Dysregulation of AMPA Receptor Trafficking and Intracellular Vesicular Sorting in the Prefrontal Cortex of Dopamine Transporter Knock-Out Rats

Dopamine (DA) and glutamate interact, influencing neural excitability and promoting synaptic plasticity. However, little is known regarding the molecular mechanisms underlying this crosstalk. Since perturbation of DA-AMPA receptor interaction might sustain pathological conditions, the major aim of our work was to evaluate the effect of the hyperactive DA system on the AMPA subunit composition, trafficking, and membrane localization in the prefrontal cortex (PFC). Taking advantage of dopamine transporter knock-out (DAT−/−) rats, we found that DA overactivity reduced the translation of cortical AMPA receptors and their localization at both synaptic and extra-synaptic sites through, at least in part, altered intracellular vesicular sorting. Moreover, the reduced expression of AMPA receptor-specific anchoring proteins and structural markers, such as Neuroligin-1 and nCadherin, likely indicate a pattern of synaptic instability. Overall, these data reveal that a condition of hyperdopaminergia markedly alters the homeostatic plasticity of AMPA receptors, suggesting a general destabilization and depotentiation of the AMPA-mediated glutamatergic neurotransmission in the PFC. This effect might be functionally relevant for disorders characterized by elevated dopaminergic activity.


Introduction
Dopamine (DA) neurotransmission in the prefrontal cortex (PFC) mediates a variety of adaptive behaviors essential for survival, such as goal-oriented behavior, reward learning, cognitive flexibility, and executive functions [1][2][3][4]. Among the neuromodulatory roles of DA in the PFC, it has been widely demonstrated that it modulates and integrates glutamatergic synaptic transmission and differently impacts the excitability of pyramidal neurons through activation of D1 and D2 dopaminergic receptors [5][6][7]. Most pyramidal neurons in the PFC express-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and are innervated by dopaminergic fibers [8,9]. Moreover, it has been recently demonstrated that in vivo pharmacological manipulations of DA receptors modulate local glutamatergic transmission via AMPA receptor phosphorylation [10]. Indeed, DA and glutamate systems have been shown to bidirectionally communicate, and interference with this interaction underlies several psychiatric disorders, such as addiction, schizophrenia, depression, posttraumatic stress disorder, and attention deficit hyperactivity disorder [5,[11][12][13][14][15].
At the synapse, AMPA receptors mediate the majority of fast excitatory neurotransmissions in the brain, depending on their number, localization, and subunit composition. In fact, the amount and composition of AMPA receptors within the postsynaptic density (PSD) of dendritic spines determine synaptic efficacy and affect neuron excitability [16].

Animals and Housing
Zinc-finger nuclease (ZNF) design, construction, in vitro validation, microinjection, and founder selection were performed, as previously described [37]. The target site for ZFN was CTCATCAACCCGCCACAGAcaccaGTGGAGGCTCAAGAG in Exon 2 of the Slc6a3 gene (NCBI Gene ID: 24898; Genomic NCBI Ref Seq: NC_005100.3; mRNA NCBI Ref Seq: NM_012694.2). The dopamine transporter (DAT) knocked-out (DAT −/− ) lines were created in the outbred Wistar Han background at SAGE Labs. Animals were housed, divided by their respective gender, in groups of three to four with water and food ad libitum. Rats were kept under standard conditions at 22 • C and on a 12 h light/dark cycle (light from 07:00 h to 19:00 h). All experiments were conducted following the guidelines established by the European Community Council (Directive 2010/63/EU of 22 September 2010) and were approved by the Belgian Ministry of Health (Neurosciences LA1500024). All efforts were pursued to minimize animal suffering and to reduce the number of animals used in the experiment.

Protein Extract Preparation and Western Blot Analysis
Adult male rats, both Wistar wild-type DAT +/+ (n = 6) and DAT −/− (n = 6), were sacrificed by decapitation, brains were collected, and, immediately after, the medial prefrontal cortices (mPFC) were dissected from 2 mm thick slices in accordance with the Rat Brain Atlas of Paxinos and Watson [38]. Then, PFCs were frozen on dry ice and stored at −80 • C for upcoming molecular analysis.
