are not detected by
these in vitro assays. For example, there may be alternative
mechanisms that alter the regulation of PLA2G6 activity, such as
changes in the binding to calmodulin or other proteins. Further
support for the loss of enzymatic function hypothesis comes from a
recent study of a Chinese population with Parkinson’s
disease, which identifies novel PLA2G6 mutations
occurring in the heterozygous state, associated with an inhibition
in the phospholipid-hydrolysing functions of PLA2G6. Moreover
another study found a genotype–phenotype correlation in
patients with infantile neuroaxonal dystrophy and
neurodegeneration with brain iron accumulation: mutations that are
predicted to lead to an absence of protein are associated with
more severe infantile neuroaxonal dystrophy-type clinical
phenotypes, while those with compound heterozygous missense
mutations correlate with the less severe phenotype of
neurodegeneration with brain iron accumulation and are predicted
to result in protein with some residual enzyme function (Kinghorn,
2015 and references therein, see below).
Kinghorn, K.J., Castillo-Quan, J.I.,
Bartolome, F., Angelova, P.R., Li, L., Pope, S., Cochemé,
H.M., Khan, S., Asghari, S., Bhatia, K.P., Hardy, J., Abramov,
A.Y. and Partridge, L. (2015). Loss of PLA2G6
leads to elevated mitochondrial lipid peroxidation and
mitochondrial dysfunction. Brain 138: 1801-1816. PubMed ID: 26001724
Abstract
The PLA2G6 gene encodes a group VIA calcium-independent
phospholipase A2 beta enzyme that selectively hydrolyses
glycerophospholipids to release free fatty acids. Mutations in PLA2G6
have been associated with disorders such as infantile neuroaxonal
dystrophy, neurodegeneration with brain iron accumulation type II
and Karak syndrome. More recently, PLA2G6 has been
identified as the causative gene in a subgroup of patients with
autosomal recessive early-onset dystonia-parkinsonism.
Neuropathological examination revealed widespread Lewy body
pathology and the accumulation of hyperphosphorylated tau,
supporting a link between PLA2G6 mutations and
parkinsonian disorders. This study shows that knockout of the Drosophila
homologue of the PLA2G6 gene, iPLA2-VIA,
results in reduced survival, locomotor deficits and organismal
hypersensitivity to oxidative stress. Furthermore, it was
demonstrated that loss of iPLA2-VIA function leads to a
number of mitochondrial abnormalities, including mitochondrial
respiratory chain dysfunction, reduced ATP synthesis and abnormal
mitochondrial morphology. Moreover, loss of iPLA2-VIA is
strongly associated with increased lipid peroxidation levels. The
findings were confirmed using cultured fibroblasts taken from two
patients with mutations in the PLA2G6 gene. Similar
abnormalities were seen including elevated mitochondrial lipid
peroxidation and mitochondrial membrane defects, as well as raised
levels of cytoplasmic and mitochondrial reactive oxygen species.
Finally, it was demonstrated that deuterated polyunsaturated fatty
acids, which inhibit lipid peroxidation, are able to partially
rescue the locomotor abnormalities seen in aged flies lacking iPLA2-VIA
gene function, and restore mitochondrial membrane potential in
fibroblasts from patients with PLA2G6 mutations. Taken
together, these findings demonstrate that loss of normal PLA2G6
gene activity leads to lipid peroxidation, mitochondrial
dysfunction and subsequent mitochondrial membrane abnormalities.
Furthermore, the iPLA2-VIA knockout fly model provides a
useful platform for the further study of PLA2G6-associated
neurodegeneration (Kinghorn, 2015).
Highlights
- Drosophila lacking iPLA2-VIA activity
display age-dependent locomotor deficits and reduced lifespan.
- Brains lacking iPLA2-VIA display severe, widespread
neurodegeneration and mitochondrial degeneration.
- Loss of iPLA2-VIA results in reduced mitochondrial
membrane potential and abnormal mitochondrial respiratory chain
activity.
