PARKIN REGULATES DRUG TAKING-BEHAVIOR IN RAT MODEL OF METHAMPHETAMINE USE DISORDER

There is no FDA-approved medication for Methamphetamine (METH) Use Disorder. New therapeutic approaches are needed, especially for people who use METH heavily and are at high risk for overdose. This study used genetically engineered rats to evaluate PARKIN as a potential target for METH Use Disorder. PARKIN knockout, PARKIN-overexpressing and wild-type young adult male Long Evans rats were trained to self-administer high doses of METH using an extended-access METH self-administration paradigm. Reinforcing/rewarding properties of METH were assessed by quantifying drug-taking behavior and time spent in a METH-paired environment. PARKIN knockout rats self-administered more METH and spent more time in the METH-paired environment than wild-type rats. Wild-type rats overexpressing PARKIN self-administered less METH and spent less time in the METH-paired environment. PARKIN knockout rats overexpressing PARKIN self-administered less METH during the first half of drug self-administration days than PARKIN-deficient rats. The results indicate that rats with PARKIN excess or PARKIN deficit are useful models for studying neural substrates underlying “resilience” or vulnerability to METH Use Disorder, and identify PARKIN as a novel potential drug target to treat heavy use of METH.


Methamphetamine (METH) Use Disorder is a world-wide health problem. In the United
States, there are more than 900,000 people who use METH and deaths from METH overdose are rapidly rising 1,2 . Despite numerous clinical trials conducted to date, there is no FDA-approved medication for METH Use Disorder. The medications tested in clinical trials have shown low efficacy in people who use METH moderately and no effect in those who use METH heavily [3][4][5] .
New therapeutic approaches are needed, particularly for people who use METH heavily, as they suffer the most from METH abuse-related neuropsychological problems [6][7][8] , are less likely to seek treatment than those using the drug moderately 9 , and are at high risk of dying from METH overdose 10 . Early intervention in METH abuse by lowering METH intake is essential not only for preventing METH overdose, but also for subsequent interventions, as greater treatment participation is achieved when METH use is low 9 .
Drug addiction is a chronic, relapsing disorder that has been characterized by compulsive seeking and escalated intake of drugs. In the current study, we used genetically engineered rats to evaluate PARKIN as a potential target for METH Use Disorder. PARKIN knockout (PKO), PARKIN-overexpressing (PO) and wild-type (WT) young adult male Long Evans rats were trained to self-administer high doses of METH using an extended-access METH selfadministration (EA METH SA) paradigm with three progressive ratio schedules. This paradigm produced an escalation of METH intake with rats working much harder to obtain METH with increasing ratio schedule and, therefore, modeled an important aspect of human drug abuse (compulsive drug taking) 32 and heavy consumption of METH seen in some people dependent on METH. Binging observed in people who use METH heavily can last 3-15 days. To approximate binging behavior, rats were allowed to self-administer high METH doses for 10 days. Rewarding properties of METH were assessed by measuring the number of lever presses for METH and METH intake during each of 10 operant sessions. Rewarding properties of METH were assessed by measuring time spent in a METH-paired environment. We demonstrate that PKO rats are predisposed to heavy METH use compared to WT counterparts whereas rats overexpressing PARKIN in the nucleus accumbens (a key area mediating rewarding/reinforcing effects) are less predisposed.

Animals
The study employed young adult male Long Evans rats (~55 days-old at the beginning of the study, N=177) of four genotypes: WT rats, PKO rats (Park2 -/-), PO rats, and PKO PO rats (Horizon Discovery, Missouri, MO/Envigo, Indianapolis, IN, USA). PO rats were generated by bilaterally overexpressing PARKIN in the nucleus accumbens of WT rats whereas PKO PO rats were generated by bilaterally overexpressing PARKIN in the nucleus accumbens of PKO rats.
The stereotaxic surgery was performed as previously published 33 , with some modifications.
Microinjections were done at a 16° angle into these coordinates: +1.8 A/P, ±3.2.M/L, -7.6 D/V from bregma. Validation of PARKIN protein loss in PKO rats is shown in Fig.2 whereas validation of parkin overexpression is shown in Fig.3. Upon arrival, animals were pair-housed and maintained under standard environmental conditions in an AAALAC-accredited vivarium.
Animals were maintained on a 12 h light/dark cycle with ad libitum access to food and water unless specified otherwise. Body weight was monitored on a daily basis before and during experiments (Suppl. Fig.S1). All experiments were approved by the Wayne State University Institutional Animal Care and Use Committee.