Proteins in the whole homogenate, post-synaptic, and extra-synaptic fractions were analyzed, as previously described [39]. Proteins from medial prefrontal cortex (mPFC) tissues were homogenized in a Teflon-glass potter using a cold buffer pH 7.4, containing 0.32 M sucrose, 0.1 mM PMSF, 1 mM HEPES, 1 mM MgCl 2 , and 1 mM NaHCO 3 in the presence of commercial cocktails of protease (cOmplete™ Protease Inhibitor Cocktail, Roche, Monza, Italy) and phosphatase (Sigma-Aldrich, Milan, Italy) inhibitors. An aliquot of the whole homogenate was kept, sonicated, and stored at −20 • C. The remaining homogenate was centrifuged at 800× g for 5 min, and the resulting supernatant was subsequently centrifuged at 13,000× g for 15 min. The pellet obtained was resuspended in a buffer containing 75 mM KCl and 1% Triton X-100 and centrifuged at 100,000× g for 1 h. The resulting supernatant, also called Triton X-100 soluble fraction (TSF, extra-synaptic fraction), was stored at −20 • C, while the pellet, also called Triton X-100 insoluble fraction (TIF, post-synaptic density fraction), was homogenized in a glass-glass potter in 20 mM HEPES, glycerol 30%, protease and phosphatase inhibitors, and stored at −20 • C. The total protein amount was measured in the homogenate, TIF, and TSF according to the Bradford Protein Assay kit from Bio-Rad (Milan, Italy), using bovine serum albumin as the calibration standard. Identical quantities of proteins from homogenate (8 mg), TIF fraction (8 mg), and TSF fraction (35 mg) were run on a sodium dodecyl sulfate 8% or 14% polyacrylamide gel under reducing conditions, and they were next transferred into a nitrocellulose membrane (GE Healthcare, Milan, Italy). Membranes were cut to allow the simultaneous detection of more proteins on one gel; then, blots were blocked for 1 h at room temperature (RT) with I-Block solution (Life Technologies Italia, Monza, Italy) in TBS 0.1% Tween-20 buffer and incubated with antibodies against proteins of interest. To allow the detection of more determinants on the same blot, the latter was stripped, blocked, and re-incubated with another antibody.
The conditions of the primary antibodies were the following: anti-vGluT1 ( Immunocomplexes were acquired by chemiluminescence using the Chemidoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA, RRID: SCR_019037) and analyzed with Image Lab TM software (Bio-Rad, RRID: SCR_014210). The full-size cropped immunoblots are presented in Supplementary Materials (Figures S1-S3). Since gels were run at least two times, the obtained results were averaged with a correction factor: correction factor gel B = average of (OD protein of interest/OD β-actin for each sample loaded in gel A)/(OD protein of interest/OD β-actin for the same sample loaded in gel B) [40].

Statistical Analysis
Data were collected in individual animals as independent determinations, and they are addressed as means ± standard errors.
Molecular changes in protein levels produced by genotype were tested for normality of residuals with the Kolmogorov-Smirnov test. Data with normal distribution were analyzed by unpaired Student's t-test (t), using as the control condition DAT +/+ animals and, as the testing condition, DAT −/− . Data with a non-normal distribution were analyzed by the Mann-Whitney test (U).
Subjects were eliminated from the final dataset if their data deviated from the mean by 2 SDs. Prism 9 (GraphPad Software Prism v9, San Diego, CA, USA, RRID: SCR_002798) was used to analyze all data. Significance for all tests was assumed at p < 0.05.

