altered between mice and humans. Control human neurons showed both increased protein
amounts and activity of calcineurin (Fig. 5, D
and E) compared to mouse WT neurons, further
suggesting that alterations in calcium homeostasis may also contribute to interspecies differences
in dopamine metabolism.
Here we have found that elevated mitochon-
drial oxidant stress in human SNc dopaminergic
neurons triggers a dopamine-dependent toxic
cascade leading to lysosomal dysfunction and
a-synuclein accumulation—causally linking three
major pathological features of PD. This discovery
complements previous work demonstrating that
lysosomal dysfunction leads to deficits in mito-
phagy and the buildup of dysfunctional mito-
chondria (24) and raises the possibility of a
pathogenic positive feedback between these
two organelles. The pathogenic cascade from
mitochondria to lysosomes was only evident in
human—not mouse—dopaminergic neurons. This
species difference was inferred to be a conse-
quence of higher amounts of dopamine in
human neurons on the basis of several lines
of evidence, including the selective accumula-
tion of neuromelanin in human neurons and
the ability of L-dopa, the synthetic precursor of
dopamine, to induce the pathological cascade in
mouse dopaminergic neurons modeling PD. In
addition, our data also suggest that increased
cytosolic dopamine contributes to elevated mito-
chondrial oxidant stress, indicating a vicious
cycle of dopamine and mitochondrial oxidation
in human midbrain neurons.
Although increased a-synuclein inhibits GCase
trafficking and lysosomal function in various cell
types (19), our data suggest that increased oxidant stress and dopamine adduct formation
modified the catalytic site of GCase and lowered
its activity preferentially in midbrain dopaminergic neurons. Because similar alterations in
oxidized dopamine and GCase were seen in neurons obtained from patients with idiopathic and
familial PD, we propose that this mechanism ap-plies across various forms of PD. In support of this
notion, we showed that elevation of a-synuclein
in the substantia nigra of DJ-1 KO mice also resulted in increased oxidized dopamine, diminished
activity of GCase, and neurodegeneration.
Our long-term midbrain cultures demonstrated
that early treatment with mitochondrial antioxidants reduced downstream accumulation of
oxidized dopamine and a-synuclein and rescued
lysosomal dysfunction, highlighting the importance of early therapeutic intervention in the
pathogenic cascade. The inherent differences
between human and mouse dopaminergic neuronal vulnerability emphasize the value of studies
in human neurons to identify pathways and targets for therapeutic development in PD and related synucleinopathies.
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Fig. 5. Alterations in calcium homeostasis and dopamine metabolism contribute to intrinsic
differences between human and mouse dopaminergic neurons. (A to C) WT and DJ-1 KO mouse
iPSC-derived dopaminergic neurons were analyzed for (A) oxidized DA at d40, d70, and d90 (50 mM
and 500 mM standards shown) or treated with L-dopa or vehicle and analyzed at d55 for (B)
oxidized DA (n = 3) and (C) lysosomal GCase activity (n = 3). (D to F) Control human and WTmouse
iPSC-derived dopaminergic neurons analyzed for (D) T-soluble calcineurin (n = 5 or 6), (E)
calcineurin activity (n = 5), and (F) DA amounts by HPLC [n = 5 (mouse); n = 10 (human)]. HPLC
chromatogram in a 4 mM standard sample is shown. Dihydroxybenzylamine (DHBA) was used as
an internal standard. (G) WT and DJ-1 KO mouse iPSC-derived dopaminergic neurons were analyzed
for total DA content at d55 (n = 5). (H) The ratio of DOPAC/DA for WTand DJ-1 KO mouse
iPSC-derived dopaminergic neurons treated with L-dopa (n = 8). Equal protein concentration was
used in nIRF assays. Error bars, means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test
(D) to (H) or one-way ANOVA with Tukey post hoc test (B) and (C). n.s., not significant.