Dopamine promotes the neurodegenerative potential of β-synuclein

Article abstract of DOI:10.1111/jnc.15134

A contribution of α-Synuclein (α-Syn) to etiology of Parkinson′s disease (PD) and Dementia with Lewy bodies (DLB) is currently undisputed, while the impact of the closely related β-Synuclein (β-Syn) on these disorders remains enigmatic. β-Syn has long been considered to be an attenuator of the neurotoxic effects of α-Syn, but in a rodent model of PD β-Syn induced robust neurodegeneration in dopaminergic neurons of the substantia nigra. Given that dopaminergic nigral neurons are selectively vulnerable to neurodegeneration in PD, we now investigated if dopamine can promote the neurodegenerative potential of β-Syn. We show that in cultured rodent and human neurons a dopaminergic neurotransmitter phenotype substantially enhanced β-Syn-induced neurodegeneration, irrespective if dopamine is synthesized within neurons or up-taken from extracellular space. Nuclear magnetic resonance interaction and thioflavin-T incorporation studies demonstrated that dopamine and its oxidized metabolites 3,4-dihydroxyphenylacetaldehyde (DOPAL) and dopaminochrome (DCH) directly interact with β-Syn, thereby enabling structural and functional modifications. Interaction of DCH with β-Syn inhibits its aggregation, which might result in increased levels of neurotoxic oligomeric β-Syn. Since protection of outer mitochondrial membrane integrity prevented the additive neurodegenerative effect of dopamine and β-Syn, such oligomers might act at a mitochondrial level similar to what is suggested for α-Syn. In conclusion, our results suggest that β-Syn can play a significant pathophysiological role in etiology of PD through its interaction with dopamine metabolites and thus should be re-considered as a disease-relevant factor, at least for those symptoms of PD that depend on degeneration of nigral dopaminergic neurons. (Figure presented.).

Full text of DOI:10.1111/jnc.15134

DR. ANUPAM RAINA (Orcid ID : 0000-0001-9995-9219)
DR. SEBASTIAN KÜGLER (Orcid ID : 0000-0002-5130-2012)
Article type : Original Article
DOPAMINE PROMOTES THE NEURODEGENERATIVE POTENTIAL OF -SYNUCLEIN
Anupam Raina a, e, Kristian Leite a, Sofia Guerin a, Sameehan U. Mahajani a, f, Kalyan S.
Chakrabarti b, g , Diana Voll a, Stefan Becker b, Christian Griesinger b, Mathias Bähr a, Sebastian
Kügler a, c ,d
a University Medicine Göttingen, Dept. of Neurology, Waldweg 33, 37073 Göttingen, Germany
b Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
c Center Nanoscale Microscopy and Physiology of the Brain (CNMPB)
d Corresponding author: Sebastian Kügler, University Medicine Göttingen, Dept. of Neurology,
Waldweg 33, 37073 Göttingen, Germany, Email: sebastian.kuegler@med.uni-goettingen.de;
Phone: 0049 551 39 8351; Fax: 0049 551 39 14476
ORCID-ID: orcid.org/0000-0002-5130-2012
e present address: Department of Molecular Biology, UT Southwestern Medical Center, Texas,
USA; f present address: Department of Pathology, Stanford University, California, USA
g present address: Division of Sciences, Krea University, Sri City, AP, India
Abbreviations:
AADC: aromatic amino acid decarboxylase; ALDH: aldehyde dehydrogenase; -Syn: alpha-
synuclein; -Syn: beta-synuclein; DA: dopamine; DAT: dopamine transporter;DCH:
dopaminochrome; DLB: Dementia with Lewy bodies; DOPAC: 3,4-dihydroxyphenylacetic acid;
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/JNC.15134
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DOPAL: 3,4-dihydroxyphenyl-acetaldehyde; EGFP: enhanced green fluorescent protein; -Syn:
gamma-synuclein; HVA: homovanillic acid; L-DOPA: 3,4-dihydroxyphenylalanine; MAO:
monoamine oxidase; Nac: N-acetyl cysteine; NAC: non-amyloidogenic component; OMM: outer
mitochondrial membrane; PD: Parkinson´s disease; RRID: Research resource identifier; ROS:
reactive oxygen species; SNpC: substantia nigra pars compacta; VMAT2: vesicular monoamine
transporter 2.
Keywords:
-Synuclein, dopamine, DOPAL, dopaminochrome, Parkinson´s disease, neurodegeneration
Abstract
A contribution of -Synuclein (-Syn) to etiology of Parkinson´s disease (PD) and Dementia with
Lewy bodies (DLB) is currently undisputed, while the impact of the closely related -Synuclein (-
Syn) on these disorders remains enigmatic. -Syn has long been considered to be an attenuator
of the neurotoxic effects of -Syn, but in a rodent model of PD -Syn induced robust
neurodegeneration in dopaminergic neurons of the substantia nigra. Given that dopaminergic
nigral neurons are selectively vulnerable to neurodegeneration in PD, we now investigated if
dopamine can promote the neurodegenerative potential of -Syn.
We show that in cultured rodent and human neurons a dopaminergic neurotransmitter
phenotype substantially enhanced -Syn-induced neurodegeneration, irrespective if dopamine is
synthesized within neurons or up-taken from extracellular space. Nuclear magnetic resonance
interaction and thioflavin-T incorporation studies demonstrated that dopamine and its oxidized
metabolites 3,4-dihydroxyphenylacetaldehyde (DOPAL) and dopaminochrome (DCH) directly
interact with -Syn, thereby enabling structural and functional modifications. Interaction of DCH
with -Syn inhibits its aggregation, which might result in increased levels of neurotoxic
oligomeric -Syn. Since protection of outer mitochondrial membrane integrity prevented the
additive neurodegenerative effect of dopamine and -Syn, such oligomers might act at a
mitochondrial level similar to what is suggested for -Syn. In conclusion, our results suggest that
-Syn can play a significant pathophysiological role in etiology of PD through its interaction with
dopamine metabolites and thus should be re-considered as a disease-relevant factor, at least for
those symptoms of PD that depend on degeneration of nigral dopaminergic neurons.
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Introduction
The synuclein family of proteins consists of three members, -Synuclein (-Syn), -Synuclein (-
Syn) and -Synuclein (-Syn). -Syn is closely linked to the etiology of PD, as mutations, enhanced
gene dosage and single nucleotide polymorphisms causing subtle increases in protein levels all
result in aggravated PD symptoms. In addition, -Syn is a major component of proteinaceous
aggregate structures, Lewy bodies and Lewy neurites, that are found in all genetic and idiopathic
cases of PD (Poewe et al. 2017; Galvin et al. 2001). -Syn accumulations have been detected in
human brains, sometimes co-localizing with -Syn (Surgucheva et al. 2014). -Syn aggregates
have not yet been detected in human brain (Spillantini & Goedert 2018), and the role of -Syn in
human brain disorders is currently enigmatic.