Extended-Access Methamphetamine Self-Administration (EA METH SA) and Conditioned Place Preference Test
In the first experiment, PKO and WT rats were trained to self-administer high doses of METH (measurement of METH reinforcement) (Fig.1A) or subjected to the conditioned place preference test (measurement of METH reward) ( Fig.2A). In the second experiment, the reinforcing/rewarding properties of METH were assessed in PO rats as compared to WT rats. In the third experiment, PARKIN was overexpressed in the nucleus accumbens of PKO rats that were subsequently compared to non-overexpressing PKO rats (phenotype rescue experiment).
Supplementary Online Data provides extensive details on stereotaxic surgery as well as the EA METH SA and conditioned place preference.

Electrophoresis and Western Blotting
Rats were sacrificed 10d after the last operant session. Nucleus accumbens was punched out of 2mm-thick coronal brain section encompassing 0.7-2.7mm from bregma. Electrophoresis and western blotting on nucleus accumbens pieces were performed as previously described 34 , utilizing anti-PARKIN and anti-β-ACTIN (1:1,000, 2132S and 3700S, respectively, Cell Signaling Technology, Danvers, MA, USA) primary antibodies. Immunoreactivities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and presented as relative optical densities normalized to control samples.

Statistical Analyses
The experimental data sets were analyzed using IBM SPSS v.25 (IBM, Armonk, NY). EA METH SA data was analyzed with two-way mixed-design ANOVA whereas METH conditioned place preference data was analyzed with two-way factorial ANOVA, both with Bonferroni correction. Before each ANOVA, the data was examined for the presence of outliers using an IQR of 3. After the removal of outlier(s) the data was examined for normality with Shapiro-Wilk's test, equality of variances (Levene's test), and sphericity (Mauchly's test). When Mauchly's test was statistically significant, a Greenhouse-Geisser correction was employed.
Group differences in PARKIN levels were assessed with the Student's t-test. To assess sizes of genotype-mediated effects and probabilities of the presence of the effects, partial eta square (hp 2 ) and power of analysis (1-b) values were calculated. Statistical significance was set at p³0.05. All data, with the exception of body weight data, is reported as mean ± SEM. Body weight data is reported as mean ± SD (Suppl. Fig.S1).

Parkin Knockout Increases Extended-Access METH Self-Administration
To determine whether PKO rats are more dependent on METH as compared to WT rats, the rats from both genotypes were subjected to the EA METH SA test (Fig.1A). Both WT and PKO rats showed an escalation of METH self-administration (Fig.1B, C) The PKO rats weighed more than WT rats but lost weight at the same rate as WT rats during EA METH SA (Fig.S1A).
Including body weight as a covariate in ANOVA analysis showed that it did not influence   8, p<0.001, hp 2 =0.729, 1-b=1.0), but there was no significant interaction between the two factors (F(3.6,71)=1.40, p=0.246). The pairwise comparisons revealed that PKO rats self-administered significantly more METH during the first two sessions (p<0.01 and p<0.001, respectively) and during the compulsive phase of METH self-administration (day 7, p<0.05 and days 8-10, p<0.01) (Fig.1C). There was no significant difference between PKO and WT rats in respect to number of presses for saline ( Fig.1B insert, n=4/genotype), confirming that METH, not the light stimulus, acted as a reinforcer.

8
The results from the EA METH SA indicate that METH exerts stronger reinforcing effects in rats lacking PARKIN than in WT controls. The effect of PARKIN knockout on METH self-administration was of large size (large hp 2 ) and significant, thus strongly suggesting PARKIN as a suppressor of reinforcing effects of METH.

