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Title: Harmane-induced selective dopaminergic neurotoxicity in C. elegans
Running title: Harmane-induced dopaminergic neurotoxicity
Authors: Shreesh Raj Sammi*,†, Zeynep Sena Agim*,†, Jason R. Cannon*,†,#
Affiliations: *School of Health Sciences, Purdue University, West Lafayette, IN 47907
†
Purdue Institute for Integrative Neurosciences, Purdue University, West
Lafayette, IN 47907
Author E-mail addresses:
Shreesh Raj Sammi: [email protected]
Zeynep Sena Agim: [email protected]
Jason R. Cannon: [email protected]
#Corresponding Author
Jason R. Cannon, Ph.D.
Associate professor of toxicology
550 Stadium Mall Drive
West Lafayette, IN 47907-2051
Phone: (765) 494-0794; Fax: (765) 496-1377
© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology.
All rights reserved. For permissions, please email: [email protected]
Abstract
Parkinson’s disease (PD) is a debilitating neurodegenerative disease. While numerous exposures
have been linked to PD etiology, causative factors for most cases remain largely unknown.
Emerging data on the neurotoxicity of heterocyclic amines suggest that this class of compounds
should be examined for relevance to PD. Here, using C. elegans as a model system, we tested
whether harmane exposure produced selective toxicity to dopamine neurons that is potentially
relevant to PD. Harmane is a known tremorigenic β-carboline (a type of heterocyclic amine)
found in cooked meat, roasted coffee beans, and tobacco. Thus, this compound represents a
potentially important exposure. In the nematode model, we observed dopaminergic neurons to be
selectively vulnerable, showing significant loss in terms of structure and function at lower doses
than other neuronal populations. In examining mechanisms of toxicity, we observed significant
harmane-induced decreases in mitochondrial viability and increased reactive oxygen species
(ROS) levels. Blocking transport through the dopamine transporter (DAT) was not
neuroprotective, suggesting that harmane is unlikely to enter the cell through DAT. However, a
mitochondrial complex I activator did partially ameliorate neurodegeneration. Further,
mitochondrial complex I activator treatment reduced harmane-induced dopamine depletion,
measured by the 1-nonanol assay. In summary, we have shown that harmane exposure in C.
elegans produces selective dopaminergic neurotoxicity that may bear relevance to PD, and that
neurotoxicity may be mediated through mitochondrial mechanisms.
Keywords: Parkinson’s disease, harmane, dopamine, neurodegeneration, C.elegans
Introduction
Parkinson’s disease (PD) is a debilitating neurodegenerative disease. The pathological hallmarks
are the loss of nigral neurons and aggregation of α-synuclein in inclusions known as Lewy
Bodies in surviving dopaminergic neurons (Spillantini et al., 1997). While numerous genetic
links have been identified and a number of environmental exposures have been linked to
etiology, the majority of the PD cases are sporadic(Cannon and Greenamyre, 2011). Given that
available treatment options do not slow disease progression, it is critical to identify and reduce
potentially causative exposures. Dietary exposures have, to date, received far less attention than
other classes of compounds, such as pesticides. Dietary compounds represent a potentially very
common and modifiable exposure. Should dietary factors be identified that contribute to disease
etiology, a reduction in intake could potentially reduce PD incidence (Agim and Cannon, 2015).
To that end, emerging data suggest that heterocyclic amines (HCAs) should be examined as
possible dopaminergic neurotoxins (Griggs et al., 2014; Louis et al., 2014). HCAs are found in
many dietary components, especially in charred meat. Interestingly, elimination of dietary red
meat has been reported to improve motor function in PD patients (Coimbra and Junqueira, 2003).
Thus, this class of compounds deserves attention with respect to potential neurotoxicity.
To date, some HCAs have been examined for potential relevance to PD. For example, we
found that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is selectively neurotoxic to
dopaminergic neurons in primary rat midbrain cultures (Griggs et al., 2014). Further, 3-amino1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole
(Trp-2) were found to interfere with dopamine metabolism (Kojima et al., 1990; Maruyama et
al., 1994).
Here, we chose to examine a specific HCA, 1-methyl-9H-pyrido[3,4-b]indole (harmane).
Harmane is a HCA belonging to the β-carboline subclass. It is a known tremorigen found in
cooked meat, fish, coffee and tobacco (Skog et al., 1997; Skog et al., 1998). Our focus on
harmane was informed by published data that higher plasma harmane levels have been found in
PD patients compared to controls (Louis et al., 2014). Prior to this study, harmane had primarily
been investigated for a role in essential tremor. For example, those suffering from essential
tremor are found to have elevated levels of harmane in blood (Louis et al., 2002). Notably,
essential tremor patients have an increased risk of PD (Benito-Leon et al., 2009). Thus, data
from PD patients and links between PD and essential tremor prompted our study.
Increased human exposure to harmane could be attributed to abundance in diet. Due to its
high lipid solubility, harmane tends to accumulate in brain tissue (Zetler et al., 1972). Ostergren
et al. also showed that harmane has high affinity binding to neuromelanin, that is abundant in
substantia nigra in humans, possibly indicating that harmane may accumulate in nigral dopamine
neurons (Ostergren et al., 2004). Taken together, there is a compelling dataset that suggests
harmane-induced neurotoxicity should be evaluated in vivo.
C. elegans is a transparent, nonpathogenic soil nematode. A major advantage of this
model system is that numerous strains are available that express fluorescent reporter systems in
specific cell populations, where cell damage and loss can easily be assessed. Thus, C. elegans as
a model system offers an outstanding opportunity to examine whether neurotoxicity is selective
to specific neuronal populations across a number of doses. Using simple nematode model, we
have identified harmane as a potential dietary toxicant responsible for dopaminergic cell loss
through mechanism coinciding with PD pathology.
Materials and Methods
Culture and Maintenance of Strains: C. elegans strains, Bristol N2, BZ555 (egIs1 [dat1p::GFP]), MT15620 (cat-2(n4547)), UA57(baIs4 [dat-1p::GFP + dat-1p::CAT-2]), CZ1632
(juIs76 [unc-25p::GFP + lin-15(+)] II), GR1366 (mgIs42 [tph-1::GFP + rol-6(su1006)]), LX929
(vsIs48 [unc-17::GFP]) and Escherichia coli OP50, were procured from Caenorhabditis Genetics
Centre, (University of Minnesota, MN, USA), grown on Nematode growth medium (NGM) and
cultured at 22˚C. A synchronized population of worms was obtained by sodium hypochlorite
treatment.
Treatment of Worms: L1 staged worms were treated with different concentrations of harmane
(Sigma Aldrich, 103276) in liquid culture as described by Pu and Le (2008) supplemented with
E. coli OP50 (Pu and Le, 2008). 100 mM stocks of harmane were prepared in DMSO and diluted
further to doses ranging from 100 to 1000 µM. The dose range was based on established C.
elegans PD models [e.g. MPP+(Pu and Le, 2008)]. Fresh stocks less than 7 days old were used
for each experiment. 70 L1 staged worms were added to 200 µl suspension in 24-well plates and
incubated at 22˚C. Since harmane is a hazardous substance, care was taken to avoid skin contact.