Degeneration of dopaminergic A9 neurons of the substantia nigra pars compacta (SNpC)
constitutes a major hallmark of Parkinson´s disease (PD). Loss of these neurons causes prominent
motor symptoms like tremor, rigidity, postural instability and inability to initiate movements. No
formal proof exists yet to declare -Syn directly responsible for degeneration of nigral
dopaminergic neurons in PD patients (Espay et al. 2019). However, the intimate link of -Syn to
etiology of PD and the specific degeneration of A9 neurons suggest a likely connection between
-Syn and the presence of dopamine (DA), which henceforth we describe as the dopaminergic
neurotransmitter phenotype of these cells. This dopaminergic neurotransmitter phenotype is a
risk factor for neurodegeneration by itself, due to the oxidative stress that results from
dopamine metabolism and auto-oxidation (Jinsmaa et al. 2018). Several studies have
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demonstrated that DA, or rather its oxidized metabolites, can interact with -Syn, thereby
enhancing the neurotoxic potential of the protein by inhibiting fibril formation and promoting
formation of disease-relevant oligomeric species (Asanuma et al. 2003; Cappai et al. 2005; Burke
et al. 2008; Pham et al. 2009; Planchard et al. 2014; Mor et al. 2019; Mor et al. 2017).
Furthermore, both enzymatic and auto-oxidation of DA results in production of cytotoxic
molecules like hydrogen peroxide and reactive aldehydes, with very high potential to oxidatively
damage proteins, lipids, and DNA.
-Syn, closely related to -Syn but partially lacking the central aggregation-promoting part
known as the NAC-domain (amino acids 61 – 95 in -Syn, amino acids 73 – 83 lacking in -Syn)
(Giasson et al. 2001), is expressed in mammalian brain to similar extent as -Syn (Kasten & Klein
2013), in a 1:1 ratio with -Syn in rodent brain synaptic boutons (Wilhelm et al. 2014). -Syn has
long been thought to serve as an anti-aggregating and potentially neuroprotective counterpart to
-Syn (Giasson et al. 2001; Uversky et al. 2002; Park & Lansbury 2003; Hashimoto et al. 2004;
Fan et al. 2006). Contrasting this view, -Syn has been shown to be capable of aggregating in cell
free assays due to macromolecular crowding and interaction with metal ions and pesticides
(Yamin et al. 2005), and due to acidic pH (Moriarty et al. 2017) and interaction with lipid vesicles
at elevated temperatures (Brown et al. 2018). In vivo, it was recently shown that -Syn forms
proteinase K resistant fibrils in dopaminergic neurons of the rat substantia nigra and causes
degeneration of these neurons to a similar extent as -Syn (Taschenberger et al. 2013). Studies
in cultured non-dopaminergic neurons have demonstrated that the general mechanism of
neurodegeneration induced by -Syn converges on permeation of the outer mitochondrial
membrane (OMM) followed by cytochrome C-mediated caspase activation (Tolo et al. 2018).
However, it remains unknown up to now, if DA and its metabolism can influence the
neuropathological profile of -Syn, which would place -Syn at a central position when trying to
understand the reasons for preferential degeneration of nigral A9 neurons in PD.
In order to study the neurodegenerative potential of -Syn in presence or absence of DA, we
generated cell cultures allowing to exploit neurons that are identical in verum and control
conditions except for the presence or absence of DA. Such cells are not naturally available.
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Genuine dopaminergic neuron cultures isolated from rodent midbrains contain only a small
percentage of dopaminergic neurons in a background of about 90% GABAergic neurons, and the
dopaminergic cell population can only be identified after fixation and immunocytochemistry.
More importantly, no controls are available for these neurons, which are differing from the
dopaminergic phenotype solely by absence of dopamine synthesis. As such, we established
rodent and human neuron cultures that express various parts of the dopamine synthesis and
uptake machinery. AAV viral vectors were used for this purpose in rodent primary neurons, and
also allowed for expression of the synucleins and fluorescent markers in both, rodent and human
neurons. These attempts created a complex, but still highly versatile cell culture system allowing
to evaluate the impact of DA on neurotoxic aspects of -Syn.
We report that both intracellular DA synthesis and uptake of extracellular DA promote -Syn-
induced neurodegeneration, and that oxidized metabolites of DA physically interact with -Syn,
allowing for modification of the aggregation propensities of -Syn. These results demonstrate
that the neuropathological profile of -Syn is substantially enhanced by DA, suggesting that an
interplay of -Syn and DA may promote loss of dopaminergic neurons in PD and DLB.
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Materials & Methods
Primary neuron culture: Primary neuron/glia co-cultures were prepared from rat embryonal E18
cortex and cultured essentially as described (Kugler et al. 2003). Briefly, cortices were
mechanically dissociated, and single cell suspensions were prepared by digestion with trypsin
and DNAse. After a short incubation in fetal calf serum to inhibit trypsin activity, cells were
triturated through a fire-polished glass capillary, debris removed by gravitational sedimentation,
cells were counted and seeded at 250.000 cells/well in 24 well plates. The cell culture medium of
750 µl/well was not exchanged or further supplemented during the course of the experiments
except for addition of L-DOPA or DA. For preparation of primary neurons from embryonic rat
brains, all experimental animal procedures in Wistar rats (RRID:RGD_13508588; provided by the
Central Animal Facility of University Medicine Göttingen) were conducted according to approved
experimental animal licenses issued by the responsible animal welfare authority
(Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, LAVES) and
controlled by the local animal welfare committee and veterinarians of University Medical Center
Göttingen. Pregnant females were about 3 – 5 months of age, weighted about 250 – 280g, and
were housed individually with ad libitum access to food and water. 55 such rats were used to
prepare primary neurons from their embryos.
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Generation of hiPSCs: The CT-03 iPSC line was derived from a healthy 57-year old female. This
line was generated from dermal skin fibroblasts, by Sendai virus-mediated transcription factor
expression. Patterning into dopaminergic neurons or glutamatergic neurons was performed
essentially as described (Kriks et al. 2011) (Vazin et al. 2014) with the modifications specified in
(Mahajani et al. 2019), as shown in Fig. 4 A, B. Cells were seeded at 150.000 cells/well in 24-well
plates. After completing the differentiation step the cell culture medium of 750 µl was not
exchanged or further supplemented during the course of the experiments.
