the effects of intra-cea parp-1 inhibition on cocaine
TRANSCRIPT
Running Head: PARP-1 INHIBITION AND COCAINE SEEKING
The effects of intra-CeA PARP-1 inhibition on cocaine reinstatement
By: Megan Brickner
Honors Thesis
Advisor: Dr. Kathryn Reissner
Department of Psychology & Neuroscience
University of North Carolina at Chapel Hill
PARP-1 INHIBITION AND COCAINE SEEKING
1
Acknowledgements
I would first like to thank my advisor and mentor, Dr. Kathryn Reissner, for supporting me as
both a professor and a research mentor. Her class and passion for the material allowed me to
translate my interests into opportunities. Thank you for seeing potential in me by investing time
and energy into my development – as a student, researcher, and person.
I am also grateful to Emily Witt who has been a fantastic graduate student mentor to me for the
past two years. Thank you for your role in shaping this thesis, as well as for teaching me so many
valuable skills that I will take with me for the rest of my research training and career. I would not
have been able to do this without you.
To the rest of the Reissner lab –Eden, Janay, Anze, and Ron – thank you for creating a
welcoming environment. I have enjoyed getting to know you all, appreciate your help, and
cherish your support.
Dr. Rachel Penton and Dr. Regina Carelli, thank you for serving on my Honors Thesis
Committee. I appreciate the time and consideration you put into my thesis and my presentation.
Finally, thank you to my family, friends, and loved ones who have supported me throughout my
time at UNC. Their love and encouragement gave me the foundation I needed to pursue research
and complete an Honors Thesis. I am so fortunate to have found such wonderful people through
the Women’s Rugby Team, Tri Sigma Sorority, and my first year dorm; a special nod to my
friends who have been in my corner since high school. To mom, dad, and Rebecca: I am so lucky
to have you by my side, thank you for providing me with so many opportunities.
This project was supported by the Gump Family Undergraduate Research Fund administered
by Honors Carolina. Additional funding was provided by the Lindquist Undergraduate Research
Award.
PARP-1 INHIBITION AND COCAINE SEEKING
2
Abstract
Currently, there are no FDA-approved treatments for cocaine or psychostimulant use disorders,
compounding the need for the development of effective interventions. The DNA repair enzyme
and transcription regulator, Poly(ADP-ribose)polymerase (PARP-1), is activated by cocaine and
contributes to mechanisms of cocaine action in the brain (Dash et al., 2017; Lax et al., 2017;
Scobie et al., 2014). While it is known that inhibition of PARP-1 with the compound PJ34 within
the central nucleus of the amygdala (CeA) reduces cocaine conditioned place preference (Lax et
al., 2017), the effects of PARP-1 inhibition on cocaine relapse after cessation of self-
administration have not been investigated. Accordingly, the goal of this project was to test the
hypothesis that pharmacological inhibition of PARP-1 in the CeA would inhibit cocaine seeking
in the rat reinstatement model of cocaine addiction. Results indicate that while vehicle-treated
rats significantly reinstated over extinction baseline, PJ34-treated rats did not, suggesting an
inhibitory effect of CeA PARP-1 inhibition against reinstatement. Future studies will better
inform the involvement of CeA PARP-1 in cocaine seeking, and guide future research toward
PARP-1 as a candidate pharmacotherapeutic intervention for cocaine use disorders.
PARP-1 INHIBITION AND COCAINE SEEKING
3
Introduction
In 2016, 1.9 million Americans were current users of cocaine, while approximately
867,000 people met the clinical criteria for a cocaine use disorder (CUD; NSDUH, 2017).
However, no FDA-approved pharmacological treatments currently exist specifically for CUD.
Accordingly, investigations into candidate pharmacotherapies are critical, and elucidation of
cellular mechanisms that drive drug seeking and relapse are essential to this endeavor. One such
mechanism may be the link between drug-paired cues and relapse. Existing research indicates
that drug use is reinforced by the addictive drugs themselves as well as the drug related cues, and
that intervention into both of these reinforcers (the drug and drug-related cues) can reduce drug
seeking in animal models (Namba, Tomek, Olive, Beckmann, & Gipson, 2018; Perry, Zbukvic,
Kim, & Lawrence, 2014).
Specifically, the rat self-administration and reinstatement model is an approach used to
model drug addiction based on the principles of operant conditioning. It provides a system in
which candidate treatments can be evaluated, and has face and construct validity as a model of
human drug use and relapse (Panlilio & Goldberg, 2007). The model we utilized has three phases:
self-administration, extinction, and reinstatement (Panlilio & Goldberg, 2007). In this model,
reinstatement to cocaine seeking following a period of extinction training can be precipitated in
numerous ways, including reintroduction of drug-paired cues (Farrell, Schoch, & Mahler, 2018).
This cue-primed reinstatement can be used to evaluate mechanisms and interventions for relapse
vulnerability.
Research in the Reissner lab has shown that chronic systemic administration of
nicotinamide (NAM), a form of vitamin B3, reduces cue-primed reinstatement to cocaine,
without effects on locomotor activity or food seeking behavior (Witt & Reissner, 2019). These
PARP-1 INHIBITION AND COCAINE SEEKING
4
results were only found in males, and not in female rats. This suggests that NAM may play a role
in modifying the salience of cocaine-associated cues in a sex-specific manner (Witt & Reissner,
2019). While the mechanism by which NAM may reduce cue-primed seeking is currently still
unknown, several known effects of NAM indicate potential mechanisms by which it may be
beneficial in combating cellular adaptations induced by cocaine self-administration.