Given the paucity of data on harmane in in vivo models, we examined the literature on
neurotoxicant PD models in C. elegans, in choosing an initial dose range, we focused more on
established MPP+ models vs. 6-OHDA, because the reported log P of MPP+ (2.7) is far closer to
that of harmane (3.6) vs. 6-OHDA (0.2) (PubChem, 2017a; PubChem, 2017b; PubChem,
2017c). In worms, it is well known that far higher doses are typically required than in cell culture
due to the nematode cuticle which exhibits strong barrier between worm and surroundings (Page
and Johnstone, 2007). While the cuticle is likely less of a barrier for nonpolar compounds, it is
worth noting that the examination of lethality for 21 compounds showed that the majority of
compounds tested produced LD50s with little variability. Those that are pH sensitive or highly
polar did exhibit marked increases in LD50s (Li et al., 2013). In considering dose, we also
reviewed suggestions for acute (12-24 hour) exposure ranges in C. elegans. Suggested dose
ranges were informed by correlation analysis between nematode, mouse, and rat data. Further,
the suggested dose ranges were based on globally harmonized system of toxicity classification
(Li et al., 2013). For harmane, the calculated suggested molarity dose ranges for lethality in C.
elegans would be ~1.2 – 250 mM (GHS, 2007; Li et al., 2013; Sigg et al., 1964). Given that we
were interested in neurotoxicity, we tested exposures at far lower doses.
Based on pilot studies and data included here, mechanistic studies were conducted in two
sets: one high dose (650 µM harmane) for 48 hours and one low dose (500 µM harmane) for 72
hours. The rationale for 2 doses was to determine if potentially protective regimens would be
effective at different doses and time-points. To determine whether mechanistic conclusions were
consistent across exposure and dose, shorter, higher dose studies were also performed (48 hours
at 650 µM).
Several chemical treatments were utilized to test potential mechanisms of toxicity and
protection. We aimed to determine whether the use mitochondrial activators such as D, L 3hydroxy butyric acid (HBA), N-acetyl-L-cysteine, and riboflavin could prevent harmane-induced
alterations in mitochondrial viability. While HBA activates complex I via complex II (Tieu et al.,
2003), riboflavin activates complex I cooperatively with complex IV (Grad and Lemire, 2006).
In contrast, NAC, a well-known antioxidant is also generalized activator of mitochondrial
complex I, IV and V (Cocco et al., 2005; Kamboj and Sandhir, 2011; Miquel et al., 1995;
Soiferman et al., 2014). For treatment with HBA, a 5M stock of sodium salt of HBA (Acros
organics, 150834), dissolved in sterile distilled water was prepared. The worms were subjected to
different doses ranging from 50 – 200 mM (for both 48 and 72-hour time-points) treatment in
liquid culture. The worms were subjected to different doses ranging from 20 to 80 µM treatment
in liquid culture. For treatment with riboflavin (RB) (Acros organics, 132350250), a 100 µg/mL
stock was prepared in 10% DMSO. The worms were subjected to doses within range 1, 2 and 3
µg/mL (Grad and Lemire, 2006). For treatment with N-acetyl L-Cysteine (NAC) (Acros
organics, 160280250), a 500 mM stock was prepared in sterile distilled water. The worms were
subjected to doses within range 5, 10 and 15 mM (Oh et al., 2015). Similarly, for treatment with
bupropion HCl (Alfa aesar, J61105), 1 mM stock was prepared in sterile distilled water, with
testing at doses of 20 - 80 µm (Felton and Johnson, 2014).
Neurodegeneration Assay: In order to determine the effect of harmane on different classes of
neurons, L1 worms expressing green fluorescence protein (GFP) in dopaminergic, serotonergic,
Gamma-amino butyric acid (GABA)ergic and cholinergic neurons were subjected to different
doses of harmane (100 to 500 µM) for 48 hours at 22ºC and evaluated using morphological
markers of neurodegeneration that have been repeatedly utilized in nematode models (Alexander
et al., 2014). Worms exhibiting neuropathological alterations such as loss, breakage in dendrites
and loss of soma were considered as affected and percentage of affected worms was calculated.
For detailed studies in dopaminergic neurons, neurodegeneration was characterized and
quantified as described by Yao et al., 2010 (Yao et al., 2010). Briefly, treated worms were
washed three times using M9 buffer, anesthetized using 100 mM sodium azide and visualized
using the fluorescence microscope (Olympus BX-53). The number of neurons were counted for
minimum of 20 worms (eight dopamine neurons per worm) per group. The percentage of
morphologically intact neurons was calculated and plotted against its respective neurons type in
control. Care was taken to keep the worm number constant and fresh stocks (no older than one
week) of harmane were used for all the studies.
Dihydroethidium Staining for ROS: Dihydroethidium (DHE) staining for reactive oxygen species
(ROS; primarily as a detector of superoxide), was performed as described by Chikka et al., 2016,
with slight modifications (Chikka et al., 2016). Briefly, worms were washed three times with M9
buffer followed by two washes with phosphate buffered saline (PBS). Approximately 50 worms
in 100 µl of PBS were mixed with equal amount of 12 µM DHE (Millipore, 309800) dissolved in
DMSO (final concentration = 0.01%). After 40 minutes of incubation in dark, worms were
washed with PBS, anesthetized using 100 mM sodium azide and imaged using fluorescence
microscope (Olympus BX-53). Approximately 20 animals per group were analyzed semiquantitatively using Image J, excluding the gut region from the region of interest (ROI) so as to
eliminate the nonspecific fluorescence.
Mitochondria staining: MitoTracker staining was performed according to the manufacturer
protocol with slight modifications. Briefly, MitoTracker Red CM-H2XRos (Life technologies,
7513) was mixed with E. coli OP50 before seeding it to the NGM plates to check the healthy
mitochondrial staining. MitoTracker Red stock solution was prepared by dissolving 50 µg
MitoTracker Red CM-H2XRos in 100 µl of DMSO. A working concentration of 4.72 µM
MitoTracker Red was fed to the worms by mixing it with E. coli OP50. The synchronous
embryos were transferred onto the MitoTracker Red containing plates and grown for 48 hours at
22ºC. Worms were washed off using M9 buffer and kept in OP50 solution for 30 minutes and
again washed three times with M9 buffer. Worms were anesthetized using 100 mM sodium azide
and observed using fluorescence microscope (Olympus BX-53).