AAV vectors: Recombinant AAV-6 vectors were produced as described (Tolo et al. 2018). Briefly,
vector genome plasmids were co-transfected with the pDP6 helper plasmid into HEK293 cells
(RRID:CVCL_6871), viral particles were harvested at 48h after transfection by three freeze-thaw
cycles, pre-purified on an iodixanol step gradient, and purified and concentrated further by
heparin affinity chromatography. After dialysis against PBS, single use aliquots were stored at -
80°C in LoBind tubes (Eppendorf). Vector genome titres were determined by qPCR and purity >
98% verified by SDS gel electrophoresis. All transgenes were expressed under control of the
human synapsin 1 gene promoter (hSyn1), which provides strict neuron-specific expression
(Kugler et al. 2003). Equal expression levels of all synucleins were verified for early time points of
the study at div9, i.e. before onset of neurodegeneration, by western blots of transduced
neurons (Fig. 1C). For the late time points of the study (i.e. after div 11, when different extents of
neurodegeneration became evident, this approach is likely to be inaccurate, due to different
numbers of surviving neurons. Thus, we also verified equal expression levels by assessing the
half-life of -, -, and -Syn in primary neurons, by exploiting a regulable vector system as
described in Supplemental Figure S1. Of note, these regulable vectors were used solely to
address stability of the synucleins, and only constitutive synuclein expression was used to study
the interactions with dopamine.
Dopaminergic neuron models
Primary neurons were transduced with AAV vectors as follows: mono-cistronic vectors
expressing AADC, VMAT2, DAT, Bcl-Xl, GCaMP6 or the nuclear mCherry fluorophore (Fig. 1 A)
were added to primary neurons at times of preparation of the cells, by mixing 9 x 108 vg each
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with 250.000 cells per well to be seeded. After incubation with gentle shaking for 30 min at 37°C,
cells were pelleted by centrifugation with 800 x g to wash off residual virus, resuspended in cell
culture medium and seeded. AAVs expressing the synucleins plus EGFP from a separate
transcription unit were added into the cell culture medium on div 3 at a titre of 6 x 109 vg/well.
For generation of a cellular model of intracellular DA production, primary rat neuron cultures
expressing AADC/VMAT2 were supplied with 10 µM L-DOPA every second day, starting from
div3. Of note, it proved essential to allow for conversion of L-DOPA into DA, as in absence of
AADC/VMAT2, L-DOPA itself caused significant neuron loss in these cultures (Supplemental
Figure S2). For generation of a cellular model of up-take of DA from extracellular space, primary
rat neurons expressing DAT/VMAT2 were supplied with 12.5 µM DA every fourth day from div7
onwards.
Human iPSC-derived dopaminergic and glutamatergic neurons were transduced on DIV 20 (i.e. at
the same time after starting the differentiation process for dopaminergic and glutamatergic
neurons). Bi-cistronic vectors expressing α-Syn and EGFP, β-Syn and EGFP, γ-Syn and EGFP or
EGFP alone (Fig. 1 A) were added to the cells at 3 x 109 vg / well.
Imaging
Live cell imaging of spontaneous neuronal activity and for determination of surviving neuron
numbers (as determined by presence of fluorescent markers) was performed on a Zeiss Observer
Z1 microscope with heated stage and CO2 incubation chamber, equipped with an AxioCam503
camera and ZEN 2.3 software. For data analysis, images were segmented in ImageJ using a
custom-made macro with two steps of thresholding. In the first step, the image was segmented
using the local thresholding method described by Phansalkar et al., with the following
parameters – Phansalkar radius of 20 pixels, parameter 1 of 0.01 and parameter 2 of 100
(Phansalkar et al. 2011). Following this, the dimmer components of the image were thresholded
again using the same method for the identification of nuclei with a low fluorescence. Then,
particles larger than 120 pixels were identified and binarized, with clusters in the binary images
separated into individual particles using the local maxima of fluorophore signals. Finally, the
resulting number of particles was counted.
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Calcium imaging exploiting the genetically encoded GCaMP6 sensor was performed essentially as
described (Tolo et al. 2018).
Western blotting
Protein lysates were obtained from transduced primary neurons by lysis in 50 mM Tris, pH 8.0,
0.5% SDS, 1 mM DTT and protease inhibitors Complete Mini, Roche). Samples were run in 12%
SDS-PAGE and were blotted to PVDF membranes (fixed with 4% formaldehyde / 0.4%
glutaraldehyde in case of detection of the synucleins), blocked in non-fat dry milk or BSA in TBS-
T, and incubated in TBS-T with the following primary antibodies at 4°C for 12h in roller tubes: anti
pan-Synuclein (Abcam ab6176; RRID:AB_305344), anti AADC (Abcam ab3905; RRID:AB_304145),
anti DAT (Abcam ab5990; RRID:AB_305226), anti VMAT2 (Everest EB06558; RRID:AB_2187855),
and anti -tubulin (Sigma T4026; RRID:AB_477577). Horseradish peroxidase-coupled secondary
antibodies were incubated for 1h at RT and signals detected and quantified after incubation in
ECL with a BioRad ChemiDoc XRS+ imager and Quantity One software.
Quantification of DA, DOPAC and HVA
Cell culture supernatant was mixed 4:1 with 2M perchloric acid (PCA) and 1% (v/v) sodium
metabisulfite (SMbS) to stabilize DA and metabolites and incubated on ice for at least 10 min.
Cells were washed with PBS, and lysed in 200 µl trichloroacetic acid (TCA) per well. The cell lysate
was mixed 9:1 with 2M PCA / 1% SMbS and incubated on ice for at least 10 min. Levels of DA,
DOPAC and HVA were then determined by HPLC with electrochemical detection essentially as
described (Tereshchenko et al. 2014). At times of harvesting cells and supernatants, neuron
numbers were counted from fluorescently labelled cells in order to allow relating
DA/DOPAC/HVA amounts to numbers of neurons present in the respective culture. As such, DA,
DOPAC, and HVA levels were expressed as ng / 104 neurons, allowing to use the same scale for
intracellular and extracellular levels from the same culture.