- Loss of iPLA2-VIA is not associated with changes in
cardiolipin composition.
- Loss of iPLA2-VIA promotes brain lipid peroxidation
and reduces whole body triacylglycerol levels.
- Human PLA2G6 mutant fibroblasts display abnormal
mitochondrial physiology.
- Mutations in PLA2G6 are associated with elevated
mitochondrial lipid peroxidation levels in human fibroblasts and
are reversed by treatment with deuterated polyunsaturated fatty
acids.
- Deuterated linoleic acid partially rescues the locomotor
abnormalities of flies lacking iPLA2-VIA.
Discussion
As the energy factories of cells, mitochondria play an essential
role in neurons, in which oxidative phosphorylation is the main
source of ATP. A previous study in a mouse model demonstrates that
knockout of Pla2g6 results in abnormal mitochondrial
membrane morphology. These findings lead to the question as to how
loss of normal PLA2G6 gene function leads to abnormal
mitochondrial morphology, and whether mitochondrial dysfunction is
an early feature of PLA2G6-associated neurodegeneration (Kinghorn,
2015).
This study demonstrates that loss of normal phospholipase A2
activity in the fruit fly and in fibroblasts taken from patients
harbouring mutations in PLA2G6, results in striking
abnormalities in both mitochondrial function and morphology.
Furthermore, it was demonstrated that mitochondrial dysfunction
precedes the mitochondrial morphological abnormalities that are
seen at the ultrastructural level. Deficits in mitochondrial
oxidative respiration are seen in iPLA2-VIA knockout
flies as young as 2 days of age, when there are no associated
mitochondrial membrane abnormalities present on electron
microscopy. Moreover, the finding that mitochondrial dysfunction
occurs early in PLA2G6-associated neurodegeneration is consistent
with the clinical features seen in infantile neuroaxonal
dystrophy- neurodegeneration with brain iron accumulation and
dystonia-parkinsonism, all of which display clear early-onset
phenotypes, that have previously been associated with
mitochondrial disease (Kinghorn, 2015).
In addition to mitochondrial dysfunction, it was also
demonstrated that loss of PLA2G6 activity is associated with
raised levels of reactive oxygen species. These species play
important roles in cell signalling, but at abnormally high levels
are detrimental to cells, ultimately leading to cell death. Lipids
are important targets of reactive oxygen species and therefore
elevated reactive oxygen species levels will lead to increases in
mitochondrial lipid peroxidation. Furthermore, it may also be that
elevated lipid peroxidation arises in the absence of phospholipase
A2 activity because some phospholipases preferentially cleave
oxidized lipids to maintain redox homeostasis. In turn, lipid
peroxides are known to trigger a cascade of events that include a
fall in mitochondrial membrane potential, mitochondrial swelling
and loss of mitochondrial matrix components. Furthermore, it has
been shown that complex I, the largest complex of the respiratory
chain, is susceptible to oxidative stress. Indeed, a decrease in
oxidative respiration in flies lacking iPLA2-VIA was
observed when substrates specific for complex I of the respiratory
chain were used. Furthermore, Lewy body disease has been shown to
be associated with reduced levels of complex I activity, further
implicating mitochondrial dysfunction in neurodegeneration, and
more specifically in PLA2G6-associated neurodegeneration, which
has been linked to parkinsonism and Lewy body pathology. In
addition, lipid peroxidation is strongly catalysed through the
Fenton reaction by iron and iron complexes and it is iron that is
often deposited in the basal ganglia of the brains of patients
affected with PLA2G6 mutations. Thus, iron-mediated
oxidative insults seem to have a crucial role in the function of PLA2G6
and in the progression of diseases associated with mutations in
this gene (Kinghorn, 2015).
Given the increase in levels of lipid peroxidation in flies
lacking iPLA2-VIA, this study looked for elevated levels
of oxidized cardiolipin, one of the most common lipids in the
inner mitochondrial membrane. It has previously been hypothesized
that PLA2G6 is involved in the remodelling of cardiolipin's fatty
acid side chains and in removing oxidized fatty acid side chains.