Nonanol Repulsion Assay: To indirectly estimate changes in dopamine levels, a dopaminedependent assay that measures 1-nonanol based repulsive behavior (Bargmann et al., 1993) was
performed, which is an established technique in this model (Kaur et al., 2012). Dopamine levels
are related to motivation, recognition, reward, memory, adaptation, hormonal regulation, and
motor control in worms (Felton and Johnson, 2014) . Any changes in dopamine levels alters the
worm behavior towards attractants and repellents (Ward, 1973). Worms with normal levels of
dopamine exhibit optimum levels of repulsive behavior towards 1-nonanol, while worms with
decreased dopamine levels exhibit extended time to exhibit the repulsive; increases in dopamine,
correspondingly decreases repulsion time (Kaur et al., 2012; Srivastava et al., 2017). The
nonanol repulsion assay for indirect quantification of dopamine levels was conducted as
previously described (Kaur et al., 2012). Briefly, treated worms were washed three times with
M9 buffer. Worms were placed on NGM plates. The poking lash dipped in 1-nonanol (Acros
Organic, AC157471000) was placed near the head region of the worms, while taking care not to
touch the worms. Time taken for the worms to exhibit the repulsive behavior was calculated
using a stop watch. Any worms coming in contact with the poking lash or 1-nonnaol were not
counted as a part of study. To further validate the relevance of the assay endpoint to dopamine
levels, we also exposed worms to the DAT inhibitor bupropion HCl and performed the assay in
worms overexpressing cat-2 (encodes tyrosine hydroxylase) and cat-2 mutants.
Thrashing Assay: The thrashing assay examines motility and locomotion defects in worms.
Thrashing assay was performed as described by Lee et al., 2008, with slight modifications (Lee
et al., 2008). Briefly, worms from the treatment plates were washed three times using M9 buffer.
The worms were then transferred to a drop of M9 buffer. After allowing adaptation for one
minute, the worms were scored for body bends for 30 seconds, where one complete sinusoidal
bend was scored as one movement.
Statistical Analysis: Statistical analysis was performed using GraphPad PRISM, Version 7.02
(1992 – 2016 GraphPad Software, Inc.). Each experiment was repeated at least three times and
normalized to untreated groups. Analysis of variance (ANOVA), followed by Tukey’s or
Dunnett’s (where comparison was only to control) post hoc tests were utilized. For all
experiments, p<0.05 was deemed to be statistically significant.
Results
Dopaminergic neurons exhibit enhanced vulnerability to harmane.
In order to determine the specific neuronal populations were selectively vulnerable to harmane,
we quantified the effect of different doses of harmane (100 - 500 µM) in worms expressing GFP
specific to dopaminergic, serotonergic, GABAergic and cholinergic neurons (BZ555, GR1366,
CZ1632 and LX929, respectively) after 48 hours of exposure. We observed that dopaminergic
neurons
were
most
vulnerable
to
harmane-induced
neurodegeneration;
exhibiting
neurodegeneration at doses as low as 100 µM harmane, where other neuronal subtypes showed
intact morphology (Figure 1). In contrast, higher doses were required to produce statistically
significant
neurodegeneration
in
serotonergic,
GABAergic
and
cholinergic
neurons.
Dopaminergic neurons exhibited a significant decrease in percentage of worms with intact
neuronal morphology at 100 µM (77.50 ± 1.11, mean ± SEM, p<0.01), 250 µM (25 ± 14.43,
p<0.001) and 500 µM (0 ± 0.00, p<0.001; n = 6 repeats/group) (Figure 1A,B). Serotonergic
neurons exhibited a significant decrease in the percentage of worms with intact neuronal
morphology at 250 µM (68.33 ± 1.66, p<0.001) and 500 µM (0 ± 0.00, p<0.001) (Figure 1C,D)
(n = 3 repeats/group). Damage to GABAergic neurons was primarily confined at inter-neuronal
commissures; where at neurodegeneration (as percentage of animals affected) was evident at 250
µM (76.66 ± 3.33, p<0.05) and 500 µM (15 ± 17.63, p<0.001) (Figure 1E,F) (n = 3
repeats/group). Cholinergic neurons, however, were found to be least susceptible to harmaneinduced neurodegeneration, where neurodegeneration was evident only at 500 µM (10 ± 5.77,
p<0.001) (Figure 1G,H) (n = 3 repeats/group). Calculated IC50s are as follows: dopaminergic
neurons (115 µM); GABAergic neurons (290 µM); serotonergic neurons (389 µM); cholinergic
neurons (492 µM). Here, the ‘inhibition’ refers to inhibiting normal neuronal morphology across
the total population for a specific neuronal population. Thus, sensitivity to harmane-induced
neurodegeneration was deemed to be dopaminergic > GABAergic ~ serotoninergic >
cholinergic. The distinction between serotonergic and GABAergic sensitivity was dependent on
whether an individual dose was examined or the calculated IC50.
Harmane induces dose dependent neurodegeneration of dopaminergic neurons.
C.elegans (hermaphrodite) possesses eight dopaminergic neurons. Of these eight neurons, there
are three dopaminergic subpopulations: four cephalic sensilla (CEP), two anterior deirid (ADE)
in the head region and two posterior deirid (PDE) in the tail region (Figure 2A). After
determining increased sensitivity of dopaminergic neurons to harmane in worms expressing GFP
tagged to dopamine transporter, we studied the effects of different doses of harmane (100 to
1000 µM) in total dopaminergic neurons and in subpopulations treated for 48 hours. In
comparison to control, the worms exhibited loss of dopaminergic neurons in a dose dependent
manner (Figure 2B). Harmane exposure resulted in various morphological changes such as
breakage, swelling or complete loss of soma and dendrites.
In terms of total dopaminergic neurons, a significant decrease in the percentage of total
neurons with typical morphology was observed in worms treated with 250 (81.85 ± 1.25,
p<0.01), 500 (36.66 ± 3.00, p<0.001), 750 (30.62 ± 5.20, p<0.001) and 1000 µM (1.25 ± 1.25,
p<0.001) harmane as compared to control (Figure 2C). ADE neurons exhibited significant
decrease in percentage of intact dopaminergic neurons at 250 (83.33 ± 2.20, p<0.01), 500 (27.5 ±
3.81, p<0.001), 750 (13.33 ± 3.63, p<0.001) and 1000 µM (0.83 ± 0.83, p<0.001) (Figure 2E).
PDE neurons exhibited considerable decrease in percentage of intact neurons at 250 (58.33 ±
11.67, p<0.01), 500 (2.5 ± 2.5, p<0.001), 750 (0.83 ± 0.83, p<0.001) and complete loss at 1000
µM (Figure 2F). CEP neurons exhibited least susceptibility to harmane exposure exhibiting
considerable decrease in percentage of intact neurons at 500 (58.33 ± 3.25, p<0.001), 750 (54.16
± 8.73, p<0.001) and 1000 µM (2.08 ± 2.08, p<0.001) (Figure 2D) (n = 3 repeats/group for all
groups in this Figure). Taken together, our results suggest that PDE neurons are most susceptible
to harmane, followed by ADE and CEP neurons.
Harmane exposures increase the levels of reactive oxygen species.
DHE staining was used to evaluate ROS levels (primarily as a measure of superoxide) following
48 hours of harmane exposure (Figure 3A). Changes in ROS levels were not detectable at 100
and 250 µM, however, a significant increase in ROS levels was observed at 750 µM (3.40 ±
0.74, p<0.05) as compared to that of untreated worms (1.00 ± 0.59) (Figure 3B). The results
imply that at higher doses harmane exposure leads to increased ROS (n = 3 repeats/group for all
groups in this Figure).