NMR
Full length recombinant 15N-labelled human synucleins were produced in BL21 E.coli in M9
minimal medium using 15NH4Cl as sole nitrogen source, purified as described (Hoyer et al. 2002)
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and adjusted to 100 µM final concentration in 20 mM sodium phosphate, pH 6.0, 2 mM tris-(2-
carboxyethyl)phosphine (TCEP), 0.05% NaN3, and 10% 2H2O. 10 mM stock solutions of DA and
DOPAL were prepared in 20 mM sodium phosphate, pH 6.0, 2mM TCEP (in order to inhibit any
auto-oxidation and thus prevent artifacts like dicatechol pyrrole adducts generated by oxidized
DOPAL (Werner-Allen et al. 2017)) and 0.05% NaN3. Chemical shift perturbations (CSPs) at
increasing DA/DOPAL concentrations were recorded on a Bruker 800 Mhz spectrometer fitted
with a CP-TCI cryoprobe. At 280K, two-dimensional 1H - 15N heteronuclear single quantum
coherence (HSQC) was recorded with 100 µM synuclein and 0.02 - 2 mM DA and DOPAL. Because
1H chemical shifts of intrinsically disordered proteins are extremely sensitive to changes in pH or
ionic strength, which may arise as an artifact due to the addition of increasing amount of higher
ligand-concentration solution into the lower ligand-concentration solution, we have focused on
the 15N chemical shifts as the reporter for ligand binding. However, the data were processed in
multiple ways, including considering both 1H and 15N chemical shifts and exclusively 15N chemical
shifts, without any significant difference in results (see Supplemental Figures S4 and S5).
Residues were assigned according to published NMR assignments for -Syn (Cho et al. 2009) and
-Syn (Bertoncini et al. 2007). Titration curves, with N chemical shift changes plotted versus
ligand concentrations were fitted by a single binding model to obtain the dissociation constant of
the interaction. Specifically, the equation
was exploited, were L is the ligand concentration, P is the protein concentration, max is the
CSP at infinite ligand concentration and Kd is the dissociation constant.
Dopaminochrome aggregation assay
Dopaminochrome (DCH) was generated by incubation of DA with sodium periodate (NaIO4)) as
described (Ochs et al. 2005). Full length human -Syn and -Syn with a C-terminal 6 x His tag
were expressed in E.coli and purified by Ni-NTA chromatography. Eluted proteins were dialyzed
against MES/MOPS buffer (20mM MES, 20mM MOPS, 100mM NaCl, pH 5.8) using 10.000 MWCO
dialysis cassettes. Finally, protein concentration of the dialysed samples were adjusted to
5mg/ml (330M) using MES/MOPS buffer. 200 l aliquot fractions were stored at -80oC. For
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fibrillation, these protein samples were incubated for 7 days at 37oC and continuous shaking at
1000 rpm. DA and DCH were added to the reaction at equimolar concentrations to the
synucleins. N-Acetyl-L-Cysteine was added at a 3-fold molar excess. DA and DCH stock solutions
were prepared fresh, directly before the start of the experiment. For the thioflavin-T (Th-T)
assay, -Syn and -Syn samples were diluted to 4.5 μM in a solution of 20 μM Th-T in PBS. This
assay was conducted at pH 7.4 in order to allow for DA auto-oxidation during incubation. To
promote aggregation 0.001% (w/w) of preformed -Syn or -Syn fibrils were added. After 7 days
of incubation the fluorescence was measured at an excitation wavelength of 440 nm and
emission of 480 nm using a microplate reader (TECAN Spark). In order to separate between
soluble and fibrillized protein, samples were centrifuged at 100,000 x g for 30 min at room
temperature. Pellets, containing fibrils and higher molecular weight aggregates, were
resuspended in MES/MOPS buffer. For CD measurements, the proteins were diluted in distilled
water to a final concentration of 0.1mg/ml. CD spectra of -Syn and -Syn samples, both
monomeric and aggregated forms, were measured between 190 to 250nm wavelength, using a
0.1mm path quartz cuvette on a ChirascanTM CD Spectrometer.
Statistical analysis
As no human and animal subjects were used for experimentation, the study was not pre-
registered and randomized. The study was exploratory insofar as due to unknown effect sizes no
pre-determination of sample sizes other than that based on scientific experiences was
performed. No exclusion criteria were pre-determined. Experimentation and statistical
evaluation were carried out by different scientists, in a way that the evaluator received blinded
data sets. Experimental data were analysed in GraphPad Prism 8 for normal data distribution by
D´Agostino-Pearson´s omnibus K2 test. Should a single extreme value suggested non-normal data
distribution, this value was not excluded (see individual symbols in Figures 2 and 3), but the
statistics was still performed under assumption of Gaussian distribution of data, using ANOVA. In
addition, the non-parametric Kruska-Wallis test with Dunn´s correction for multiple comparisons
was performed, in order to confirm validity of the statistical analysis under the assumption that
data were not normally distributed. For all data analysed, ANOVA and Kruska-Wallis test
delivered identical statistical outcomes. As no data points were excluded from analysis, distinct
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test for outliers were not performed. Thus, as shown in the figures of this study, statistically
significant differences between multiple groups were evaluated by 1-way ANOVA with Dunnett’s
post hoc test for multiple comparisons, or, in case of pairwise comparisons, by unpaired, 2-tailed
Student´s T-test. Bar diagrams show mean ± standard deviation (SD), box plots are composed
according to Tukey´s (box showing Q1 to Q3 and the median, whiskers including values within
3/2 times the interquartile range of Q1 and Q3, and individual dots of outliers).
Statistical powers of all comparisons were analysed by G*Power3.1 (Faul et al. 2009) with the
following settings: test family = t-tests (2-tailed); statistical tests = difference of means between 2
independent groups; type of power analysis = post hoc; effect size d computed from means and
standard deviations of groups to compare; α error probability = 0.05; with given sample sizes the
power of the statistical assessment was computed as (1 –  error probability). A reasonable
statistical power of the respective statistical analysis was assumed at (1 –  error probability) >
0.8.
Results
Generation of a primary neuron cell culture system to study the dopamine-dependent neurotoxic
potential of -Syn
In order to generate neurons with a dopaminergic neurotransmitter phenotype and appropriate
controls, we aimed for cells that are identical in all aspects but differ only in their capacity to
synthesize or to take up DA. As a basis, we used cortical neurons isolated from embryonal rat
brains. These cultures survive for long-term due to astroglial support, develop an unstimulated
synchronized firing activity (Murphy et al. 1992) and can serve to study a wide variety of
physiologic effects related to the synucleins (Tolo et al. 2018). The genuine neurotransmitter
phenotype of these cells (85% are glutamatergic, 15% are GABAergic) was not erased, but
persisted while adding the dopaminergic neurotransmitter phenotype. All necessary
dopaminergic factors and the synucleins were expressed in these cultures by means of
recombinant AAV vectors under control of a strictly neuron-specific promoter (Fig 1 A-C).
