Mitochondrial dysfunction in genetic parkinsonism: what muscles can tell us about the brain

Parkinson*s disease (PD) is the second most common neurodegenerative disease
after Alzheimer*s disease, affecting approximately 0.3% of the general
population, and 1% of the population over the age of 60. Worldwide 6.3 million
people have PD. Due to aging of the general population, these numbers of
affected individuals are expected to double by the year 2030.

PD is characterized by loss of dopaminergic neurons in the substantia nigra and
the accumulation of intracellular protein aggregates called Lewy bodies.
Symptomatic treatment strategies with dopamine replacement therapy (including
levodopa and dopamine agonists) or stereotactic deep brain surgery have
dramatically improved the clinical status of patients. However, PD remains a
progressive disease and despite optimal medical management, patients become
increasingly disabled, causing an immense burden not only for affected
patients, but also for their relatives and society.

Optimized treatment strategies are, therefore, urgently needed, in particular
approaches that delay or perhaps even prevent the disease altogether, but this
calls for a better insight into the pathophysiological mechanisms underlying
PD. Oxidative stress and mitochondrial dysfunction are among the leading
candidates as causes of nigral degeneration in PD. Indeed, various studies have
tested mitochondrial integrity in individuals with idiopathic PD, and reported
a possible correlation between PD and mitochondrial respiratory chain
dysfunction in the substantia nigra. Especially, NADH-ubiquinone reductase
(complex I) activity was significantly reduced in post-mortem nigral neurons of
patients with PD. However, clinical trials in the late nineties, administering
antioxidants like Q10 to patients with PD were not convincingly successful, and
the *mitochondrial hypothesis* lost some of its initial attraction. However, in
the last two years, attention for the possible role of mitochondrial
dysfunction has, again, greatly increased because of fascinating observations
on genetic variants of parkinsonism. Initially thought to be a sporadic
disorder, PD has now been associated with at least five monogenetic forms (two
autosomal dominant and three recessive forms). At least two of the recessive
forms, called parkin and pink1, are associated with abnormal protein products
that appear to kill neurons via mitochondrial dysfunction. This evidence thus
far stems exclusively from preclinical studies. For example, recent studies
have used a Drosophila model of autosomal recessive juvenile PD and showed that
flies with both the parkin and pink1 mutant protein exhibit locomotor defects
caused by apoptotic cell death of specific muscles. Mitochondrial pathology
leading to oxidative stress was the earliest manifestation of muscle
degeneration. Transcriptional alterations occurring during this muscle
degeneration indicated that oxidative stress response components are induced in
parkin mutants and that loss-of-function mutations in oxidative stress
components enhance the parkin mutant phenotypes. This indicates that oxidative
stress and mitochondrial dysfunction may play a fundamental role in the
etiology of autosomal recessive PD.

An important next step will be to translate these preclinical findings to
patients with PD. Here, we propose an initial study to further examine
mitochondrial dysfunction in patients with PD, using - for the first time -
muscle biopsies of affected patients with a genetic form of PD.

Can mitochondrial dysfunction (especially reduced complex I activity) be
observed in skeletal muscle samples from patients with autosomal recessive PD
(in this pilot study, parkin parkinsonism)?

The project is expected to (a) clarify the possible role of mitochondrial
dysfunction in PD; and (b) to define the molecular mechanisms underlying this
mitochondrial dysfunction, which will be crucial for the future development of
possible neuroprotective agents.

For this initial study, 10 patients with proven autosomal recessive PD carrying
the parkin mutation (AR-PD) will be included. Diagnosis of autosomal recessive
PD will be established according to the UK brain bank criteria, together with
genetic DNA analyses confirming a mutation in the parkin gene. Patients are
maintained on their usual treatment. The severity of the disease will be
established using the Hoehn-Yahr (H/Y) scale in the on-phase, and the UPDRS,
part III. Furthermore, patients are asked whether they experience any symptoms
of myalgia, exercise in-tolerance, cramps, or fixed weakness of certain muscles
(e.g. the extraocular muscles).
Blood and urine samples will be taken to measure creatine kinase, lactate and
pyruvate levels.
A needle-biopsy from the quadriceps muscle of the left leg is performed and
immediately processed for histopathological, electronmicroscopical and
mitochondrial studies, including the respiratory chain complexes I to V. This
procedure is performed according to a specified method, using a unique set of
incubations with 3 carboxyl-14C labeled substrates, in combination with
measurement of ATP production and enzyme activity of complex I to V in intact
muscle mitochondria. This method has been developed and performed in our
institution for many years and gives maximum information about the
mitochondrial energy-generating system. Control values are obtained from
previously obtained muscle tissue of 43 healthy individuals, in which the same
procedure was performed.
Additionally, cultures of both skeletal muscle cells and fibroblasts will be
set-up for further analyses and determination of, amongst others, the
anti-oxidant glutathione. Reduced levels of glutathione have been associated
with dopaminergic cell loss in PD, and restoration of glutathione in culture
may protect cells from apoptosis due to oxidative stress.

Statistical analyses
Data obtained from the skeletal muscle biopsies will be analyzed using an
analysis of variance (ANOVA) for multiple comparisons, including mitochondrial
respiratory chain complexes I-V. Furthermore, descriptive analyses of skeletal
muscle biopsies by histology and electron microscopy are performed.

The main burden for the patient will consist of pain during the needle biopsy,
lasting in general for 1 day.
This pain is comparable with a bruise, and is usually well tolerable.
Furthermore, there is a very small change of developing a large haematoma after
the needle biopsy and venous puncture.