Harmane treatment decreases mitochondrial viability.
The effects of 48 hours of harmane exposure on mitochondria proton gradients were evaluated
using a reduced form of MitoTracker stain, which stains only mitochondria that have an intact
proton gradient, deemed to be ‘viable’. We observed a dose dependent loss of mitochondria with
intact proton gradients in worms exposed to harmane (Figure 3C). Semi-quantitative analysis
revealed a significant loss of mitochondria with intact proton gradients in worms treated with
100 µM (0.34 ± 0.04, p<0.001), 250 µM (0.14 ± 0.02, p<0.001), 500 µM (0.05 ± 0.00, p<0.001)
and 750 µM (0.10 ± 0.00, p<0.001) as compared to that of control (1.00 ± 0.00) (Figure 3D) (n =
3 repeats/group for all groups in this Figure), implying that harmane leads to loss of
mitochondrial viability.
Harmane exposure leads to decrease in dopamine dependent response towards 1-nonanol.
Dopamine modulates various behavioral functions in C. elegans, any alteration in levels of
dopamine leads to altered response to 1-nonanol (Kaur et al., 2012). This assay has been
extensively used in this system to estimate dopamine levels. To further validate the relationship
between the endpoint and dopamine levels, we also showed that DAT inhibition, a strategy to
increase synaptic dopamine, lowered repulsion time, indicative of increased dopamine levels
(Supplemental Figure 1) (n = 3 repeats/group for all groups in this Figure). We studied the
effect on chemo-repulsive behavior in worms exposed to harmane in doses ranging from 50 to
500 µM. In comparison to untreated worms (1.00 ± 0.00), we observed that the lower doses i.e.
50 µM (0.61 ± 0.03, p<0.01) and 100 µM (0.61 ± 0.03, p<0.001) resulted in statistically
decreased repulsion time (indicative of higher dopamine levels), whereas higher doses, 250 µM
(1.54 ± 0.03) and 500 µM (2.41 ± 0.06) showed significantly increased repulsion time (indicative
of lower dopamine levels). These results indicate that the harmane exposure at or above 250 µM
results in decreased dopamine levels (Figure 3E).
Neuroprotection studies on harmane-induced dopaminergic neurotoxicity
In general, mechanistic studies aimed at protection showed that harmane toxicity was generally
more amenable to modulation under a lower dose (500 µM), longer exposure time (72 hours).
Thus, mechanistic studies were primarily conducted under this regimen (Figures 4-6).
Nonetheless, for consistency with toxicity experiments and to also evaluate mechanistic
modulation under higher-dose (650 µM), shorter exposure time (48 hours), and mechanistic
studies were also conducted under this regimen (Supplemental Figures 2-4).
Inhibition of dopamine transporter does not alter harmane-induced neuronal loss.
In order to investigate if harmane-induced neurodegeneration is mediated through DAT uptake, a
common entry mechanism for DA neurotoxicants, we co-treated harmane-exposed worms with
bupropion HCl (BP), a known DAT inhibitor. The studies were divided into two groups, by
exposure 48 or 72 hour and 500 or 650 µM harmane. In 72 hour studies, we did not show
detectable changes induced by BP treatment in neuronal loss (Figure 4A) for total, CEP, ADE
and PDE neurons (Figure 4B - E) (n = 3 repeats/group for all groups in this Figure). Similarly,
in 48 hour studies, we did not observe any alterations in harmane-induced neurodegeneration
upon treatment with BP (20, 40 & 80 µM) (Supplementary figure 2) (n = 3 repeats/group for all
groups in this Figure). Similarly, the above results imply that harmane-induced neuronal loss is
not mediated through DAT uptake.
Mitochondrial complex I modulation partially-ameliorates neurotoxicity.
To determine if mitochondrial complex I may modulate harmane neurotoxicity, we assessed the
effect of mitochondrial complex I activator, HBA, on harmane-induced neurodegeneration.
Similar to the studies on DAT inhibitor, the study design employed both 72 hour/low dose and
48 hour/high dose studies with doses of HBA ranging from 50 to 200 mM. In pilot studies,
worms were exposed to HBA alone at concentrations up to 200 mM, which did not produce any
evidence of neurodegeneration (data not shown).
In 72 hour studies at a lower dose of harmane, the rescuing effect of HBA was relatively
more pronounced (Figure 5A). In terms of total neurons, a significant increase in percentage of
intact neurons was observed in harmane treated worms (500 µM), when subjected to treatment
with 100 mM (58.33 ± 6.61, p<0.05), 150 mM (61.66 ± 3.23, p<0.01) and 200 mM HBA (64.16
± 5.20, p<0.01) as compared to 500 µM harmane alone (35.20 ± 3.68) (Figure 5B). In
dopaminergic subpopulations, HBA was not protective in PDE neurons (Figure 5C). However,
HBA partially ameliorated neurodegeneration in other subpopulations. In CEP neurons, HBA
treatment produced an increase in the percentage of intact neurons at 100 mM (79.16 ± 9.79,
p<0.05), 150mM (85.00 ± 3.30, p<0.01) and 200 mM (89.58 ± 5.41, p<0.01) as compared to
harmane 500 µM alone (52.91 ± 4.80) (Figure 5D). In ADE neurons, HBA treatment increased
the percentage of intact neurons at 100 mM (75.00 ± 8.03, p<0.05), 150 mM (74.16 ± 8.81,
p<0.05) and 200 mM (77.50 ± 10.00, p<0.05) as compared to harmane 500 µM alone (35.00 ±
5.77) (Figure 5E) (n = 3 repeats/group for all groups in this Figure). The results above indicate
that mitochondrial complex I activator treatment provides protection from harmane-induced
dopaminergic neurodegeneration. In 48 hour studies, we found that HBA treatment partially
rescued harmane-induced neurodegeneration, to a somewhat lesser extent than the lower dose,
longer term exposure regimen (Supplementary Figure 3) (n = 3 repeats/group for all groups in
this Figure).
Mechanisms of harmane-induced alterations of mitochondrial viability.
To confirm that HBA treatment improved mitochondrial function and that neuroprotection was
likely to be mediated through this mechanism, we assessed the effect of HBA on mitochondria
with intact proton gradients using MitoTracker at 100 –200 mM (Figure 6A). In comparison to
untreated worms (1.00 ± 0.00), we observed a significant increase in mitochondria with intact
proton gradients in worms treated with 100 mM (2.13 ± 0.16, p<0.001) and 150 mM (1.81 ±
0.05, p<0.001) HBA, while 200 mM HBA did not produce significant differences from control
(Figure 6B) (n = 3 repeats/group for all groups in this Figure), suggesting that HBA improves
mitochondrial viability, particularly at the lower two doses tested.