Two different cultures of dopaminergic neurons were developed: intracellular dopamine
synthesis was enabled by expression of aromatic amino acid decarboxylase (AADC; which
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converts L-DOPA into DA) and vesicular monoamine transporter 2 (VMAT2; which transports DA
into synaptic vesicles), and initiated by addition of 3,4-dihydroxyphenylalanine (L-DOPA) into the
cell culture medium (Fig 1 D, E). Alternatively, take up and re-release of DA was enabled by
expression of the dopamine transporter (DAT; which re-uptakes secreted DA from the synaptic
cleft), VMAT2, and initiated by addition of DA into the cell culture supernatant (Fig 1 F, G).
Levels of DA and its relatively stable and non-cytotoxic metabolites, 3,4-dihydroxy-phenylacetic
acid (DOPAC) and homovanillic acid (HVA) that were achieved by the two strategies are shown in
Fig 1 E, G for cell cultures at 15 days of in vitro culture (div 15), both in cell lysates and in the cell
culture supernatant. At the same time, neuron numbers were counted, and the levels of DA,
DOPAC and HVA are presented as nanograms per 10.000 neurons, in order to allow for the same
scale for intra- and extracellular levels.
The DA synthesis capacity of our cultures relates to that of genuine A9 dopaminergic neurons
according to the following considerations: a level of 100 ng HVA / 10.000 cultured neurons
detected in supernatant of AADC/VMAT2 expressing neurons means that these cells have
synthesized and released at least 100 ng of DA, which is likely to be an underestimation due to
the unknown amounts of DA that became auto-oxidized. In the rat brain about 14.000
dopaminergic neurons project into the striatum, where 7-9 ng DA per 40 mg wet weight can be
detected (Cheng et al. 2018), corresponding to about 200-250 ng of DA / 10.000 neurons (i.e. 20
– 25 pg DA/neuron). Thus, we consider our dopaminergic neurons to be able to synthesize DA in
the same order of magnitude as genuine A9 neurons. Notably, brain synapses are much better
shielded from the environment as compared to the synapses of our 2D cultures, which lose most
of the synthesized DA into the cell culture supernatant due to endogenous electrical activity of
the cortical neurons. Detection of intracellular DOPAC demonstrated that a certain fraction of
the synthesized DA was also available for metabolism within the neuronal cytoplasm, most likely
representing the activity of neuronal monoamine oxidase (MAO) and aldehyde dehydrogenase
(ALDH), that catabolize DA either en route to synaptic vesicles, or degrade DA leaking from
synaptic vesicles (Meiser et al. 2013).
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Impact of a dopaminergic neurotransmitter phenotype on the neurodegenerative potential of -
Syn in primary neurons
Synuclein-induced neurodegeneration in presence or absence of DA and its metabolites was
studied by expression of either -Syn, -Syn, -Syn or EGFP in both types of dopaminergic neuron
cultures. Expression levels of the synucleins (Fig 1 C) were adjusted in a way that their expression
in absence of DA caused only minor (if any) neurodegeneration on top of the normal cell loss
occurring in primary cultures over time (Lesuisse & Martin 2002). We performed baseline cell
counts at div 11, in order to allow for generation of stable conditions in terms of neuronal
maturation, DA production, growth of astrocytes and establishment of endogenous network
activity. Cell counts to determine ongoing neurodegeneration were then performed at div 15 and
div 19, and surviving neuron numbers are presented as the percentage of cells compared to
baseline counts at div 11 (Fig 2 and 3).
Figure 2 shows the impact of a dopaminergic neurotransmitter phenotype on neurons expressing
EGFP, -Syn, -Syn, or -Syn for the condition in which DA is synthesized intracellularly (Fig. 2A).
Neurons that expressed only EGFP did not show any aggravation of neurodegeneration at div 15
or 19 due to the presence of DA (Fig. 2 B). Neurons that expressed -Syn showed a moderate
decline in surviving neuron numbers at div 15 when synthesizing DA, and a significant
degeneration due to presence of DA at div 19 (Fig. 2C). The impact of DA synthesis on survival of
-Syn expressing neurons took place with a somewhat faster pace (Fig. 2D): here, a significant
cell loss due to presence of DA was detected already at div 15, and at div 19 the majority of -Syn
expressing dopaminergic neurons had degenerated. -Syn expressing neurons were considered
as an additional control that might be better suited as compared to EGFP, as -Syn did not cause
evident neurotoxicity in previous studies (Taschenberger et al. 2013; Tolo et al. 2018). However,
although there was no significant cell loss detected in these neurons upon DA synthesis, a trend
towards reduced neuron counts was evident, suggesting that under our experimental conditions
even -Syn might tend to develop certain neurotoxic properties in presence of DA (Fig 2 E).
Figure 3 shows the impact of a dopaminergic neurotransmitter phenotype on neurons expressing
EGFP, -Syn, -Syn, or -Syn for the condition in which DA is absorbed from the extracellular
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space (Fig. 3A). Neurons that express only EGFP did not show any enhancement of
neurodegeneration due to up-take of DA from the cell culture medium (Fig. 3B). Neurons that
express -Syn did not show enhanced neurodegeneration due to DA up-take at div 15, but only
at div 19 (Fig. 3 C). Neurons expressing -Syn showed a faster progressing impact of DA on
neurodegeneration: we detected a significantly enhanced neuron loss due to DA already at div
15, which was even more pronounced at div 19 (Fig. 3D). In addition, -Syn expressing neurons
were the only cultures which demonstrated some neurodegeneration due to the sole presence
of DA within the cell culture medium, in absence of AADC and VMAT2 expression at div 19 (p =
0.04, power (1- err. prob.) = 0.96). If this effect is due to a low-level up-take of DA through
other transport systems than DAT remains unknown. Neurons expressing -Syn behaved
essentially as those expressing EGFP and did not present with any DA-induced aggravation of
neurodegeneration (Fig. 3 E).
Impact of the dopaminergic neurotransmitter phenotype on neurodegeneration induced by -
Syn in human iPSC-derived neurons
Modified primary neurons allowed to study the impact of DA on the neurotoxic potential of -
Syn in cells that were identical on the genomic level in verum and control conditions, but might
be criticized as not having a native dopaminergic neurotransmitter phenotype. Thus, we sought
to confirm our results in human iPSC-derived neurons that were differentiated into either a
dopaminergic (Fig 4 A) or a glutamatergic neurotransmitter phenotype (Fig 4 B). Dopaminergic
cultures generated in this paradigm produce DA levels detectable by HPLC with electrochemical
detection only when supplied with exogenous L-DOPA. However, in absence of exogenously
added L-DOPA the final metabolite HVA becomes detectable in the supernatant of these cultures
at late stages (i.e. > div 35; 1.05 ± 0.6 ng/104 cells), demonstrating that they produce low levels of
DA endogenously. It seems reasonable to speculate that this low-level DA production is due to
the self-limiting control that DA exerts on TH activity (Meiser et al. 2013).