Results
We started by analyzing the levels of the vesicular glutamate transporter type 1 (vGluT1), which is responsible for the packaging of glutamate into synaptic vesicles for its exocytotic release [41,42]. Figure 1a shows a significant increase in the expression of vGluT1 in the mPFC of DAT −/− rats (Figure 1a: +26% vs. DAT +/+ , t = 3.667, p = 0.0043). We then analyzed the expression of the glial glutamate transporter, GLT-1, which removes the extracellular glutamate from the synaptic cleft, thus regulating synaptic glutamate levels [43]. Figure 1b  Results obtained in the whole homogenate are expressed as mean percentage ± mean standard error from six independent determinations for each experimental group. Unpaired Student's t-test *p < 0.05, **p < 0.01 vs DAT+/+ rats.
We next investigated the expression of AMPA receptor subunits and their coupled scaffolding proteins [44]. To dissect the effect of DA overactivity on protein translation GLT-1, vGluT1, and β-Actin. Results obtained in the whole homogenate are expressed as mean percentage ± mean standard error from six independent determinations for each experimental group. Unpaired Student's t-test * p < 0.05, ** p < 0.01 vs. DAT +/+ rats.
We next investigated the expression of AMPA receptor subunits and their coupled scaffolding proteins [44]. To dissect the effect of DA overactivity on protein translation from their availability at synaptic and extra-synaptic sites, we evaluated the expression of AMPA receptor subunits in the whole homogenate (Homo), synaptic (PSD), and extra-synaptic (Extra-syn) fractions, respectively. We found a significant decrease in GluA1 and GluA2 protein levels in the whole homogenate ( representative immunoblots for GluA1, GluA2, GluA3, and β-Actin are shown for each fraction evaluated. Results are expressed as mean percentage ± mean standard error from six independent determinations for each experimental group. Unpaired Student's t-test * p < 0.05, ** p < 0.01 vs. DAT +/+ rats.
In parallel to AMPA receptor subunits, we also measured the expression of the specific anchoring proteins of GluA1, SAP97, GluA2, and GRIP. Both these scaffolding proteins were reduced in both homogenate and post-synaptic density of the mPFC of DAT −/− rats (SAP97 Figure    representative immunoblots for GluA1, GluA2, GluA3, and β-Actin are shown for each fraction evaluated. Results are expressed as mean percentage ± mean standard error from six independent determinations for each experimental group. Unpaired Student's t-test * p < 0.05, ** p < 0.01 vs. DAT +/+ rats. In parallel to AMPA receptor subunits, we also measured the expression of the specific anchoring proteins of GluA1, SAP97, GluA2, and GRIP. Both these scaffolding proteins were reduced in both homogenate and post-synaptic density of the mPFC of DAT −/− rats (SAP97 Figure  whole homogenate, in the synaptic, and in the extra-synaptic fractions. The ratio GluA1/GluA2 (panel d) and GluA2/GluA3 ratio (panel e) were measured in the synaptic fraction. In (panel f), representative immunoblots for GluA1, GluA2, GluA3, and β-Actin are shown for each fraction evaluated. Results are expressed as mean percentage ± mean standard error from six independent determinations for each experimental group. Unpaired Student's t-test * p < 0.05, ** p < 0.01 vs. DAT +/+ rats.

Discussion
In this study, we reveal that DA overactivity regulates the translation of cortical AMPA receptors and their localization at synaptic sites through, at least in part, the impairment of the related intracellular vesicular sorting pathway ( Figure 6). It appears, Results are expressed as mean percentage ± mean standard error from six independent determinations for each experimental group. Unpaired Student's t-test * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DAT +/+ rats.

Discussion
In this study, we reveal that DA overactivity regulates the translation of cortical AMPA receptors and their localization at synaptic sites through, at least in part, the impairment of the related intracellular vesicular sorting pathway ( Figure 6). It appears, thus, that a physiological concentration of DA is critical for the maintenance of AMPA receptor trafficking at the PSD. Our findings represent an aberrant form of neuroplasticity that, via hyperactive ascending DAergic projections to the cortical glutamatergic synapses, might underly working memory and executive function impairments previously observed in both rodents and humans under elevated dopaminergic activity [6,53,54]. First, we found that DA overactivity alters the expression of some critical determinants of the cortical glutamate synapse, i.e., vGluT1 and GLT-1. As suggested by the upregulation of vGluT1, the increase in glutamate release may be buffered by the increased expression of the glial transporter, GLT-1, which may remove the excess of extracellular glutamate and prevent its excitotoxicity. These changes indicate that DA overactivity has caused adaptive rearrangements of the cortical synapse, further strengthening the notion of the tight relationship between DA and glutamate.