Parkin Knockout Increases Preference for METH-Paired Compartment
Compulsive drug use is maintained by the rewarding properties of the drug and by the loss of control over drug intake. To determine whether PARKIN knockout increases the rewarding effects of METH, PKO and WT rats underwent the conditioned place preference test (Fig.1D).
Though the conditioned place preference test has limitations 35  Pairwise comparisons revealed that there was no significant difference in baseline preference for a compartment between PKO and WT rats (p=0.834) (Fig.1E). Both genotypes showed a significant METH-induced conditioned place preference (p<0.001), with the PKO rats displaying significantly higher preference for METH-paired compartment than the WT rats (p<0.05) ( Fig.1F). ANCOVA determined that body weight had no influence on the results (not shown).
The results from the conditioned place preference test suggest that METH-paired contextual cues are more rewarding for PKO rats than for WT rats.

Accumbens
We previously determined that METH binge decreases PARKIN levels in the striatum via oxidative damage 36 . To determine whether exposure of WT rats to EA METH SA paradigm decreases PARKIN levels in the nucleus accumbens, PARKIN levels were assessed in rats that self-administered METH at 10 days after the last operant session and compared to PARKIN levels in rats exposed to saline. The nucleus accumbens tissue samples from PKO rats were included in the analysis to ascertain the lack of PARKIN protein in these samples. METHexposed rats had significantly lower levels of accumbal PARKIN compared to saline-exposed rats (-24%, t=3.02, df=15, p<0.01, n=8-9) (Fig.2B).

Validation of Parkin Overexpression
Viral vector microinjections took place 3 weeks before METH self-administration or conditioned place preference experiments to allow for maximal PARKIN overexpression ( Fig.3A). Investigation of the microinjection sites using Trypan blue dye confirmed that during microinjections, the needle reached approximately the middle of the nucleus accumbens (Fig.3B) and that 2µL of the dye did not spread beyond the borders of the nucleus accumbens (Fig.3B, C).
We next assessed whether non-coding AAV2/6 or microinjection surgery has an effect on the outcome of EA METH SA or METH conditioned place preference test. Rats were microinjected with non-coding AAV or underwent sham microinjections with 2uL saline. These groups were  (Fig.3G). Similarly, these experimental groups did not significantly differ in respect to time spent in the saline-paired compartment (p>0.1). There was no statistically significant interaction between treatment and different microinjections on METH-paired place preference (p>0.1). The NC-AAV and SM groups did not differ from non-microinjected WT rats in respect to self-administered METH or degree of preference for the METH-paired compartment. Comparison of the three conditions are summarized in Fig.3H and Fig.3I. This data indicated that neither non-coding AAV2/6 nor sham microinjections influenced the outcomes of EA METH SA or METH conditioned place preference test.

Time in METH-Paired Compartment than Wild-type Controls
Since WT rats subjected to EA METH SA had lower PARKIN levels in the nucleus accumbens than saline controls, we next investigated whether overexpression of PARKIN in the nucleus accumbens of WT rats would result in attenuated METH self-administration as compared to non-overexpressing WT rats. Two concentrations of PARKIN-AAV2/6 were utilized to overexpress PARKIN -2×10 7 or 6.5×10 7 TUs/side (4-6 or 15-20-fold excess of PARKIN). The PO rats weighed slightly less than WT controls (Fig.S1B); however, body weight did not have a significant effect on METH self-administration as a covariate (F(1,9)  WT rats overexpressing PARKIN at high levels spent less time in METH-paired compartment in the conditioned preference test than non-overexpressing WT controls (Fig.4F).
Two-way ANOVA revealed the main effect of treatment on METH preference (F(1,20) to the saline-paired compartment, was detected in WT rats (p<0.0001) but not in PO rats (p=0.123), however, there was a difference between genotypes in METH group (p<0.05) (Fig.4F). The ANCOVA analysis determined that body weight had no influence on the results (not shown).
These results further support the conclusion that PARKIN negatively regulates reinforcing/rewarding properties of METH.