We used these cultures without addition of exogenous L-DOPA, aiming to investigate if even very
low levels of DA synthesis would have detectable pathophysiological effects in combination with
the synucleins. Both types of neurons were transduced with AAV vectors that co-express -Syn,
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-Syn or -Syn with EGFP, or expressed EGFP alone. The number of EGFP-expressing neurons was
quantified at 20 days after transduction (dpt 20, corresponding to div 40) with synuclein-
expressing vectors, in order to allow for a similar time frame as used in primary neurons.
Neurodegeneration induced by both -Syn and -Syn was moderately, but significantly
aggravated in neurons of the dopaminergic neurotransmitter phenotype as compared to neurons
of the glutamatergic neurotransmitter phenotype (Fig. 4 C). However, the effect size, i.e. the
difference in neurodegeneration between glutamatergic and dopaminergic neurons, was less
pronounced as compared to modified rodent neurons with and without DA production,
presumably due to the lower levels of DA generated by human neurons. Neurons of both
neurotransmitter phenotypes showed no neurodegeneration after expression of -Syn or EGFP.
Thus, results obtained in human-derived neurons in principle support our findings from modified
primary rat neurons, that a dopaminergic neurotransmitter phenotype promotes the
neurodegenerative potential of -Syn, even if levels of DA synthesis are low.
Impact of -Syn on synchronized neuronal network activity in absence and presence of DA
Besides its neurodegenerative properties, -Syn has been shown to attenuate spontaneous
synchronized network activity when overexpressed in aged cultured cortical neurons (Tolo et al.
2018). Thus, we now evaluated if -Syn overexpression would also cause this functional defect,
and if a dopaminergic neurotransmitter phenotype would have any aggravating effect in this
regard. We compared spontaneous synchronized activity in primary cortical neurons that
overexpressed -Syn, -Syn or -Syn, either in unmodified neurons or in neurons provided with a
dopaminergic neurotransmitter phenotype by AADC and VMAT2 expression and supplied with L-
DOPA. All cultures also expressed the genetically encoded calcium sensor GCaMP6 to record
network activity, and in addition the anti-apoptotic factor Bcl-Xl, which had been shown to
protect primary neurons from neurodegeneration by preventing synuclein-induced damage of
the outer mitochondrial membrane in absence of DA (Tolo et al. 2018) (Fig 5 A). If Bcl-Xl would
prevent neurodegeneration in presence of DA, this would be indicative for DA enhancing
mitochondrial outer membrane damage.
In absence of DA -Syn expressing cultures showed a reduced spontaneous burst frequency (Fig
5 B) and a reduced number of neurons participating in this activity (Fig 5 D), as compared to
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controls expressing -Syn, which behave equal to native neurons (Tolo et al. 2018). Presence of
DA significantly enhanced the synchronized burst frequency of -Syn expressing cultures
(without DA: 0.5 ± 0.5 bursts/min, with DA: 1.4 ± 0.9 bursts/min; mean ± SD, p = 0.006; unpaired,
2-tailed t-test) (Fig 5 B versus 5 C). In contrast to -Syn expressing cultures, -Syn expressing
cultures did not show significantly reduced network activity as compared to controls in absence
of DA (Fig 5B), and also no diminishment of neurons contributing to this activity (Fig 5 D). Again,
presence of DA rather enhanced than reduced neuronal activity in -Syn expressing cultures (Fig
5 C, E).
Protection of mitochondrial outer membrane integrity prevents the neuropathological impact of
-Syn and DA
No confounding effect on network activity through neurodegeneration was detectable in this
experiment, as Bcl-Xl expression kept neuron numbers constant in both absence (Fig 5 F) and
presence of DA (Fig 5 G).Thus, our current data show that Bcl-Xl was able to provide the same full
neuroprotection against -Syn and -Syn in DA producing neurons as compared to non-DA
producing neurons, suggesting that DA promotes the neurotoxic potentials of both -Syn and -
Syn by a mechanism that depends at least to a significant extent on damaging the integrity of the
outer mitochondrial membrane.
Interaction of -Syn with DA
Given that DA promoted the neurotoxic potential of -Syn, we next asked the question if this
could be due to a direct interaction of DA with -Syn. Therefore, we used NMR to investigate the
interaction of -Syn with DA and its metabolite DOPAL. This metabolite was chosen since i)
DOPAL was demonstrated to be directly neurotoxic to nigral dopaminergic neurons (Burke et al.
2008) and ii) DOPAL is produced by MAO at the outer mitochondrial membrane (Meiser et al.
2013), a sub-cellular location with high vulnerability for synuclein toxicity (Tolo et al. 2018). For
comparison, interaction of -Syn with DA and DOPAL was investigated under identical
conditions. Experiments were carried out in solution containing a strong anti-oxidant in order to
prevent auto-oxidation of DA or DOPAL. Experimental conditions were chosen to maintain the
synucleins within a monomeric state, which was proven by absence of chemical shift
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perturbation (CSP) intensity decline over the time course of the measurements, indicating that
no monomers have been consumed into oligomers or aggregates. The intensity of the protein
NMR peaks was monitored throughout the experiments to rule out any time dependent
depletion of monomer species.
The dissociation constant (kD) for DA and -Syn was determined to be 866 ± 98 µM, as compared
to 96 ± 75 µM for DA and -Syn (Fig 6 A, B). For the interaction of DOPAL with the synucleins we
determined kDs of 170 ± 37 µM for -Syn, and > 3 mM for -Syn (Fig. 6 C, D) These data
demonstrate that DA and DOPAL show a highly divergent interaction potential with either -Syn
or -Syn, in that DA has a 10-fold higher affinity for -Syn, while DOPAL has a more than 10-fold
higher affinity for -Syn. While none of these kD values suggest an ultra-high affinity between
synucleins and DA or metabolites of DA, they are still in a physiological relevant range, given the
high intracellular concentrations of synucleins and DA (see Discussion).