The effect of HBA on mitochondria with intact proton gradients was then tested in
harmane-treated worms. As with the previous experiments, we performed MitoTracker staining
for low (500 µM) and high doses (650 µM, Supplementary Figure S4 A, B) (n = 3 repeats/group
for all groups in this Figure) of harmane. Here, detectable changes were only observed in the low
dose group (Figure 6C). The effect on mitochondria with intact proton gradients in worms
treated with 150 mM HBA (0.13 ± 0.00, p<0.05) was significantly enhanced in comparison to
that of worms treated with 500 µM harmane (0.08 ± 0.00) (Figure 6D). Riboflavin treatment did
not modulate mitochondrial viability in worms exposed to harmane (Figure 6E,F). However,
NAC treatment provided significant protection from loss of mitochondrial proton gradient in
worms treated with 500 µM harmane at doses 10 mM (0.22 ± 0.019, p<0.05) and 15 mM (0.36 ±
0.023, p<0.001) as compared to that of worms treated with harmane (0.12 ± 0.00) (Figure
6G,H). The above results indicate additional contribution of complex V towards amelioration of
mitochondrial viability in worms challenged with harmane.
HBA enhances dopamine-dependent response towards 1-nonanol in untreated and
harmane-treated worms.
HBA treatment was also evaluated for effects on harmane-induced dopamine-dependent
behaviors, again using the 1-nonanol assay. We observed significant enhancement in dopamine
responsiveness evident as decreased repulsion time in worms treated with 100 mM (0.73 ± 0.06,
p<0.01), 150 mM (0.64 ± 0.02, p<0.001) and 200 mM (0.67 ± 0.04, p<0.01) HBA as compared
to repulsion time in control (1.00 ± 0.00) (Figure 7A) (n = 3 repeats/group for all groups in this
Figure). Similar to the previous experiments treatment with lower doses of harmane i.e. 100 µM
(0.58 ± 0.02, p<0.001) and 150 µM (0.78 ± 0.06, p<0.05) resulted in a decrease in repulsion
time.
In worms treated with 250 µM harmane, the repulsion time was increased relative to
controls (1.81 ± 0.02). Reduced repulsion times by HBA treatments suggest amelioration of
dopamine depletion: 150 mM (0.92 ± 0.03, p<0.001) and 200 mM (0.93 ± 0.11, p<0.001).
Similarly, in worms treated with 500 µM harmane, repulsion times also suggest neuroprotection
(2.82 ± 0.04), 100 mM (1.97 ± 0.17, p<0.001), 150 mM (1.22 ± 0.02, p<0.001) and 200 mM
(1.01 ± 0.09, p<0.001) HBA (Figure 7A). We also performed this assay in cat-2/TH mutant
(MT15620) and worms over-expressing cat-2/TH (UA57) to validate the findings. As expected,
we observed a significant increase in repulsion time in cat-2/TH mutant (1.97 ± 0.02, p<0.001)
and worms over-expressing cat-2/TH (0.53 ± 0.02, p<0.001) compared to that of control (1.00 ±
0.00) (Figure 7B). The above results established that HBA restores the dopamine function in
untreated and harmane-treated worms.
HBA treatment does not alter the motility in C. elegans.
Since muscles are also rich in mitochondria, increased mitochondrial viability is expected to
interfere with motility, biasing the results of 1-nonanol assay. Hence, we performed thrashing
assay to see if HBA treatment alters the motility in worms. We observed that HBA treatment at
100, 150 and 200 mM failed to produce significant alterations in motility as compared to that of
control (Figure 6C), implying that HBA treatment does not alter motility in worms.
Discussion
A large body of data implies a significant role for environmental exposures in the etiology of PD.
Yet, many linked compounds are rarely encountered and may not influence a significant number
of cases. Here, we aimed to investigate whether harmane, a dietary toxin, may produce selective
neurodegeneration in dopamine neurons. Harmane is a toxin formed in heating of biological
matter. While harmane is a known dietary toxin and tremorigen that has been found to be present
in increased levels in PD patients (Louis et al., 2014), detailed in vivo neurotoxicity assessments
have yet to be conducted. To conduct our studies, we chose the nematode model C. elegans,
which has been extensively used in PD research and allowed us to assess selectivity of
neurotoxicity across a wide range of doses and treatment times. Here, we have shown that
dopaminergic neurons were found to be most susceptible to harmane-induced neurodegeneration,
with non-dopaminergic neurons requiring higher doses to detect neurodegeneration. Dopamine–
dependent behaviors were affected by harmane treatment and evidence of increased ROS and
decreased mitochondrial viability were observed. Treatment with the mitochondrial complex I
activator, HBA was protective, ameliorating morphological changes consistent with
neurodegeneration and many neurochemical changes associated with harmane exposure. The
findings presented here are expected to set the stage for follow-up pathological and mechanistic
studies in higher order species.
C. elegans strains expressing fluorophores in distinct neuronal populations are ideal tools
in assessing selective neurotoxicity. Our findings showed that dopaminergic neurons are more
susceptible to damage by harmane at doses as low as 100 µM; followed by
serotonergic/GABAergic and then cholinergic neurons. In performing detailed studies at 100 to
1000 µM, we observed complete loss of neurons at doses close to 1000 µM.
Interestingly, we also observed differential sensitivity in dopaminergic neuronal
subpopulations, with PDE neurons being most vulnerable, followed by ADE and CEP neurons.
The differential sensitivity order is essentially the reverse of what has been reported in 6hydroxydopamine (Nass et al., 2002) and MPP+ models exhibiting dopamine neuron sensitivity
as ADE > CEP > PDE (Wang et al., 2007). Differences in overall and subpopulation sensitivity
observed between toxicants could possibly arise from DAT-dependence or strain differences. 6hydroxydopamine and MPP+ uptake into dopamine neurons is mediated through DAT (Cannon
and Greenamyre, 2010). Thus, DAT level expression differences and resultant uptake differences
occurring in these populations could mediate subpopulation sensitivity for DAT-dependent
toxicants. While DAT inhibition has been shown to prevent MPP+-mediated dopaminergic loss
in C.elegans (Pu and Le, 2008), such inhibition did not modulate neurotoxicity in our studies.
Thus, our data suggest that harmane does not enter dopaminergic neurons through DAT.
Interestingly, DAT inhibition appeared to increase toxicity, although not significantly. It is
possible that DAT inhibition may increase the production of unpackaged reactive dopamine and
dopamine metabolites, which may be toxic (Jinsmaa et al., 2009). Further work will focus on
whether harmane alters dopamine neurotransmission and whether increased oxidized dopamine
metabolites may act directly on mitochondria. Relative DAT levels in dopaminergic
subpopulations have yet to be determined in C. elegans. Thus, it is possible that the relatively
high lipophilicity of harmane allows it to enter cells in a DAT-independent manner as our data
indicate.
Increased ROS (Dias et al., 2013) and mitochondrial dysfunction (Reddy, 2009; Schon
and Manfredi, 2003; Young, 2009) have been repeatedly linked to the pathogenesis in PD. We
observed considerable increases in ROS (superoxide) at 750 µM. Mitochondrial viability
changes were observed at far lower doses (100 µM) than required to elicit ROS increase,
indicating that mitochondrial loss is likely an early event in harmane-induced neurodegeneration.