First, NAM is a known inhibitor of poly(ADP-ribose) polymerase (PARP) (Javle &
Curtin, 2011; Riklis, Kol, & Marko, 1990). PARP is a family of enzymes composed of at least 17
members, each with a specific function and specialization (Amé, Spenlehauer, & de Murcia,
2004). Of the 17 PARP family members, PARP-1 is the most active and readily studied enzyme
(Morales et al., 2014). In general, PARPs function by attaching ADP-ribose units onto substrate
proteins in order to regulate the cell’s response to oxidative stress conditions such as DNA
damage to aid in DNA repair (Malanga & Althaus, 2005; Vyas, Chesarone-Cataldo, Todorova,
Huang, & Chang, 2013). This process is known as PARylation. DNA in the brain can be
damaged after cocaine exposure (Alvarenga et al., 2010), indicating an important role for PARP-
mediated repair. Secondly, PARPs also regulate transcription (Kraus & Hottiger, 2013) by
binding to gene promoter regions to regulate gene expression (Gupte, Liu, & Kraus, 2017), and
cocaine has been found to modulate this PARP-mediated transcription (Lax et al., 2017; Scobie
et al., 2014). Additionally, PARP-1 activation can trigger the mitochondria to release apoptosis-
inducing factor, which can lead to apoptosis (Yu et al., 2002; Du et al., 2003). This suggests that
cocaine use may be harmful to the brain, as PARP-1 expression is increased in the nucleus
accumbens (NAc) after cocaine exposure (Dash et al., 2017).
This cocaine exposure-induced increased PARP-1 expression within the NAc (Dash et al.,
2017) may also promote behaviors associated with drug seeking (Scobie et al., 2014). For
PARP-1 INHIBITION AND COCAINE SEEKING
5
example, experimenter-induced overexpression of PARP-1 in mice resulted in increased cocaine
self-administration, increased motor responses to cocaine, and increased conditioned place
preference (CPP) – a behavioral paradigm used to assess of cocaine-associated memories and
seeking (Scobie et al., 2014). PARP-1 is also activated during retrieval of cocaine-associated
contextual memory in the central nucleus of the amygdala (CeA), indicating a role of PARP-1 in
the retrieval of cocaine-associated contextual memory (Lax et al., 2017). Further, inhibition of
PARP-1 in the CeA with PJ34 reduced CPP (Lax et al., 2017). However, the effects of intra-CeA
PARP-1 inhibition on cocaine reinstatement following self-administration and extinction have
not been investigated.
The CeA is one of a number of sub-nuclei within the amygdala (Rasia-Filho, Londero, &
Achaval, 2000). The amygdala is a prominent and key structure in the brain’s motivation-related
circuitry and helps create and strengthen relationships between stimuli, significance, and
motivation (for review, see Kim, 2013). In relation to addiction, the amygdala is activated by
drug cues that can promote drug craving and seeking in humans (Tang, Fellows, Small, &
Dagher, 2012). In animal models, the CeA in particular has been found to be involved in cue-
related drug craving (Lu et al., 2005; Lu, Uejima, Gray, Bossert, & Shaham, 2007). For example,
optogenetic stimulation of the CeA augmented the motivation for and intake of cocaine, while
optogenetic inhibition of the CeA attenuated both the acquisition of cocaine self-administration
and cocaine intake in female rats (Warlow, Robinson, & Berridge, 2017). This CeA stimulation
also increased the attractiveness of the cocaine-associated cues themselves as the rats displayed
biting behavior towards the nose port that pairs the CeA stimulating laser with intravenous
cocaine, indicating amplified incentive salience towards the cue (Warlaw et al., 2017). As
mentioned, inhibition via intra-CeA PJ34 administration reduced cocaine CPP (Lax et al., 2017).
PARP-1 INHIBITION AND COCAINE SEEKING
6
This suggests that PARP-1 activation may play a role in cocaine-associated memory retrieval
(Lax et al., 2017) and as such, PARP-1 inhibition within the CeA may lead to similar reductions
in cocaine seeking behaviors after a self-administration and extinction paradigm.
PARP inhibition has not been examined in a self-administration and extinction model of
reinstatement; however, I hypothesized that the reported effects of NAM against reinstatement
may occur through an effect of PARP inhibition. If this is true, direct inhibition of PARP should
inhibit reinstatement. Moreover, because PARP activation in the CeA plays a particularly salient
role in responsiveness to drug-paired cues, I hypothesized that NAM might inhibit cue-primed
reinstatement through inhibition of PARP in the CeA. Accordingly, the goal of this project was
to determine if pharmacological inhibition of PARP via intra-CeA administration of PJ34 would
inhibit cocaine seeking in the rodent reinstatement model of cocaine seeking. .
PARP-1 INHIBITION AND COCAINE SEEKING
7
Methods
Subjects
This study utilized thirty-two adult male Sprague-Dawley rats purchased from Envigo.
They were housed individually in temperature and humidity controlled cages. Rats were exposed
to a 12-hour light-dark cycle, where the dark period lasted from 7 AM to 7 PM. All experiments
were performed during the dark cycle at the same time daily. All experiments were approved by
the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina
at Chapel Hill, and were in accordance with the guidelines of the American Association for the
Accreditation of Laboratory Animal Care (AAALAC) and the National Research Council’s
Guide for Care and Use of Laboratory Animals.
Surgery
Rats were anesthetized with ketamine (100 mg/kg, i.m.) and xylazine (7 mg/kg, i.m.).
Catheters were then implanted into the right jugular vein with an exit on the back to allow for
intravenous (i.v.) administration of cocaine. Intracranial cannulas were then implanted via
stereotaxic surgery into the CeA according to the coordinates of Paxinos and Watson: AP -2.56
mm, ML +4 mm, DV -7 mm (Lax et al., 2017). Upon completion of the surgeries, rats received
meloxicam analgesia (4 mg/ml) for 3 days during post-surgical recovery and care. Catheters were
flushed daily with gentamicin antibiotic (0.1 mL; 5 mg/mL), and an anticoagulant, heparinized
saline (0.1 mL; 100U/mL), throughout surgery recovery as well as cocaine self-administration in
order to support catheter patency. Twenty-four hrs before self-administration began, catheter
patency was validated with propofol (1 mg/0.1 mL, i.v.).
PARP-1 INHIBITION AND COCAINE SEEKING
8
Behavioral Training
All operant conditioning was performed in standard rat operant boxes (Med Associates).
