| Abstract |
Parkinson's disease was first described more than 200 years ago and yet
the aetiology of the disease is not fully understood. Cellular phenotypes
include mitochondrial impairment, lysosomal clearance dysfunction with
alpha-synuclein accumulation and intracellular trafficking alterations. In
most cases PD develops sporadically and the causes are yet unknown. However,
in 10% of the cases, PD is inherited and the cause is genetic. Currently, 23
PARK loci have been identified within genes causing PD. Here, we focus on
PARK17, where a point mutation leading to an amino acid exchange p.D620N
in VPS35 has been found to cause an autosomal-dominant form of PD.
Fibroblasts from a patient carrying the p.D620N mutation and from two age
and gendermatched control were derived from a skin biopsy. Functional
analyses of these fibroblasts revealed that mitochondrial membrane
potential (MMP) was decreased in the patient cells compared to controls
without any alteration of mitochondrial morphology. Next, the fibroblasts
were reprogrammed into induced pluripotent stem cells (iPSCs), that were
characterised in detail. The iPSCs were first differentiated in small
molecule neuronal precursor cells (smNPCs), where we analysed
mitochondrial function and lysosomal clearance capacity. We found no
significant difference in MMP between patient and control smNPCs. However,
we observed a decreased autophagic flux and lower levels of mature
cathepsin D protein, a lysosomal hydrolase responsible for the degradation
of a-synuclein. Nonetheless, we found no difference in a-synuclein protein
level. In order to study the impact of p.D620N on PD-related neuronal
phenotypes we differentiated the smNPCs in this more disease-relevant cell
population. We generated a neuronal culture enriched in dopaminergic
neurons. We found that the mitochondrial network was fragmented with
smaller mitochondria and less branching. Mitochondria had lower MMP and
increased intra-mitochondrial reactive oxygen species (ROS) levels. In
addition, mitochondrial respiration was impaired which resulted in lower
production of ATP. After CCCP treatment, mitophagy was induced in the
patient neurons to the same level as in the control neurons. However,
while in the control neurons the autophagosomes containing mitochondrial
fragments were successfully cleared, in the patient neurons they
accumulated and were not cleared properly. In line with this observation,
the autophagic flux was decreased and late endosome/lysosomal mass was
decreased. This decreased autophagic flux was accompanied by an increase
of alpha-synuclein protein levels.
We further wanted to pinpoint how p.D620N VPS35 caused mitochondrial
impairment in patient neurons. Accumulation of a-synuclein has been shown
to induce similar mitochondrial alterations. Therefore, we measured alpha-
synuclein protein levels in the mitochondrial and the cytosolic fraction
and found that it was increased in both fractions to the same ratio.
However, when we knocked down alpha-synuclein to the levels of the
controls, it was not sufficient to rescue the decreased MMP and increased
ROS level in the patient neurons. We conclude that alpha-synuclein
accumulation in the mitochondrial fraction was not sufficient to cause the
observed phenotype.
Recently, an increased LRRK2 kinase activity was identified in p.D620N
VPS35 mutant monocytes and erythrocytes from patients, as well as brains
from homozygous mice. Therefore, we hypothesized that pathological LRRK2
kinase activity may induce mitochondrial impairment. We measured the
phosphorylation levels of Rab10, one of LRRK2 kinase substrates, however,
no significant difference between patient and control neurons was observed.
In this study, we show for the first time that p.D620N VPS35 causes PD-
related cellular phenotypes in patient-derived neurons. The patient-
derived neurons displayed impaired mitochondria and lysosomal clearance,
with accumulation of alpha-synuclein.
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