We observed behavior indicative of increased dopamine levels at low doses and behavior
indicative of reduced dopamine levels (increased repulsion time) at higher doses (250 and 500
µM). The nonanol assay for higher doses was not conducted since worms showed a little
movement. Interestingly, harmane is known to inhibit monoamine oxidase inhibitory activity,
which could potentially increase DA levels (Herraiz and Chaparro, 2006). Dopamine itself is
reactive and neurotoxic (Hastings and Zigmond, 1994). Thus, it is possible that at higher doses,
cell loss and eventual dopamine depletion result from dopamine-dependent toxicity.
Mitochondrial complex I dysfunction is a critical pathogenic event in PD, with multiple
PD-relevant toxicants acting through this mechanism to lesion dopamine neurons (Betarbet et al.,
2000; Cannon and Greenamyre, 2010). Thus, given that our data showed harmane exposure
reduced viable mitochondria, we tested whether a complex I activator would be protective.
Previous studies on Streptomyces venezuelae induced neurodegeneration in worms have shown
that 50 mM HBA, a mitochondrial complex I activator, prevents dopaminergic cell loss (Ray et
al., 2014). Thus, we treated worms with HBA at 50 to 200 mM. We observed significant
amelioration of cell loss in response to HBA. The efficacy of HBA varied in terms of doses and
neuronal class. In 48 hour/high dose studies, HBA exhibited considerable amelioration in total
neurons at doses 100 and 150 mM. Amongst the dopaminergic neuronal subclasses, CEP
neurons did not exhibit significant protection, perhaps because this population was more
sensitive to harmane exposure. We also studied the effect of HBA on mitochondrial proton
gradients. We observed a significant increase in mitochondria with intact proton gradients at 100
and 150 mM HBA, corroborating our findings on the beneficial effect of HBA. Specifically, we
studied the effect of HBA on mitochondrial viability in worms treated with 650 and 500 µM
harmane. Although we did not observe significant amelioration in the high dose group (650 µM),
a significant increase in mitochondria with intact proton gradients was observed in low dose
group (500 µM) when treated with 150 mM HBA. In light of the observed protective action of
HBA, a possible explanation for a marginal effect on viable mitochondria could be that the
effects of HBA are greater in neuronal mitochondria, rather than total mitochondria. Given that
riboflavin treatment did not modulate mitochondrial viability in worms exposed to harmane, but
that NAC treatment provided significant protection, it is possible that activation of other
complexes may be protective (i.e. complex V). These studies, while suggestive of a critical role
for mitochondria, especially complex I in harmane-induced neurotoxicity will need to followed
by studies in systems amenable to directly detecting effects on mitochondrial complex activity.
The protective effects of HBA in harmane-treated worms were also evident in dopamineassociated behavior. In harmane treated worms, which exhibited increased dopamine-dependent
repulsion time (indicative of low dopamine levels), we observed a significant improvement with
HBA treatment. We also used cat-2 mutant (MT15620) and worms over expressing cat-2
(UA57) as positive and negative controls respectively to validate the experimental findings by
showing that the dopamine system can be modulated and can be assessed for protection and
potentiation. Significant elevation and decline in repulsion time in case of MT15620 and UA57
confirmed our findings and experimental validity. Since the nonanol assay is a behavior assay
and is scored on the basis of repulsive behavior, variations in motility could bias the results of
the study. Notably muscles are also rich in mitochondria. Thus, any substance enhancing the
viability of mitochondria could also positively affect muscular functioning raising a possibility
that the response could partially be due to the improved motility. Therefore, we addressed this
question by performing the thrashing assay, where we observed that motility in worms treated
with HBA was statistically insignificant to untreated worms, confirming that the observed effects
in nonanol assay were solely due to improved dopamine levels.
The doses of harmane used in these studies are far higher than that a human would likely
be exposed to. For example, we used 100 µM harmane for many studies. In humans, the mean
log blood harmane in PD cases has been reported at 0.59 g−10/mL (roughly double controls)
(Louis et al., 2014). Brain concentrations are elevated in essential tremor cases and are also
roughly 2.5 times that in blood (Louis et al., 2013). While comparisons to more polar molecules
are not directly relevant, it is worth noting that in general, far higher doses are typically required
in C. elegans than other in vivo models for most neurotoxicants and our doses are within
suggested nematode testing ranges (Li et al., 2013). Further, in mammalian systems, harmane
accumulates in the brain with concentrations ~ 6.5 times greater than peripheral tissue in
laboratory animals, making brain as a primary target organ (Zetler et al., 1972). Selective
accumulation across the blood-brain-barrier into the parenchyma as well as chronic exposures
are not possible in C. elegans.
Our studies for the first time show that harmane exposure is selectively neurotoxic to
dopamine neurons in C. elegans. Mechanistic studies indicated that cell entry is unlikely through
DAT and that the mitochondria may be a primary target. Our results suggest that future studies
focused on epidemiological links between harmane and clinical PD, as well as mechanistic
studies in higher order species should be conducted to establish the potential relevance of
harmane exposure to PD etiology.
Supplementary data description
DAT inhibition alters repulsion time in response to 1-nonanol (Supplementary Figure 1).
Treatment with bupropion HCl (DAT-1 inhibitor) led to significant decrease in repulsion time in
a dose dependent manner.
Inhibition of dopamine transporter does not alter harmane-induced neuronal loss
(Supplementary Figure 2). DAT inhibition by bupropion HCl did not alter harmane-induced
neuronal loss.
Mitochondrial complex I activation lessens harmane-induced neurotoxicity (48 hour
studies) (Supplementary Figure 3). HBA treatment decreases harmane-induced dopaminergic
neurodegeneration.
D, L 3-Hydroxy butyric acid treatment does not alleviate mitochondrial viability at high
doses of harmane (Supplementary Figure 4). Treatment with D, L 3-Hydroxy butyric acid did
not significantly alter mitochondrial viability.
Funding
This work was supported by the National Institute of Environmental Health Sciences at the
National Institutes [R01ES025750 to J.R.C.].
Acknowledgement
Strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure
Programs (P40 OD010440). We would also like to acknowledge Dr. Andrea Kasinski and
Kaushik Muralidharan for providing an initial strain to our laboratory.