For -Syn we found that DA interacted predominately with all amino acids in the C-terminus of
the protein (Fig 6 E), including but clearly extending the 125YEMPS129 motif considered important
for the DA-mediated inhibition of -Syn fibrillation (Herrera et al. 2008). For -Syn a similar
tendency was obtained, although with an about 3-fold lower affinity for C-terminal amino acid
residues (Fig 6 F). DOPAL interacted specifically with two amino acids in the N-terminus of -Syn
(A17 and T27), but its interaction with the corresponding amino acids of -Syn was substantially
weaker (Fig 6 G). Since residue 27 is A in -Syn but T in -Syn, this finding suggests that individual
amino acids that are not conserved between both synucleins may indeed alter the affinity for
ligands such as DOPAL. Furthermore, DOPAL induced the biggest chemical shift change on Q127
in the C-terminus of -Syn (Fig 6 H). Unfortunately, the spectrum of Q134, the corresponding
amino acid of -Syn, was consistently overlapped, making it impossible to assign a CSP value to
this residue.
Fibrillation of -Syn is inhibited by the oxidized DA metabolite dopaminochrome (DCH)
Finally, we investigated if DA and/or oxidized metabolites could modify the aggregation
properties of -Syn. We incubated -Syn under conditions allowing it to form fibrils in vitro, with
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either DA, DA plus the antioxidant N-acetyl-cysteine (Nac) or with the oxidized metabolite
dopaminochrome (DCH), and recorded fibril formation by the thioflavin-T (Th-T) assay. DCH was
chosen as an oxidized metabolite because it is able to inhibit fibrillation of -Syn (Norris et al.
2005) and is directly neurotoxic to nigral dopaminergic neurons (Touchette et al. 2016). Our data
demonstrate that incubation with DA partially inhibited fibrillation of -Syn, most likely due to
the partial oxidation of DA during the incubation process. When this oxidation of DA was
prevented by Nac, -Syn fibrillation was promoted as compared to incubation with DA alone. In
contrast, incubation of -Syn with DCH almost completely prevented fibrillation of -Syn (Fig 7
A). These findings were almost identical to those obtained for incubation of -Syn with DA, DA +
Nac, or DCH (Fig 7 B). Thus, neurotoxic metabolites of DA can interact with -Syn in the same
principal way as with -Syn, in that they prevent formation of protein fibrils and potentially
enhance the concentration of toxic oligomers. At the endpoint of the aggregation assay fibrillar
components were pelleted by centrifugation, and were analyzed for secondary structure
elements alongside the soluble protein fractions by circular dichroism (CD). These analyses
confirmed that the soluble protein fractions under all conditions present with a predominant
random coiled secondary structure, as demonstrated by the minimum close to 200 nm in molar
residue ellipticity (MRE) (Fig 7 C - F, solid lines). In contrast, the pellet fraction contained
predominately -sheet rich secondary structures (MRE maximum at 200 nm, MRE minimum at
225 nm; dashed lines in Fig 7 C - E), except for samples derived from incubation of -Syn with
DCH, which did not contain any proteinaceous components with defined secondary structure
(dashed line in Fig. 7 F). These results obtained by end-point analysis of the aggregation assay
were confirmed by analysis of early fibrillation kinetics, analysis of protein species in pellet and
supernatant of the sedimentation assay and by electron microscopy (Suppl. Fig. S7).
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Discussion
Our data demonstrate that -Syn can substitute for -Syn in an aspect that is considered central
for the etiology of PD, i.e. the selective vulnerability of dopaminergic neurons for
neurodegeneration. Several studies have already suggested that DA and -Syn act cooperatively
as culprits in models of PD (Mor et al. 2019; Alvarez-Fischer et al. 2008; Mosharov et al. 2009; Xu
et al. 2002; Ulusoy et al. 2012; Mor et al. 2017), but no such evidence was available for -Syn up
to now. Our recent finding that -Syn can aggregate in dopaminergic neurons of the rat
substantia nigra in vivo, and causes neurodegeneration in these neurons to similar extent as
compared to -Syn, already indicated that -Syn has a neuropathological potential very similar to
that of -Syn (Taschenberger et al. 2013). However, this resemblance was also found in cultured
non-dopaminergic neurons (Taschenberger et al. 2013; Tolo et al. 2018), and thus the relative
impact of the dopaminergic neurotransmitter phenotype was elusive so far. Our current results
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obtained in three independent cell culture models now clearly demonstrate that -Syn and -Syn
possess an almost identical neuropathological profile in terms of potentiation of
neurodegeneration through a dopaminergic neurotransmitter phenotype. The fact that -Syn
has not yet been detected in Lewy bodies (Spillantini et al. 1997; Spillantini & Goedert 2018;
Shults 2006) does not contradict an important contribution of -Syn to etiology of PD, given that
not fibrils, but oligomers are considered to be the major neurotoxic entities in synucleinopathies
(Rockenstein et al. 2014; Karpinar et al. 2009). Thus, while substantial amounts of -Syn might
be sequestered in Lewy bodies to render them non-toxic, it might be that -Syn persists within
the cytoplasm as more or less soluble oligomers, which might cause e.g. mitochondrial lesions as
described (Tolo et al. 2018). Therefore, future modelling of synucleinopathies should consider
both -Syn and -Syn as potential culprits, and probably neuropathological impacts of one
synuclein cannot be considered independently from the other synuclein.
With respect to the physical interaction of DA and its oxidized, neurotoxic metabolites with -Syn
we describe both commonalities and differences to -Syn: the inhibition of -Syn`s aggregation
by DCH demonstrates that the functional interaction of oxidized DA metabolites with -Syn
follows the same principal pathway as for -Syn. In solution and in absence of seeds, -Syn forms
fibrils only at slightly acidic pH of 5.8, which can be taken representative for the endocytic and
secretory compartments of cells (late endosomes, Golgi, secretory and synaptic vesicles)
(Demaurex 2002). A slightly acidic environment can also be found in the intermembrane space of
mitochondria, where proton traps inside cristae microdomains might generate a pH significantly
below 7 even under normal physiological conditions (Santo-Domingo & Demaurex 2012). The
fact that -Syn forms proteinase K-resistant fibrils in dopaminergic neurons in vivo illustrates that
it can also aggregate in a cellular environment (Taschenberger et al. 2013). Thus, our data show
that the aggregation state of -Syn can be modified by oxidized metabolites of DA, resulting in
formation of potentially more neurotoxic oligomeric species. However, the precise nature of the
oligomeric/aggregates species of -Syn generated under influence of DA in a cellular context
remain to be evaluated.
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Differences between interactions of the two synucleins with DA and metabolites of DA were
evident as well. The kds determined for interaction of -Syn with DA and DOPAL vary by almost
one order of magnitude from those determined for -Syn and DA/DOPAL. Interestingly, -Syn
has a much higher affinity for DOPAL and a much lower affinity for DA as compared to -Syn. It
remains to be determined if further oxidized metabolites of DA such as dopamine-o-quinone
(Asanuma et al. 2003), 5,6-Dihydroxyindole (Pham et al. 2009), Indole-5,6-quinone (Bisaglia et al.