More evident changes were observed at the AMPA receptors level, in line with previous findings demonstrating that DA receptors modulate synaptic plasticity by altering AMPA receptors' expression and surface delivery in cortical neurons [28]. In fact, we demonstrated that DA overactivity dysregulates the glutamate synapse in the mPFC by reducing translation of GluA1 and GluA2, as well as synaptic and extra-synaptic availability of GluA1, GluA2, and GluA3 AMPA receptor subunits. The reduced GluA1 and GluA2 translation might be due to reduced transcription of the different subunits to the modulatory effect of miRNAs or post-translational changes, such as SUMOylation or ubiquitination [55]. Since a physiological communication among DAergic and glutamatergic systems contributes to adaptive behavior fundamental for survival [56], the overall AMPA downregulation herein described suggests a previously undescribed DAdriven maladaptive plasticity that might underly a depotentiation of the post-synaptic strength and responsiveness. Interestingly, our hypothesis aligns with previous data showing that specific DAT inhibitors and elevated DA levels in cortical slices from naïve rats impaired long-term depression, thus inducing abnormal PFC activity [57]. Moreover, it is known that experiences, especially those involving a rise of DA, such as addictive behaviors, are likely to generate or re-program the excitatory synapse toward immature or silent synapse in which AMPA receptors are either absent or highly unstable [58,59], thus regulating drug-associated memories [60]. Moreover, since silent synapses are abundant during the early stage of development [61], we can hypothesize that DA overactivity, via alteration of AMPA abundance and trafficking, might maintain the First, we found that DA overactivity alters the expression of some critical determinants of the cortical glutamate synapse, i.e., vGluT1 and GLT-1. As suggested by the upregulation of vGluT1, the increase in glutamate release may be buffered by the increased expression of the glial transporter, GLT-1, which may remove the excess of extracellular glutamate and prevent its excitotoxicity. These changes indicate that DA overactivity has caused adaptive rearrangements of the cortical synapse, further strengthening the notion of the tight relationship between DA and glutamate.
More evident changes were observed at the AMPA receptors level, in line with previous findings demonstrating that DA receptors modulate synaptic plasticity by altering AMPA receptors' expression and surface delivery in cortical neurons [28]. In fact, we demonstrated that DA overactivity dysregulates the glutamate synapse in the mPFC by reducing translation of GluA1 and GluA2, as well as synaptic and extra-synaptic availability of GluA1, GluA2, and GluA3 AMPA receptor subunits. The reduced GluA1 and GluA2 translation might be due to reduced transcription of the different subunits to the modulatory effect of miRNAs or post-translational changes, such as SUMOylation or ubiquitination [55]. Since a physiological communication among DAergic and glutamatergic systems contributes to adaptive behavior fundamental for survival [56], the overall AMPA downregulation herein described suggests a previously undescribed DA-driven maladaptive plasticity that might underly a depotentiation of the post-synaptic strength and responsiveness. Interestingly, our hypothesis aligns with previous data showing that specific DAT inhibitors and elevated DA levels in cortical slices from naïve rats impaired long-term depression, thus inducing abnormal PFC activity [57]. Moreover, it is known that experiences, especially those involving a rise of DA, such as addictive behaviors, are Biomolecules 2023, 13, 516 9 of 14 likely to generate or re-program the excitatory synapse toward immature or silent synapse in which AMPA receptors are either absent or highly unstable [58,59], thus regulating drug-associated memories [60]. Moreover, since silent synapses are abundant during the early stage of development [61], we can hypothesize that DA overactivity, via alteration of AMPA abundance and trafficking, might maintain the cortical excitatory synapses in a molecular composition that might prevent synapse maturation and that might remodel cortical neurocircuitry toward a vulnerable endophenotype.