Before undergoing surgical procedures, each rat was trained in operant responding for food.
During this session, presses on the active lever in operant box were paired with a release of a 45
mg food pellet, while presses on the inactive had no consequence. Food training sessions ended
after at least 100 pellets were received or after 6 hours had elapsed. Surgery was performed as
described above before beginning the cocaine self-administration paradigm.
At the start of each daily 2 hr session, the house light was illuminated and two levers
were extended: an active and inactive lever. Throughout the self-administration sessions, under a
fixed-ratio 1 (FR1) schedule, active lever presses resulted in an infusion of cocaine (0.75
mg/kg/infusion), as well as presentation of a light and tone cue for 5 s. Reward and cue delivery
were followed by a 20 s time out, during which time presses were recorded but had no effect.
Presses on the inactive lever had no programmed consequences throughout the session. On the
first two self-administration days, all rats were restricted to a maximum of 40 cocaine infusions;
all following sessions were unrestricted. To qualify as a successful self-administration session,
the animal must have received 10 or more infusions of cocaine per session.
Following 12 successful self-administration sessions of 10 or more infusions, the
extinction period began and continued for 15 days. Extinction sessions began similarly to self-
administration sessions with house light illumination and extension of levers; however, cocaine,
tone, and light stimuli were no longer presented following active lever presses.
Half of the rats were randomly assigned to receive the PARP-1 inhibitor, PJ34 in
artificial cerebrospinal fluid (aCSF; n=16), while the other half received a vehicle solution, aCSF
(n=16), as a control. PJ34 (50μM) or vehicle was administered bilaterally via microinjection
PARP-1 INHIBITION AND COCAINE SEEKING
9
(0.5L per hemisphere; 1L/min) directly into the CeA as described by Lax et al. (2017).
Microinjections occurred 1mm below the base of the cannula. After the infusion, the
microinjectors were kept in the guide cannulas for 60s, to allow for diffusion. Microinjections
were administered 30 min prior to reinstatement testing, based on the methods employed by Lax
et al. (2017).
Thirty min after the microinjection, animals were placed into the operant boxes for the 2
hour cue-primed reinstatement test. During reinstatement, the active lever was once again paired
with the light and tone cues, while the inactive lever continued to have no programmed
consequences. Cocaine was not reintroduced.
Histology
Following the cue-primed reinstatement session, animals were anesthetized with
pentobarbital and transcardially perfused with 4% paraformaldehyde in phosphate buffer. The
brains were extracted and cryoprotected in a 30% sucrose solution prior to cryostat sectioning.
100 m brain slices of the CeA were collected for Cresyl Violet staining for histological
confirmation of cannula placement.
Data Analysis
Behavioral data were collected on lever presses during each phase of training and testing,
and were analyzed using GraphPad Prism. A repeated-measures two-way analysis of variance
(ANOVA) test was used to compare the number of cocaine infusions between groups during
self-administration, as well as active lever responses during cocaine self-administration and
extinction. Reinstatement responding was also analyzed using an two-way repeated measures
PARP-1 INHIBITION AND COCAINE SEEKING
10
ANOVA between groups (PJ34 vs. vehicle) comparing operant behavior on the last day of
extinction with reinstatement behavior via mean levels of active lever presses. Sidak’s test of
multiple comparisons was used to examine the direction of mean difference in lever pressing by
condition. Results are reported as mean ± variance (SEM), where p < 0.05 was considered
significant.
PARP-1 INHIBITION AND COCAINE SEEKING
11
Results
No significant differences were found between future treatment groups for active lever
presses or infusions during cocaine self-administration (lever presses: Fig. 1A; F(1, 30) = 0.68, p
= .42; infusions: Fig. 1B; F(1, 30) = 0.29, p = .59). The number of active lever presses
throughout extinction was also not significantly different between future treatment groups (Fig.
1A; F(1, 30) = 0.10, p = .76). However, there was a significant main effect of session across self-
administration (Fig. 1A; F(11, 330) = 6.99, p < 0.0001) and extinction (Fig. 1A; F(15, 540) =
73.71, p < 0.0001 ), indicating that the number of lever presses was significantly different based
on the day of the paradigm. There was also a significant effect of session across self-
administration when comparing cocaine infusions (Fig. 1B; F(11, 330) = 10.59, p < 0.0001),
indicating that the number of cocaine rewards significantly differed based on the session number
of the self-administration phase.
Two-way repeated measures ANOVAs were also utilized to analyze cue-primed
reinstatement responding, to determine whether active lever presses during the final extinction
session and the reinstatement test differed as a function of treatment group. A significant main
effect was found for session, indicating a significant difference in active lever presses during the
last day of extinction and reinstatement (Fig. 2; F(1, 30) = 35.84, p < .0001). Sidak’s test of
multiple comparisons indicated that the active lever presses were significantly higher during the
cue-primed reinstatement than during the last day of extinction for both the PJ34 (Fig. 2; p
= .002) and vehicle group (Fig. 2; p < .0001). No main effect of treatment group observed for
lever pressing between the PJ34 and vehicle groups during reinstatement (Fig. 2; F(1, 30)= 0.88,
p= .36).
Histology of the brains was performed to examine cannula placement and PJ34 delivery
PARP-1 INHIBITION AND COCAINE SEEKING
12
to the CeA. Analysis indicated that 20 of the 32 rats had cannula tracts in the proper positions, 5
bilateral hits and 15 unilateral hits. The data from the other 12 rats were removed, and analysis
was repeated to include only the hits. Note that while histological analysis has been completed,
plans to generate placement maps were disrupted due to SARS-CoV-2 interruption of research
activities.