2007), or salsolinol (do Carmo-Goncalves et al. 2018), are also interacting with -Syn and -Syn
with significantly different affinities. If this would be the case, then not only the levels of oxidized
metabolites of DA and of the synucleins may be important for the etiology of synucleinopathies,
but also cellular conditions which favor generation of the one over the other metabolite of DA.
Dissociation constants for the synucleins and DA/DOPAL were found to be in the higher
micromolar range, and thus comparable to kDs that were found for the interaction of -Syn with
metal ions like copper (Miotto et al. 2014). These kDs are orders of magnitude higher than those
of high affinity interactions with immediate biological relevance, for example the binding of
steroids to plasma carrier proteins or interactions of pro- and anti-apoptotic proteins, with kDs
that are in the range of 1 - 10 nM (Zheng et al. 2016). None the less, given the high
concentrations of DA within dopaminergic neurons and the high levels of -Syn and -Syn
especially at synaptic sites, biologically relevant interactions are still very likely. Assuming that
about 50% of a typical neuronal volume of 6 pL (Hosseini-Sharifabad & Nyengaard 2007; Healy-
Stoffel et al. 2012; Howard et al. 1993) is available for cytoplasmic distribution of DA and
metabolites, 20 pg DA/neuron corresponds to a concentration of 43 mM. The majority of DA is
sequestered within synaptic vesicles, reaching concentrations up to 300 mM (Pothos et al. 1998).
However, only 1% of the intracellular DA that would not be sequestered in vesicles but be
available for interaction with synucleins, still represents a cytoplasmic concentration of about
400 µM. For levels of the synucleins, we consider cellular concentrations of about 300 µM after
overexpression and about 60 µM for endogenous levels. This was deduced from western blots as
shown in Fig 1 C, where the protein equivalent of 4000-5000 neurons, corresponding to a
cytoplasmic volume of 12nL, was loaded alongside known amounts of recombinant synucleins.
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These calculations suggest that kDs in the higher micromolar range for the interaction of DA or
metabolites of DA with the synucleins are clearly physiologically relevant.
Alternatively, or probably additionally, the aggravated pace of neurodegeneration caused by -
Syn and -Syn in the dopaminergic neurotransmitter phenotype might depend on damage of
neurons through the enhanced oxidative burden due to DA catabolism, which generates
considerable amounts of reactive oxygen species (ROS). Primarily, hydrogen peroxide is
produced as a by-product of MAO-mediated oxidative deamination of DA at the outer
mitochondrial membrane (OMM) (Meiser et al. 2013). Recent studies in primary neurons have
revealed that -Syn overabundance amplifies the oxidative burden within mitochondria, and that
degeneration of -Syn and -Syn expressing neurons eventually proceeds due to damage of the
OMM (Tolo et al. 2018). Considering DA metabolism as an additional oxidative stressor that
might attack integrity of mitochondrial membrane components, it seems likely that this additive
attack results in faster or more pronounced neuronal death, probably even independent from
synuclein oligomerization or aggregation. The fact that Bcl-Xl, the major watchdog of OMM
integrity, was able to prevent neurodegeneration caused by the combined activity of -Syn and
-Syn expression and dopaminergic metabolism, argues in favor of this line of interpretation.
Nonetheless, ROS production and synuclein oligomerization may both converge their detrimental
impact on the outer mitochondrial membrane, thereby suggesting a reasonable mechanism of
how the dopaminergic neurotransmitter phenotype might aggravate the neurotoxic potential of
-Syn.
Conclusion
In conclusion, we have demonstrated that a dopaminergic neurotransmitter phenotype robustly
promotes the neurodegenerative potential of -Syn, supporting the hypothesis that -Syn can
act as a neurodegenerative culprit specifically in dopaminergic neurons. We also show that -Syn
physically interacts with DA and neurotoxic metabolites of DA, which can affect the aggregation
propensities of -Syn. Thus, it seems warranted to reconsider the role of -Syn in the etiology of
synucleinopathies like PD and DLB, at least when it comes to the influence of -Syn on
degeneration of dopaminergic neurons.
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--Human subjects --
Involves human subjects:
If yes: Informed consent & ethics approval achieved:
=> if yes, please ensure that the info "Informed consent was achieved for all subjects, and the
experiments were approved by the local ethics committee." is included in the Methods.
ARRIVE guidelines have been followed:
Yes
=> if it is a Review or Editorial, skip complete sentence => if No, include a statement in the "Conflict of
interest disclosure" section: "ARRIVE guidelines were not followed for the following reason:
"
(edit phrasing to form a complete sentence as necessary).
=> if Yes, insert in the "Conflict of interest disclosure" section:
"All experiments were conducted in compliance with the ARRIVE guidelines." unless it is a Review
or Editorial
Acknowledgements:
We are grateful for the excellent technical support of Sonja Heyrodt and Monika Zebski, to
Supriya Pratihar for help with NMR, and to Filippo Favretto for help with CD. We thank Cristiane
Klein (University of Lübeck, Germany) for the CT-03 iPSC line and Luigi Bubacco (University of
Padova) for providing DOPAL.
Funding: This work was supported by the German Research Council funded Center for Nanoscale
Microscopy and Physiology of the Brain (CNMPB), the Max Planck Society and the German
Research Council under Germany´s Excellence Initiative Strategy - EXC 2067/1-390729940.
Competing interests: The authors declare that they have no competing interests. Mathias Bähr
serves as an Editor for the Journal of Neurochemistry.
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Data statement: All data generated or analyzed during this study are included in this published
article.
Open Science Badges
This article has received a badge for *Open Materials* because it provided all relevant
information to reproduce the study in the manuscript. More information about the Open Science
badges can be found at https://cos.io/our-services/open-science-badges/.
Author contributions (according to CRediT taxonomy):
AR: investigation, methodology, formal analysis, visualization
KL: investigation, methodology, formal analysis, visualization
SG: investigation, methodology, formal analysis, visualization
SUM: investigation, methodology, formal analysis, visualization
KSC: investigation, methodology, formal analysis, visualization
DV: Investigation, formal analysis, visualization
SB: resources
CG: resources, conceptualization, supervision, validation, funding acquisition
MB: resources, funding acquisition, supervision
SK: conceptualization, supervision, validation, funding acquisition, visualization, writing
All authors discussed and approved the manuscript.
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