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Figure Legends:
Figure 1. Dopaminergic neurons are selectively sensitive to harmane exposure. Worms were
treated with harmane for 48 hours and neurodegeneration was assessed. Dopaminergic neurons
(Strain: BZ555) exhibited heightened sensitivity to harmane, with neurodegeneration observed at
doses as low as 100 µM (A,B). Exposing worms to 100 µM, 250 µM and 500 µM harmane
produced quantifiable neurodegeneration (n = 6). Other neuron types i.e. serotonergic (Strain:
GR1366; C,D), GABAergic (Strain: CZ1632; E,F) and cholinergic (Strain: LX929; G,H) were
comparatively less susceptible to harmane-induced neurodegeneration. The three other neuronal
cell populations examined were devoid of any neurodegeneration at 100 µM. Serotonergic
neurons exhibited significant neurodegeneration at 250 µM and 500 µM (n = 3). GABAergic
neurons showed affliction in neuronal damage at 250 µM and 500 µM, with damage majorly
restricted to inter-neuronal commissures (n = 3). Cholinergic neurons were found to be least
susceptible, displaying neuronal damage only at 500 µM (n = 3). Data presented as mean ±
S.E.M. Percentage of intact neurons was calculated by counting the total number of neurons per
worm for 20 animals in each experimental group. Data analyzed by one-way ANOVA followed
by Dunnett’s post hoc test. *p<0.05, ***p<0.001.
Figure
2.
Harmane
treatment
produces
dose-dependent
neurodegeneration
of
dopaminergic (DA) neurons. Representative images of dopaminergic neurons in C. elegans
show four cephalic sensilla (CEP), two anterior deirid (ADE) in head region along with two
posterior deirid (PDE) neurons in tail region. Tail ray neurons are present in males only (A).
Treatment of worms with harmane for 48 hours produces dose-dependent degeneration of DA
neurons exhibiting morphological changes such as breakage or loss of dendrites (arrows),
swelling (filled arrowheads) and loss of soma (open arrows) (B). The percentage of neuronal loss
was calculated specific to DA neuronal subtype: total (C), CEP (D), ADE (E) and PDE (F). Data
presented as mean ± S.E.M. Percentage of intact neurons was calculated by counting the total
number of neurons per worm for 20 animals in each experimental group. Data analyzed by oneway ANOVA followed by Tukey’s post hoc test. **p<0.01, ***p<0.001. n = 3. Scale bar
represents 20 µm.
Figure 3. Harmane-induced neurochemical changes. Harmane treatment (48 hours) produces
ROS, reduces mitochondrial viability, and alters dopamine levels. Treatment of worms with
harmane produced elevated ROS levels (A). Statistically significant changes were detectable at
750 µM (B). Harmane treatment led to decreased mitochondrial viability (C). Statistically
significant decreases were quantifiable at doses 250 µM and above (D). Harmane treatment at 50
to 500 µM produced alterations in DA function as measured through nonanol repulsion assay
(E). Lower doses of harmane (50 and 100 µM) significantly elevated dopamine responsiveness
in terms of reduced repulsion time, whereas higher doses (250 and 500 µM) exhibited significant
delay in responsiveness, indicative of dopamine depletion (E). Data presented as mean ± S.E.M.
Repulsion time was calculated for 18 animals in each experimental group. Worms were analyzed
semi-quantitatively for approximately 20 animals per treatment group. For quantification of
fluorescence images (A, C), the gut was excluded from the region of interest (ROI) so as to
eliminate nonspecific fluorescence from the study. Data analyzed by one-way ANOVA followed
by Tukey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001. n = 3. Scale bar represents 50 and 20
µm for (A) and (C), respectively.
Figure 4. Harmane induced DA neurotoxicity is unlikely to be mediated through dopamine
transporter (DAT) uptake. Lower-dose, longer harmane treatment times (500 µM for 3 days)
also produced neurodegeneration that was unaffected by blocking DAT (A). The percentage of
neuronal loss was calculated with respect to DA neuronal subtype: total (B), CEP (C), ADE (D)
and PDE (E). Data presented as mean ± S.E.M. Percentage of intact neurons was calculated by
counting the total number of neurons per worm for 20 animals in each experimental group. Data
analyzed by one-way ANOVA followed by Tukey’s post hoc test. n = 3/group. Scale bar
represents 20 µm.
Figure 5. D, L 3-Hydroxy butyric acid (HBA) treatment provides protection from
harmane-induced neurodegeneration. Neuroprotection was evaluated at multiple doses.
Treatment with HBA, a mitochondrial complex I activator treatment resulted in a significant
decrease in dopaminergic neurodegeneration 500 µM harmane, over a longer exposure time (72
hours) (A). Here, the percentage of neuronal loss was also calculated with respect to DA
neuronal subtype: total (B), CEP (C), ADE (D) and PDE (E). Data are presented as mean ±
S.E.M. The percentage of intact neurons was calculated by counting the total number of neurons
per worm for 20 animals in each experimental group. Data analyzed by one-way ANOVA
followed by Tukey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001. n = 3. Scale bar represents
20 µm.
Figure 6. Treatment of harmane-exposed worms with D, L 3-Hydroxy butyric acid (HBA)
and N-acetyl-L-cysteine (NAC) improves mitochondrial viability. HBA treatment led to
significant increases in mitochondrial viability (A). At 100 and 150 mM doses, increased
viability was quantified, while at a higher dose (200 mM) changes in viability were not
detectable (B). At a lower dose (500 µM), longer exposure time (72 hours), a modest but
significant increase was detected upon treatment with HBA (C, D). At a lower dose (500 µM),
longer exposure time (72 hours), treatment with riboflavin was devoid of any effect on
mitochondrial viability (E, F). At a lower dose (500 µM), longer exposure time (72 hours), a
significant dose dependent increase was detected upon treatment with NAC (G, H). Data
presented as mean ± S.E.M. Mitochondrial viability was semi quantitatively analyzed through
image J for approximately 20 animals per group. The gut was excluded from the region of
interest (ROI) so as to eliminate nonspecific fluorescence from the study. Data analyzed by one-
way ANOVA followed by Tukey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001. n = 3. Scale
bar represents 20 µm.
Figure 7. Treatment of harmane-exposed worms with D, L 3-hydroxy butyric acid (HBA)
rescues dopamine levels. Treatment with lower dose of harmane (50, 100 and 150 µM) and
HBA (100, 150 & 200 mM) exhibited increase in dopamine levels (as indicated by lowered
repulsion time) as compared to untreated control; HBA treatment was found to significantly
alleviate dopamine levels in worms treated with 250 and 500 µM harmane (A), MT15620 (cat-2
mutant) exhibited lowered dopamine levels, whereas worms overexpressing cat-2 (UA57)
exhibited significantly increased dopamine levels (B), Worms treated with HBA were devoid of
any alteration in motility as elucidated through thrashing assay (C). Data presented as mean ±
S.E.M. Repulsion time and number of thrashes (with number of thrashes representing one
complete sinusoidal movement) were calculated for 18 animals in each experimental group. Data
analyzed by two-way ANOVA for grouped analysis and by one-way ANOVA followed by
Tukey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001. n = 3.