Behavioral analysis was repeated, using only data from rats with either bilateral or
unilateral hits of the CeA. The updated analyses revealed no significant differences between the
PJ34 (n=9) and the vehicle (n=11) future treatment groups for the number of active lever presses
throughout self-administration or extinction (self-administration: Fig. 3A; F(1, 18) = 1.98, p
= .18; extinction: Fig. 3A; F(1, 18) = 0.16, p = .70), nor infusions during cocaine self-
administration (Fig. 3B; F(1, 18) = 0.55, p = .47).There was a significant main effect of session
across self-administration (Fig. 1A; F(11, 198) = 4.93, p < 0.0001) and extinction (Fig. 1A; F(15,
270) = 48.88, p < 0.0001 ). There was also significant effect of session across self-administration
when comparing cocaine infusions (Fig. 1B; F(11, 198) = 8.38, p < 0.0001).
Additionally, when comparing active lever presses during the last day of extinction and
the cue-primed reinstatement test, a significant main effect was found for session (Fig. 4; F(1, 18)
= 18.46, p = .0004), such that the active lever presses were significantly higher during the cue-
primed reinstatement than the last day of extinction for the vehicle group (Fig. 4; p < .0026).
However, there were no significant differences between the active lever presses during the last
extinction session and the reinstatement test for the PJ34 group (Fig. 4; p = .059). Finally, there
was no main effect of treatment group observed for lever pressing during reinstatement between
the PJ34 and vehicle groups (Fig. 4; F(1, 18)= 0.53, p= .48).
PARP-1 INHIBITION AND COCAINE SEEKING
13
Figure 1. (A) Comparison of active lever presses throughout the self-administration and
extinction phases for all rats, separated by future treatment groups. Analyses showed no
significant difference between the active lever presses in either phase between the treatment
groups. (B) Cocaine infusions during self-administration sessions for vehicle (open circles) and
PJ34 (filled circles) future groupings. No significant differences in cocaine infusions were found
between the experimental and control groups.
A)
B)
PARP-1 INHIBITION AND COCAINE SEEKING
14
Figure 2. Active lever pressing during extinction and cue-primed reinstatement test for
vehicle (open bars) and PJ34-treated rats (filled bars) for all rats used in the study. Post-hoc
analyses indicated significant reinstatement in both the vehicle-treated and PJ34-treated group.
However, there was no main effect of treatment group. *=p<0.05, indicating a main effect of
session.
PARP-1 INHIBITION AND COCAINE SEEKING
15
Figure 3. (A) Comparison of active lever presses throughout the self-administration and
extinction phases of the paradigm for the rats with bilateral or unilateral cannula hits, separated
by future treatment groups (Vehicle n=11; PJ34 n=9). Analyses showed no significant difference
between the active lever presses in either phase between the treatment groups. (B) Cocaine
infusions during self-administration sessions for vehicle (open circles) and PJ34 (filled circles)
future groupings. No significant differences in cocaine infusions were found between the
experimental and control groups.
A)
B)
PARP-1 INHIBITION AND COCAINE SEEKING
16
Figure 4. Active lever pressing during extinction and cue-primed reinstatement test for
vehicle (open bars) and PJ34-treated rats (filled bars). Post-hoc analyses indicated significant
reinstatement in the vehicle-treated, but not PJ34-treated group. * indicates main effect of
session.
11 9
PARP-1 INHIBITION AND COCAINE SEEKING
17
Discussion
This study investigated the role of intra-CeA PARP-1 in cue-primed reinstatement via
intra-CeA administration of PJ34 in a rat reinstatement model of cocaine seeking. While CeA
PARP-1 has been shown to mediate cocaine conditioned place preference (Lax et al., 2017), the
involvement of PARP-1 in reinstatement to cocaine seeking is unknown. I hypothesized that
administration of a 50 μM (total volume 0.5μL/hemisphere) dose of PJ34 delivered 30 min
directly into the CeA before cue-primed reinstatement would reduce reinstatement responding.
When analyzing only unilateral and bilateral hits confirmed by histology, only the vehicle group
significantly reinstated as compared to the final day of extinction, while the PJ34 group exhibited
no significant difference in active lever presses when comparing the last day of extinction and
the cue-primed reinstatement test. However, there was no significant difference between the
vehicle and control groups in active lever presses during reinstatement. This suggests that while
the treatment of PJ34 was not effective in significantly reducing seeking behaviors when
compared to the vehicle group, PJ34 has the potential to reduce the salience of light and tone
cues to inhibit seeking behaviors. More studies will be required to confirm this result and more
effectively power the study.
Strengths & Weaknesses of Current Study
A strength of the study was the ability to assess cue-induced reinstatement. In humans,
drug-associated cues can cause cravings, induce seeking behaviors, and initiate relapse
(Childress et al., 1993), suggesting that targeting cue-primed reinstatement is clinically relevant.
Past studies have shown drug seeking behaviors can be profoundly influenced by drug cues for a
long period following cessation of drug use in both animal models (Lu, Grimm, Hope, &
PARP-1 INHIBITION AND COCAINE SEEKING
18
Shaham, 2004; Madangopal et al., 2019) and humans (Parvaz, Moeller, & Goldstein, 2016).
Secondly, the PARP-1 inhibitor used in this study, PJ34, is a highly specific and potent inhibitor.
Use of PJ34 results in 10,000 times more PARP inhibition than other PARP inhibitors such as 3-
AB (Abdelkarim et al., 2001). PJ34 also exhibits more specific inhibition, via IC50 values, of
PARP-1 than inhibition of TNKS1 and PARP-2 (Wahlberg et al., 2012).
However, a limitation of this study was intra-CeA cannula placements. Histological
analysis confirmed that only 20 cannulas were positioned correctly above the CeA, indicating
that those other 12 animals did not have PJ34 or vehicle delivery to the CeA during
microinjections. Elimination of rats with inaccurate cannula placement changed our results to
suggest an inhibitory effect of CeA PARP-1 inhibition against reinstatement. However, while the
rats in the PJ34 group did not significantly reinstate, the vehicle-treated rats did. With proper
cannula placement and PJ34 administration to the CeA, it is possible the results would have
confirmed our hypothesis: intra-CeA PARP-1 inhibition would result in a significant reduction of
reinstatement behaviors in comparison to a control group, as previously observed for cocaine
CPP (Lax et al., 2017).