Parkin Knockout Rats Overexpressing PARKIN Self-administer Less METH During the First Half of Drug Self-administration Compared to PARKIN-deficient Rats.
The next experiment aimed to determine whether overexpression of PARKIN in the nucleus accumbens of PKO rats would attenuate METH self-administration as it did in nucleus accumbens of WT rats. High levels (15-20-fold) of PARKIN were overexpressed in the nucleus accumbens of PKO rats (n=6/group), while control rats were microinjected with saline (sham microinjection surgery). PKO and PKO PO rats did not differ in respect to body weight during 13 the 10 days of METH SA (Supp. Fig.1C). There was a significant main effect of time (F(1.2,11)=192, p<0.0001, hp 2 =0.838, 1-b=1.0) and a weaker main effect of genotype (F(1,9)=5.15, p=0.05, hp 2 =0.364, 1-b=0.525) on lever presses for METH between the genotypes (Fig.5A).
There was no statistically significant interaction between the time and genotype (p>0.1).
Examination of METH intake data produced similar results: a significant main effect of time

DISCUSSION
METH Use Disorder is a disorder without a pharmacotherapy and, therefore, in need of new drug targets for development of new drugs. In this study, we used genetically engineered rats to evaluate PARKIN as a potential target for METH Use Disorder. We found that PARKIN knockout rats self-administered more METH and spent more time in the METH-paired environment than WT rats whereas WT rats overexpressing PARKIN in the nucleus accumbens self-administered less METH and spent less time in the METH-paired environment. We also found that PARKIN knockout rats overexpressing PARKIN in the nucleus accumbens selfadministered less METH during the first half of drug self-administration days than PARKIN-deficient rats. The results indicate that rats with PARKIN excess or PARKIN deficit are useful models for studying neural substrates underlying "resilience" or vulnerability to METH Use Disorder, and identify PARKIN as a novel potential drug target to treat heavy use of METH.

The Role of PARKIN in METH-Taking Behavior
Drug-taking behavior is a consequence of reinforcing effects of the drug of abuse, mediated by goal-directed and habit systems in the brain. At the initial stages of drug self-administration, the goal-directed system directs the relationship between action (lever pressing) and outcome (drug infusion and experiencing sought-after drug effects). After prolonged drug taking, instrumental action becomes compulsive and habitual 37 . Escalation of drug self-administration is a hallmark of compulsive drug taking and drug dependence 32 . Hence, escalation of METH selfadministration by both PKO and WT rats despite increasing effort required to obtain METH (from 1 to 5 presses needed to obtain METH injection) suggests increasing motivation to obtain the drug, leading to METH dependence. Compared to the WT controls, the PKO rats pressed significantly more for METH and, consequently, consumed more METH than WT rats during the initial stage (FR1) and during the FR5 stage (compulsive intake) of EA METH SA. In contrast, the PO rats pressed significantly less for METH and consumed more of the drug at these stages. Two conclusions can be reached based on these findings. First is that PARKIN may be involved in negative modulation of reinforcing/rewarding effects of METH within the nucleus accumbens, which is the main brain region mediating rewarding/reinforcing effects 38 . This conclusion is supported by the results from the conditioned place preference test. The conditioned place preference test and self-administration measure different processes, namely drug reward and context-drug associative learning vs. acquisition of a drug reinforced operant response, respectively. However, nucleus accumbens involvement and reward are common and there is reasonable concordance between drugs that produce conditioned place preference and drugs that are self-administered 35 . Because both paradigms involve associative learning 35,39 and because the nucleus accumbens plays a role in this type of learning 37,40,41 , PARKIN may have a role in processes associating METH with stimuli and context. In addition to the aforementioned functions, the nucleus accumbens controls goal-directed behavior during instrumental conditioning 37,[42][43][44][45] . Consequently, the second conclusion is that accumbal PARKIN may negatively regulate goal-directed behavior during METH self-administration. We demonstrated that the volume spread after the microinjections was largely confined to the nucleus accumbens.
Therefore, it is unlikely that the difference in METH self-administration observed between PO and WT rats was due to unspecific PARKIN overexpression in the adjacent dorsomedial striatum, which is a key brain area regulating goal-directed behavior 37,46-48 , but rather to overexpression of PARKIN in the nucleus accumbens, which has a role in action selection 45 .
Habitual drug taking is mediated by the dorsolateral striatum 37,46,49,50 and it is not known whether striatal PARKIN affects this behavior. Overexpression of PARKIN in the nucleus accumbens likely resulted in its excess not only in accumbal perikarya, but also in terminals located in brain areas to which the nucleus accumbens projects to 33 . Consequently, PARKIN could have presynaptically modulated METH taking in these brain areas. Even though PKO and PO rats displayed opposite addictive behaviors, molecular mechanisms underlying these behaviors may be different due to compensatory mechanisms that occurred in the brains of PKO but not in PO rats during development 51 . Such changes might explain the smaller effect of PARKIN overexpression on METH taking in PKO PO rats as compared to PO rats.