Oxidation of mitochondrial phospholipids such as cardiolipin leads
to cytochrome c release and apoptosis. To determine whether
cardiolipin is oxidized in PLA2G6 loss-of-function, the
cardiolipin composition of iPLA2-VIA−/− fly
heads was analyzed using mass spectrometry. It was demonstrated
that mitochondrial membrane disintegration and mitochondrial
dysfunction occur in the absence of any significant differences in
cardiolipin amount or side chain composition in fly brains lacking
iPLA2-VIA. Furthermore, oxidized cardiolipin was not
detected in significant amounts. This may be related to the types
of fatty acids present in Drosophila cardiolipin.
Palmitic acid is the major component of cardiolipin side chains in
Drosophila and is more resistant to oxidation than
linoleic acid, the main cardiolipin side chain in most mammalian
tissues. However, it could also point towards redundancy in the
cardiolipin remodelling enzymes. There are a number of enzymes
involved in cardiolipin remodelling and it may be the case that
fatty acid remodelling of the side chains, in particular removal
of oxidized fatty acids, can be performed by other enzymes
involved in cardiolipin remodelling such as tafazzin (Kinghorn,
2015).
It was found that loss of normal PLA2G6 function is strongly
associated with elevated mitochondrial lipid peroxidation.
Furthermore lipid peroxidation is known to induce mitochondrial
injury, leading to a decrease in oxidative respiratory chain
function, reduced mitochondrial membrane potential, reduced
cellular ATP levels and raised reactive oxygen species levels, all
features that were demonstrated in flies and human fibroblasts
lacking normal PLA2G6 gene activity. This in turn would
be predicted to lead to cell death and neurodegeneration. As lipid
peroxides are harmful mediators of oxidative stress, strategies
focused on quenching, recycling or protecting such products could
be highly effective in the treatment of the disease (Kinghorn,
2015).
In addition to showing that lipid peroxidation is increased in
both fly brains lacking iPLA2-VIA and human PLA2G6
mutant fibroblasts, it was demonstrated that D-PUFAs reduce the
rates of lipid peroxidation in two lines of PLA2G6
mutant fibroblasts and restore mitochondrial membrane potential.
This suggests that D-PUFAs may have potential therapeutic roles in
PLA2G6-associated neurodegeneration and its associated
mitochondrial dysfunction (Kinghorn, 2015).
Overall, these data promote Drosophila as a useful
model for studying PLA2G6-associated neurodegeneration, and for
manipulating pathways and therapeutic targets in order to reverse
or prevent the deleterious effects of lipid peroxides. Loss of iPLA2-VIA
gene function results in reduced lifespan and locomotor ability,
increased sensitivity to oxidative insults as well as hypoxia,
osmotic and starvation stressors. Thus flies lacking iPLA2-VIA
display phenotypes that can be used as markers in genetic
modifying screens or in the screening of potential therapeutic
compounds. To test the validity of the fly model for drug
screening and to confirm the benefits of D-PUFAs in vivo in an
organism lacking iPLA2-VIA, the effects of D-PUFAs on the
locomotor deficits of iPLA2-VIA−/− flies
were examined. This demonstrated that treatment with D2-linoleic
acid partially rescues the climbing abnormalities in aged iPLA2-VIA
knockout flies. Therefore this approach of using compounds, which
act to reduce the autoxidation of lipids, may represent a future
therapeutic strategy for patients with PLA2G6-associated
neurodegeneration and warrants further investigation (Kinghorn,
2015).
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Reviews
Ramanadham, S., Ali, T., Ashley, J. W., Bone, R. N., Hancock, W. D. and Lei, X. (2015). Calcium-Independent Phospholipases A2 (iPLA2s) and their Roles in Biological Processes and Diseases. J Lipid Res. PubMed ID: 26023050
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