Before repeating this study with adjusted stereotaxic cannula placements, it may be
beneficial to conduct an experiment to see the effects of a systemic PARP-1 inhibitor. Past
research on the use of systemic NAM, a known PARP inhibitor, found that cue-primed
reinstatement can be reduced with chronic administration of NAM (Witt & Reissner, 2019). The
current experiment was designed as a follow-up study to investigate the CeA based on previous
findings involving PARP-1 inhibition and the CeA (Lax et al., 2017) as well as the experimental
overexpression of PARP-1 (Scobie et al., 2014). An experiment utilizing a systemic PARP-1
inhibitor could provide evidence that, when applied across the whole brain, PARP-1 inhibition
PARP-1 INHIBITION AND COCAINE SEEKING
19
has the desired effect on cocaine seeking during a reinstatement paradigm without limiting PJ34
to one, specific brain area.
The Relationship of PARP and NAD+
As well as a PARP inhibitor, NAM is also a precursor for oxidized nicotinamide adenine
dinucleotide (NAD+), which may be beneficial in reversing the neurodegeneration associated
with drug use, as it is a coenzyme necessary for many cell reactions – including metabolism.
Further, NAD+ and PARP-1 affect each other. PARP-1 consumes NAD+ in order to respond to
DNA breakage and complete cellular repair tasks within the nucleus (Morales et al., 2014).
PARPs aid in the repair mechanism for damaged DNA by synthesizing poly-ADP-ribose chains
on proteins, which in turn, signal for DNA repair enzymes to recruit to and mend the breakage
point (Yelamos, Farres, Llacuna, Ampurdanes, & Martin-Caballero, 2011).
While it is involved in DNA repair, too much PARP activation can result in diminished
levels of NAD+ (Zhang et al., 2019) and ATP (Ha & Snyder, 1999). This depletion of NAD+ by
PARP-1 during routine and drug-induced repair can lead to lack of cellular repair and
neurodegeneration (Liu et al., 2009). For this study, these findings were interpreted to suggest
that inhibiting PARP-1, and therefore helping maintain NAD+ levels after drug exposure, may
aid in DNA repair, as DNA in the brain and other organs can be damaged after just a single
exposure to cocaine (Alvarenga et al., 2010). However, the results of systemic administration of
NAM being associated with reduced cue-induced reinstatement (Witt & Reissner, 2019) may be
due to NAM’s role as an NAD+ precursor. It may be advantageous to investigate the effects of
increasing NAD+ levels in the CeA or NAc as the protective nature of NAD+ by NAM
PARP-1 INHIBITION AND COCAINE SEEKING
20
suppresses DNA fragmentation throughout both healthy and necrotic cell death (Klaidman,
Mukherjee, Hutchin, & Adams, 1996).
Brain Regions Involved in Cocaine Seeking
While the CeA’s involvement in motivation and stimuli makes this brain region a
promising target for pharmacotherapies to reduce cocaine seeking, the NAc may also be a
promising target for PARP-1 inhibition for reducing seeking behaviors, especially if systemic
PARP inhibition proves successful. The NAc is a central nucleus in the brain’s mesolimbic
pathway (Adinoff, 2004). Past research has found an increase of PARP-1 levels in the CeA
during CPP (Lax et al., 2017), but similar findings have been found following cocaine exposure
in the NAc (Dash et al., 2017; Scobie et al., 2014). After cocaine exposure in rats, the NAc
undergoes other cocaine-induced adaptions thought to contribute to long-lasting cocaine seeking
(Kalivas, 2009; Scofield et al., 2016). Neuropathological adaptions include changes in
methylation of DNA that alters gene expression (for review: Vaillancourt, Ernst, Mash, &
Turecki, 2017), while subsequent behavioral adaptions include cocaine-induced behavioral
sensitization (Anier, Malinovskaja, Aonurm-Helm, Zharkovsky, & Kalda, 2010), incubation of
cocaine craving (Massart et al., 2015), and addiction (Sadri-Vakili, 2015). Within the self-
administration paradigm, the presentation of cocaine-paired cues trigger rapid synaptic plasticity
in the NAc, which makes the neurons hypersensitive to the incoming excitatory signals (Gipson
et al., 2013). In turn, this sensitization promotes the physical seeking behaviors due to the
outputs from the NAc to the brain’s motor circuits (Gipson et al., 2013). Accordingly,
pharmacologically targeting PARP within the NAc to attenuate cue-induced relapse may be a
worthwhile next step in this study.
PARP-1 INHIBITION AND COCAINE SEEKING
21
Role of Gene Transcription
As addressed above, PARP-1 activity is important for regulation of gene transcription.
PARP-1 can promote the activation of transcription through engagement with gene promoter and
enhancer complexes (Kraus & Lis, 2003). In the NAc, chronic cocaine increased the association
between PARP-1 and NF-kB, a transcription factor, suggesting that cocaine exposure may
enhance PARP-1’s involvement in activating transcriptional complexes (Scobie et al., 2014).
PARP-1 can also repress transcription in certain genes (Kraus, 2008). For example, within the
CeA, PARP-1 may play a role in the transcription regulation involved in cocaine CPP (Lax et al.,
2017). A new gene, D3ZLJ1, was discovered and while it is unknown how D3ZLJ1 is exactly
involved in drug addiction, PARP-1 binds to its promoter to repress gene expression, which in
turn, augments cocaine CPP memory retrieval (Lax et al., 2017). Administration of PJ34 inhibits
this PARP-1 binding to D3ZLJ1, which is associated with increased D3ZLJ1 expression. The
subsequent upregulation of D3ZLJ1 and/or downregulation of PARP-1 reduced cocaine-CPP
memory retrieval (Lax et al., 2017).
Additionally, PARP-1 can affect transcription by preventing Histone H1 from binding to
PARP-1 regulated promotors (Krishnakumar et al., 2008). H1 modulates the chromatin
arrangement (Woodcock, Skoultchi, & Fan, 2006) and is a transcription regulator that can
repress or enhance the transcription of genes (Hergeth & Schneider, 2015). Blocking H1 from
binding has implications for proper regulation of gene expression and chromatin structure
(Krishnakumar et al., 2008). Following chronic cocaine administration, increased expression of
PARP-1 in the mouse NAc is associated with increased levels of PARylated H1, which changes
the chromatin structure to be ready for transcription (Scobie et al., 2014).