Figure 1. Dopaminergic neurons are selectively sensitive to harmane exposure. Worms were treated with
harmane for 48 hours and neurodegeneration was assessed. Dopaminergic neurons (Strain: BZ555)
exhibited heightened sensitivity to harmane, with neurodegeneration observed at doses as low as 100 µM
(A,B). Exposing worms to 100 µM, 250 µM and 500 µM harmane produced quantifiable neurodegeneration
(n = 6). Other neuron types i.e. serotonergic (Strain: GR1366; C,D), GABAergic (Strain: CZ1632; E,F) and
cholinergic (Strain: LX929; G,H) were comparatively less susceptible to harmane-induced
neurodegeneration. The three other neuronal cell populations examined were devoid of any
neurodegeneration at 100 µM. Serotonergic neurons exhibited significant neurodegeneration at 250 µM and
500 µM (n = 3). GABAergic neurons showed affliction in neuronal damage at 250 µM and 500 µM, with
damage majorly restricted to inter-neuronal commissures (n = 3). Cholinergic neurons were found to be
least susceptible, displaying neuronal damage only at 500 µM (n = 3). Data presented as mean ± S.E.M.
Percentage of intact neurons was calculated by counting the total number of neurons per worm for 20
animals in each experimental group. Data analyzed by one-way ANOVA followed by Dunnett’s post hoc test.
*p<0.05, ***p<0.001.
184x279mm (300 x 300 DPI)
Figure 2. Harmane treatment produces dose-dependent neurodegeneration of dopaminergic (DA) neurons.
Representative images of dopaminergic neurons in C. elegans show four cephalic sensilla (CEP), two anterior
deirid (ADE) in head region along with two posterior deirid (PDE) neurons in tail region. Tail ray neurons are
present in males only (A). Treatment of worms with harmane for 48 hours produces dose-dependent
degeneration of DA neurons exhibiting morphological changes such as breakage or loss of dendrites
(arrows), swelling (filled arrowheads) and loss of soma (open arrows) (B). The percentage of neuronal loss
was calculated specific to DA neuronal subtype: total (C), CEP (D), ADE (E) and PDE (F). Data presented as
mean ± S.E.M. Percentage of intact neurons was calculated by counting the total number of neurons per
worm for 20 animals in each experimental group. Data analyzed by one-way ANOVA followed by Tukey’s
post hoc test. **p<0.01, ***p<0.001. n = 3. Scale bar represents 20 µm.
184x109mm (300 x 300 DPI)
Figure 3. Harmane-induced neurochemical changes. Harmane treatment (48 hours) produces ROS, reduces
mitochondrial viability, and alters dopamine levels. Treatment of worms with harmane produced elevated
ROS levels (A). Statistically significant changes were detectable at 750 µM (B). Harmane treatment led to
decreased mitochondrial viability (C). Statistically significant decreases were quantifiable at doses 250 µM
and above (D). Harmane treatment at 50 to 500 µM produced alterations in DA function as measured
through nonanol repulsion assay (E). Lower doses of harmane (50 and 100 µM) significantly elevated
dopamine responsiveness in terms of reduced repulsion time, whereas higher doses (250 and 500 µM)
exhibited significant delay in responsiveness, indicative of dopamine depletion (E). Data presented as mean
± S.E.M. Repulsion time was calculated for 18 animals in each experimental group. Worms were analyzed
semi-quantitatively for approximately 20 animals per treatment group. For quantification of fluorescence
images (A, C), the gut was excluded from the region of interest (ROI) so as to eliminate nonspecific
fluorescence from the study. Data analyzed by one-way ANOVA followed by Tukey’s post hoc test. *p<0.05,
**p<0.01, ***p<0.001. n = 3. Scale bar represents 50 and 20 µm for (A) and (C), respectively.
181x279mm (600 x 600 DPI)
Figure 4. Harmane induced DA neurotoxicity is unlikely to be mediated through dopamine transporter (DAT)
uptake. Lower-dose, longer harmane treatment times (500 µM for 3 days) also produced neurodegeneration
that was unaffected by blocking DAT (A). The percentage of neuronal loss was calculated with respect to DA
neuronal subtype: total (B), CEP (C), ADE (D) and PDE (E). Data presented as mean ± S.E.M. Percentage of
intact neurons was calculated by counting the total number of neurons per worm for 20 animals in each
experimental group. Data analyzed by one-way ANOVA followed by Tukey’s post hoc test. n = 3/group.
Scale bar represents 20 µm.
184x224mm (300 x 300 DPI)
Figure 5. D, L 3-Hydroxy butyric acid (HBA) treatment provides protection from harmane-induced
neurodegeneration. Neuroprotection was evaluated at multiple doses. Treatment with HBA, a mitochondrial
complex I activator treatment resulted in a significant decrease in dopaminergic neurodegeneration 500 µM
harmane, over a longer exposure time (72 hours) (A). Here, the percentage of neuronal loss was also
calculated with respect to DA neuronal subtype: total (B), CEP (C), ADE (D) and PDE (E). Data are
presented as mean ± S.E.M. The percentage of intact neurons was calculated by counting the total number
of neurons per worm for 20 animals in each experimental group. Data analyzed by one-way ANOVA followed
by Tukey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001. n = 3. Scale bar represents 20 µm.
184x259mm (300 x 300 DPI)
Figure 6. Treatment of harmane-exposed worms with D, L 3-Hydroxy butyric acid (HBA) and N-acetyl-Lcysteine (NAC) improves mitochondrial viability. HBA treatment led to significant increases in mitochondrial
viability (A). At 100 and 150 mM doses, increased viability was quantified, while at a higher dose (200 mM)
changes in viability were not detectable (B). At a lower dose (500 µM), longer exposure time (72 hours), a
modest but significant increase was detected upon treatment with HBA (C, D). At a lower dose (500 µM),
longer exposure time (72 hours), treatment with riboflavin was devoid of any effect on mitochondrial
viability (E, F). At a lower dose (500 µM), longer exposure time (72 hours), a significant dose dependent
increase was detected upon treatment with NAC (G, H). Data presented as mean ± S.E.M. Mitochondrial
viability was semi quantitatively analyzed through image J for approximately 20 animals per group. The gut
was excluded from the region of interest (ROI) so as to eliminate nonspecific fluorescence from the study.
Data analyzed by one-way ANOVA followed by Tukey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001. n =
3. Scale bar represents 20 µm.
184x299mm (300 x 300 DPI)
Figure 7. Treatment of harmane-exposed worms with D, L 3-hydroxy butyric acid (HBA) rescues dopamine
levels. Treatment with lower dose of harmane (50, 100 and 150 µM) and HBA (100, 150 & 200 mM)
exhibited increase in dopamine levels (as indicated by lowered repulsion time) as compared to untreated
control; HBA treatment was found to significantly alleviate dopamine levels in worms treated with 250 and
500 µM harmane (A), MT15620 (cat-2 mutant) exhibited lowered dopamine levels, whereas worms
overexpressing cat-2 (UA57) exhibited significantly increased dopamine levels (B), Worms treated with HBA
were devoid of any alteration in motility as elucidated through thrashing assay (C). Data presented as mean
± S.E.M. Repulsion time and number of thrashes (with number of thrashes representing one complete
sinusoidal movement) were calculated for 18 animals in each experimental group. Data analyzed by twoway ANOVA for grouped analysis and by one-way ANOVA followed by Tukey’s post hoc test. *p<0.05,
**p<0.01, ***p<0.001. n = 3.
184x220mm (300 x 300 DPI)
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