Molecular Determinants of Altered METH-Taking Behavior in PKO and PO Rats
In relation to the present investigation, studies in young adult PKO mice (3-4-month-old) reported no change in the levels of striatal dopamine or dopamine receptors as compared to WT littermates 52,53 , with dopamine metabolites reported unchanged or elevated 54,55 . Our previous study in young adult PKO rats (2-month-old) similarly found no change in dopamine and dopamine metabolites but reported that PKO rats had lower levels of glycosylated form of postsynaptic D2 receptor than WT rats in the dorsal striatum 34 . Low D2/D3 receptor availability in striatal regions, including the nucleus accumbens, is considered a risk factor for METH Use Disorder because it is a molecular feature of impulsivity; animal as well as human data support this notion 56,57 . Furthermore, it has been demonstrated that D2-expressing medium spiny neurons in the nucleus accumbens antagonize the reinforcing and rewarding effects of another psychostimulant, cocaine 58 . Parkin gene knockout results in augmented glutamatergic neurotransmission and altered levels of glutamate receptors as well as GABAB receptors in mice 31 . This data suggests the involvement of PARKIN protein in regulation of these receptors.
METH Use Disorder has been linked to alterations in energy metabolism and cytoskeletal arrangement as well as to oxidative stress and inflammation 11-21,65-67 . For example, METHinduced inflammation in the nucleus accumbens was shown to mediate METH reinforcing properties 11,65 . In the EA METH SA paradigm, large doses of METH were self-administered, likely causing oxidative stress and inflammation in multiple brain areas, including the nucleus accumbens 11, 68 . PARKIN has been linked to energy metabolism, and cytoskeletal arrangement as well as to protection against oxidative stress, and inflammation 25,26,[29][30][31] . Therefore, PARKIN is well-positioned to regulate responses to METH and could have decreased METH intake in rats via any of the aforementioned mechanisms.
Addiction circuitry includes multiple brain areas 69 , all of which have likely undergone neuroadaptive changes in the PKO rats during development 51 . We hypothesize that because of these changes and lack of PARKIN in addiction circuitry, overexpression of PARKIN in the nucleus accumbens only had smaller effect on METH self-administration in PKO rats than WT rats. Overexpression of PARKIN in the nucleus accumbens and prefrontal cortex and/or dorsal striatum may be necessary to suppress the addictive phenotype in PKO rats. Data from PKO PO rats suggest that PARKIN has stronger effect on molecular mechanisms underlying reinforcing properties than on those underlying loss of control over METH intake in these rats. It remains to be determined whether PARKIN overexpression in the nucleus accumbens of PKO rats would have more pronounced effect on METH self-administration in a paradigm with short-access to the drug (e.g. 1h), consequently leading to low METH intake.

Limitations and Methodological Considerations
A limitation of using PKO rats is that developmental adaptations to the absence of the parkin gene may have compensated for an early loss of gene function. As a result, such adaptations may have obscured the analysis of the effects of PARKIN protein per se. Nevertheless, overexpression of PARKIN resulted in behaviors opposite to those observed in PKO rats, thus validating the role of PARKIN in METH self-administration. High level of PARKIN overexpression was needed for a desired effect, which can be viewed as non-translational.
However, since we employed a model of very high METH intake, lower levels of PARKIN overexpression will likely work with models of moderate METH intake.

Conclusions
To date, pharmacological approaches have failed as treatments for METH Use Disorder. We demonstrated for the first time that PARKIN plays important role in this disorder. Specifically, we demonstrated that PARKIN modulates the rewarding/reinforcing properties of high-dose METH. The role of PARKIN in drug use disorder has not been previously studied; therefore, our study is novel. From the public health point of view, the most important finding of this investigation is that overexpression of PARKIN in the nucleus accumbens alone was sufficient to attenuate intake of very high doses of METH by WT rats, making PARKIN a novel potential drug target to treat heavy use of METH. In addition, we demonstrated that rats with PARKIN excess or PARKIN deficit are useful models for studying neural substrates underlying "resilience" or vulnerability to METH Use Disorder.