Perhaps PARP-1’s modulatory role in transcription contributes to cocaine addiction by
PARP-1 INHIBITION AND COCAINE SEEKING
22
activating transcription on genes that promote drug seeking or repressing transcription on genes
within the brain that help suppress those behaviors. The expression of the PARP-1 gene itself
may be regulated by the binding of PARP-1 to hairpin loops in the DNA molecules (Soldatenkov
et al., 2001), suggesting that cocaine mediated PARP-1 increases may also serve as a feedback
loop that modulates PARP-1 levels within the brain. However, future research should investigate
and build upon these claims.
Conclusion
There is no current evidence to suggest that PARP-1 activity increases in the human brain
after cocaine use. Past research on rodent models, however, provide promising indications that
PARP-1 plays a role in cocaine addiction, which can presumably be translated into the human
model. In support of this translational utility, PARP inhibitors are currently used as treatments
for cancers that are caused by mistakes in the DNA repair genes (for a review paper on clinical
trials see Chen, 2011). PARP inhibition stops the cancer cells from being repaired, leading to cell
death and tumor size reduction (Chen, 2011). In the example of breast cancer, the BRCA DNA
gene is faulty. If the cancerous cells only have PARP to repair the DNA because the BRCA is
compromised, the DNA repair will be incomplete but the compromised cell can still replicate
(Chen, 2011). This can lead to tumor growth as usually cells with broken DNA will trigger their
own death. Inhibiting PARP would cause the cancer cells with broken DNA to undergo
apoptosis and stunt tumor growth (Chen, 2011). An increase of PARP activity has also been
found in inflammation-related diseases, such as neurodegenerative diseases (Pazzaglia & Pioli,
2019).
PARP-1 INHIBITION AND COCAINE SEEKING
23
Presently, there are no FDA-approved pharmacological treatments for CUD or other
psychostimulant use disorders. The most common treatment plans revolved psychosocial
therapies such as contingency management and cognitive behavioral therapy, but none of the
latest therapeutic approaches are particularly effective for most patients (Kampman, 2019). It is
important to find better, more effective treatments for cocaine use. This can be accomplished by
discovering the complex neural mechanism that underlies cocaine addiction and relapse. Strides
have been made to further develop pharmacological treatments, as the effects of cocaine on the
brain reward circuitry are being discovered. Accordingly, the dissemination and conclusions
drawn from this study have the potential to be of importance in pharmacological research,
specifically uncovering the role of PARP-1 in drug addiction.
PARP-1 INHIBITION AND COCAINE SEEKING
24
References
Abdelkarim, G., Gertz, K., Harms, C., Katchanov, J., Dirnagl, U., Szabo, C., & Endres, M.
(2001). Protective effects of PJ34, a novel, potent inhibitor of poly(ADP-ribose)
polymerase (PARP) in in vitro and in vivo models of stroke. International Journal of
Molecular Medicine. doi: 10.3892/ijmm.7.3.255
Alvarenga, T.A., Andersen, M.L., Ribeiro, D.A., Araujo, P., Hirotsu, C., Costa, J.L., Battisti,
M.C. and Tufik, S. (2010), BRIEF REPORT: Single exposure to cocaine or ecstasy
induces DNA damage in brain and other organs of mice. Addiction Biology, 15: 96-99.
doi:10.1111/j.1369-1600.2009.00179.x
Amé, J. C., Spenlehauer, C., & de Murcia, G. (2004). The PARP superfamily. BioEssays, 26,
882–893. doi: 10.1002/bies.20142
Amiri, K. I., Ha, H. C., Smulson, M. E., & Richmond, A. (2006). Differential regulation of CXC
ligand 1 transcription in melanoma cell lines by poly(ADP-ribose) polymerase-
1. Oncogene, 25(59), 7714–7722. doi: 10.1038/sj.onc.1209751
Anier, K., Malinovskaja, K., Aonurm-Helm, A., Zharkovsky, A., & Kalda, A. (2010). DNA
Methylation Regulates Cocaine-Induced Behavioral Sensitization in
Mice. Neuropsychopharmacology, 35(12), 2450–2461. doi: 10.1038/npp.2010.128
Chen, A. (2011). PARP inhibitors: its role in treatment of cancer. Chinese Journal of Cancer,
463–471. doi: 10.5732/cjc.011.10111
Childress, A. R., Hole, A. V., Ehrman, R. N., Robbins, S. J., McLellan, A. T., and O’Brien, C. P.
(1993). Cue reactivity and cue reactivity interventions in drug dependence. NIDA Res.
Monogr. 137, 73–95.
PARP-1 INHIBITION AND COCAINE SEEKING
25
Dash, S., Balasubramaniam, M., Rana, T., Godino, A., Peck, E. G., Goodwin, J. S., Villalta, F.,
Calipari, E. S., Nestler, E. J., Dash, C., & Pandhare, J. (2017). Poly (ADP-Ribose)
Polymerase-1 (PARP-1) Induction by Cocaine Is Post-Transcriptionally Regulated by
miR-125b. eNeuro, 4(4). doi:10.1523/ENEURO.0089-17.2017
Du, L.; Zhang, X.; Han, Y.Y.; Burke, N.A.; Kochanek, P.M.; Watkins, S.C.; Graham, S.H.;
Carcillo, J.A.; Szabo, C.; Clark, R.S. (2003) Intra-mitochondrial poly(ADP-ribosylation)
contributes to NAD+ depletion and cell death induced by oxidative stress. J. Biol. Chem.
278, 18426–18433
Farrell, M. R., Schoch, H., & Mahler, S. V. (2018). Modeling cocaine relapse in rodents:
Behavioral considerations and circuit mechanisms. Progress in Neuro-
Psychopharmacology and Biological Psychiatry, 87, 33–47. doi:
10.1016/j.pnpbp.2018.01.002
Gipson, C. D., Kupchik, Y. M., Shen, H., Reissner, K. J., Thomas, C. A., & Kalivas, P. W.
(2013). Relapse Induced by Cues Predicting Cocaine Depends on Rapid, Transient
Synaptic Potentiation. Neuron, 77(5), 867–872. doi: 10.1016/j.neuron.2013.01.005
Gupte, R., Liu, Z., & Kraus, W. L. (2017). PARPs and ADP-ribosylation: recent advances
linking molecular functions to biological outcomes. Genes & Development, 31(2), 101–
126. doi: 10.1101/gad.291518.116
Ha, H.C.; Snyder, S.H. Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by
ATP depletion. Proc. Natl. Acad. Sci. USA 1999, 96, 13978–13982.
Hergeth, S. P., & Schneider, R. (2015). The H1 linker histones: multifunctional proteins beyond
the nucleosomal core particle. EMBO Reports, 16(11), 1439–1453. doi:
10.15252/embr.201540749
PARP-1 INHIBITION AND COCAINE SEEKING
26
Javle, M., & Curtin, N. J. (2011). The role of PARP in DNA repair and its therapeutic
exploitation. Br J Cancer, 105(8), 1114-1122. Retrieved from
https://www.ncbi.nlm.nih.gov/pubmed/21989215. doi:10.1038/bjc.2011.382
Kampman, K. M. (2019). The treatment of cocaine use disorder. Science Advances, 5(10). doi:
10.1126/sciadv.aax1532
Kim, S.-I. (2013). Neuroscientific Model of Motivational Process. Frontiers in Psychology, 4.
doi: 10.3389/fpsyg.2013.00098
Klaidman, L. K., Mukherjee, S. K., Hutchin, T. P., & Adams, J. D. (1996). Nicotinamide as a
precursor for NAD+ prevents apoptosis in the mouse brain induced by tertiary-
butylhydroperoxide. Neurosci Lett, 206(1), 5-8.
Kraus, W. L. (2008). Transcriptional control by PARP-1: chromatin modulation, enhancer-
binding, coregulation, and insulation. Current Opinion in Cell Biology, 20(3), 294–302.
doi: 10.1016/j.ceb.2008.03.006
Kraus, W. L., & Hottiger, M. O. (2013). PARP-1 and gene regulation: progress and puzzles.
Molecular Aspects of Medicine, 34(6), 1109-1123. doi:10.1016/j.mam.2013.01.005
Kraus, W., & Lis, J. T. (2003). PARP Goes Transcription. Cell, 113(6), 677–683. doi:
10.1016/s0092-8674(03)00433-1
Krishnakumar, R., Gamble, M. J., Frizzell, K. M., Berrocal, J. G., Kininis, M., & Kraus, W. L.
(2008). Reciprocal Binding of PARP-1 and Histone H1 at Promoters Specifies
Transcriptional Outcomes. Science, 319(5864), 819–821. doi: 10.1126/science.1149250
Lax, E., Friedman, A., Massart, R., Barnea, R., Abraham, L., Cheishvili, D., Zada, M., Ahdoot,
H., Bareli, T., Warhaftig, G., Visochek, L., Suderman, M., Cohen-Armon, M., Szyf, M.,
& Yadid, G. (2017). PARP-1 is required for retrieval of cocaine-associated memory by
PARP-1 INHIBITION AND COCAINE SEEKING
27
binding to the promoter of a novel gene encoding a putative transposase inhibitor. Mol
Psychiatry, 22(4), 570-579. doi:10.1038/mp.2016.119
Liu, D., Gharavi, R., Pitta, M., Gleichmann, M., & Mattson, M. P. (2009). Nicotinamide prevents
NAD+ depletion and protects neurons against excitotoxicity and cerebral ischemia:
NAD+ consumption by SIRT1 may endanger energetically compromised neurons.
Neuromolecular Med, 11(1), 28-42. doi:10.1007/s12017-009-8058-1
Lu, L., Grimm, J. W., Hope, B. T., & Shaham, Y. (2004). Incubation of cocaine craving after
withdrawal: a review of preclinical data. Neuropharmacology, 47, 214–226. doi:
10.1016/j.neuropharm.2004.06.027
Lu, L., Hope, B. T., Dempsey, J., Liu, S. Y., Bossert, J. M., & Shaham, Y. (2005). Central
amygdala ERK signaling pathway is critical to incubation of cocaine craving. Nature
Neuroscience, 8(2), 212–219. doi: 10.1038/nn1383
Lu, L., Uejima, J. L., Gray, S. M., Bossert, J. M., & Shaham, Y. (2007). Systemic and Central
Amygdala Injections of the mGluR2/3 Agonist LY379268 Attenuate the Expression of
Incubation of Cocaine Craving. Biological Psychiatry, 61(5), 591–598. doi:
10.1016/j.biopsych.2006.04.011
Malanga, M., & Althaus, F. R. (2005). The role of poly(ADP-ribose) in the DNA damage
signaling network. Biochemistry and Cell Biology, 83(3), 354–364. doi: 10.1139/o05-038
Madangopal, R., Tunstall, B. J., Komer, L. E., Weber, S. J., Hoots, J. K., Lennon, V. A., …
Hope, B. T. (2019). Author response: Discriminative stimuli are sufficient for incubation
of cocaine craving. ELIFE. doi: 10.7554/elife.44427.015
Massart, R., Barnea, R., Dikshtein, Y., Suderman, M., Meir, O., Hallett, M., … Yadid, G. (2015).
Role of DNA Methylation in the Nucleus Accumbens in Incubation of Cocaine
PARP-1 INHIBITION AND COCAINE SEEKING
28
Craving. Journal of Neuroscience, 35(21), 8042–8058. doi: 10.1523/jneurosci.3053-
14.2015
Morales, J., Li, L., Fattah, F. J., Dong, Y., Bey, E. A., Patel, M., … Boothman, D. A. (2014).
Review of poly (ADP-ribose) polymerase (PARP) mechanisms of action and rationale for
targeting in cancer and other diseases. Critical reviews in eukaryotic gene
expression, 24(1), 15–28. doi:10.1615/critreveukaryotgeneexpr.2013006875
Namba, M. D., Tomek, S. E., Olive, M. F., Beckmann, J. S., & Gipson, C. D. (2018). The
Winding Road to Relapse: Forging a New Understanding of Cue-Induced Reinstatement
Models and Their Associated Neural Mechanisms. Frontiers in Behavioral
Neuroscience, 12. doi: 10.3389/fnbeh.2018.00017
NIDA. (2018, July 13). Cocaine. Retrieved from
https://www.drugabuse.gov/publications/drugfacts/cocaine.
Parvaz, M. A., Moeller, S. J., & Goldstein, R. Z. (2016). Incubation of Cue-Induced Craving in
Adults Addicted to Cocaine Measured by Electroencephalography. JAMA
Psychiatry, 73(11), 1127. doi: 10.1001/jamapsychiatry.2016.2181
Pazzaglia, S., & Pioli, C. (2019). Multifaceted Role of PARP-1 in DNA Repair and
Inflammation: Pathological and Therapeutic Implications in Cancer and Non-Cancer
Diseases. Cells, 9(41). doi: 10.3390/cells9010041
Perry, C. J., Zbukvic, I., Kim, J. H., & Lawrence, A. J. (2014). Role of cues and contexts on
drug-seeking behaviour. British Journal of Pharmacology, 171(20), 4636–4672. doi:
10.1111/bph.12735
Rasia-Filho, A. A., Londero, R. G., & Achaval, M. (2000). Functional activities of the amygdala:
an overview. Journal of psychiatry & neuroscience : JPN, 25(1), 14–23.
PARP-1 INHIBITION AND COCAINE SEEKING
29
Riklis, E., Kol, R., & Marko, R. (1990). Trends and developments in radioprotection: the effect
of nicotinamide on DNA repair. Int J Radiat Biol, 57(4), 699-708. Retrieved from
https://www.ncbi.nlm.nih.gov/pubmed/1969902.
Sadri-Vakili, G. (2015). Cocaine triggers epigenetic alterations in the corticostriatal
circuit. Brain Research, 1628, 50–59. doi: 10.1016/j.brainres.2014.09.069
Scobie, K. N., Damez-Werno, D., Sun, H., Shao, N., Gancarz, A., Panganiban, C. H., Dias, C.,
Koo, J., Caiafa, P., Kaufman L., Neve, R. L., Dietz, D. M., Shen, L., & Nestler, E. J.
(2014). Essential role of poly(ADP-ribosyl)ation in cocaine action. Proc Natl Acad Sci U
S A, 111(5), 2005-2010. doi:10.1073/pnas.1319703111
Soldatenkov, V. A., Chasovskikh, S., Potaman, V. N., Trofimova, I., Smulson, M. E., &
Dritschilo, A. (2001). Transcriptional Repression by Binding of Poly(ADP-ribose)
Polymerase to Promoter Sequences. Journal of Biological Chemistry, 277(1), 665–670.
doi: 10.1074/jbc.m108551200
Tang, D., Fellows, L., Small, D., & Dagher, A. (2012). Food and drug cues activate similar brain
regions: A meta-analysis of functional MRI studies. Physiology & Behavior, 106(3),
317–324. doi: 10.1016/j.physbeh.2012.03.009
Vaillancourt, K., Ernst, C., Mash, D., & Turecki, G. (2017). DNA Methylation Dynamics and
Cocaine in the Brain: Progress and Prospects. Genes, 8(5), 138. doi:
10.3390/genes8050138
Vyas, S., Chesarone-Cataldo, M., Todorova, T., Huang, Y.-H., & Chang, P. (2013). A systematic
analysis of the PARP protein family identifies new functions critical for cell
physiology. Nature Communications, 4(1). doi: 10.1038/ncomms3240
PARP-1 INHIBITION AND COCAINE SEEKING
30
Wahlberg, E., Karlberg, T., Kouznetsova, E., Markova, N., Macchiarulo, A., Thorsell, A.-G., …
Weigelt, J. (2012). Family-wide chemical profiling and structural analysis of PARP and
tankyrase inhibitors. Nature Biotechnology, 30(3), 283–288. doi: 10.1038/nbt.2121
Warlow, S. M., Robinson, M. J., & Berridge, K. C. (2017). Optogenetic Central Amygdala
Stimulation Intensifies and Narrows Motivation for Cocaine. The Journal of
Neuroscience, 37(35), 8330–8348. doi: 10.1523/jneurosci.3141-16.2017
Witt, E. A., & Reissner, K. J. (2019). The effects of nicotinamide on reinstatement to cocaine
seeking in male and female Sprague Dawley rats. Psychopharmacology. doi:
10.1007/s00213-019-05404-y
Woodcock, C. L., Skoultchi, A. I., & Fan, Y. (2006). Role of linker histone in chromatin
structure and function: H1 stoichiometry and nucleosome repeat length. Chromosome
Research, 14(1), 17–25. doi: 10.1007/s10577-005-1024-3
Yu, S.W. Wang, H., Poitras, M.F., Coombs, C., Bowers, W.J., Federo, H.J., Poirier, G.G.,
Dawson, T.M., & Dawson, V.L. (2002). Mediation of poly(ADP-ribose) polymerase-1-
dependent cell death by apoptosis-inducing factor. Science, 297, 259–263.
Zhang, D.; Hu, X.; Li, J.; Liu, J.; Baks-te Bulte, L.; Wiersma, M.; Malik, N.-u.-A.; van Marion,
D.M.S.; Tolouee, M.; Hoogstra-Berends, F.; et al. DNA damage-induced PARP1
activation confers cardiomyocyte dysfunction through NAD+ depletion in experimental
atrial fibrillation. Nat. Commun. 2019, 10, 1307.