In addition to surface delivery, the distribution of membrane AMPA receptors could be rapidly modified by lateral diffusion via constitutive or regulated pathways [62,63]. The overall reduction in AMPA receptors at both synaptic and extra-synaptic sites, as observed in DAT −/− rats, further corroborates the hypothesis that a hyperdopaminergic state might affect not only AMPA receptor abundance at the membrane, but also AMPA receptor recruitment at the active site of the synapse and the laterally diffusing AMPA receptor pool. Such depotentiation is strengthened by a defective anchoring of these receptors at the post-synaptic membrane, as shown by the reduced expression of their main scaffolding proteins, SAP97 and GRIP, indicating that AMPA receptors are less retained at the PSD of dendritic spines and, thereby, less stable, thus affecting the ability to dynamically remodel the synapse in response to various form of plasticity [19]. Interestingly, such alterations are paralleled by altered synaptic localization of structural markers, such as Neuroligin-1 and nCadherin, which glue together pre-and postsynaptic terminals to sustain physiological neurotransmission [64]. The reduced Neuroligin-1 and nCadherin expression in the PSD further points to unstable synapses and a compromised glutamatergic transmission driven by hyperdopaminergia. Further, the reduced expression and synaptic localization of CRMP-2, a structural marker that facilitates synaptic AMPA receptor trafficking [65], add evidence to the impaired molecular composition of the cortical synapse in DAT −/− rats. Since the reduced localization of Neuroligin-1 at synapse might fail to assemble and anchor the AMPA receptor in the active nanodomain in dendritic spines [66], the increased expression and localization at the PSD of the scaffold protein PSD95 may be interpreted as an attempt to capture labile AMPA receptors [67] or as a maladaptive mechanism that further occludes synaptic transmission [68]. To counteract such synaptic structural instability, the increased translation of Neuroligin-1 and nCadherin, observed in the homogenate of DAT −/− rats, might reflect an adaptive response or an attempt to restore a physiological synaptic communication.
In parallel with the altered trafficking of AMPA receptors, DA overactivity modifies the subunit composition of the remaining receptors in the PSD. In fact, the reduction of the GluA1/GluA2 ratio in DAT −/− rats suggests a less excitable synaptic network, since it is well established that GluA1/GluA2, containing AMPA receptors, are inserted into the PSD as consequence of synaptic plasticity to sustain neuronal activity [69]. In addition, the DA overactivity-induced switch toward GluA2-containing AMPA receptors suggests a weakened synapse and, therefore, indicates the generation of silent synapses, as previously observed [60,70]. Conversely, the unaltered GluA2/GluA3 ratio is indicative of unaffected constitutive recycling at the synaptic level in DAT −/− rats [69], suggesting that DA overactivity primarily affects the activity-dependent trafficking pathway.
Interestingly, when neuronal activity is chronically enhanced, depotentiation of AMPA receptor-mediated transmission occurs via increased internalization of the receptors, a process able to modify the synaptic strength, known as synaptic scaling [18]. Such internalization occurs through exocytic and endocytic events [71] and involves Arc/Arg 3.1. Upon intense synaptic stimulation, Arc/Arg 3.1 rapidly accumulates at the PSD [72], promoting AMPA internalization by interacting with the endocytic machinery [23,73,74]. Accordingly, in parallel with reduced membrane AMPA receptor expression, we observed increased Arc/Arg3.1 levels in the PSD, suggesting that DA hyperactivity regulates homeostatic scaling and shapes synaptic strength by inducing neuroadaptations that resemble the molecular profile of cocaine-exposed rats [75,76].
Another novel finding of the present manuscript derives from the evidence that DA hyperactivity alters the mechanisms subserving endosomal sorting. Interestingly, the increased expression of Rab5, which through a vesicle fusion process regulates AMPA receptor endocytosis and sorting in the early endosomes [24,77], further corroborates the increased AMPA receptor internalization in the mPFC of DAT −/− rats. Following AMPA entry into the endosomal system, Rab5 overexpression generally drives endosomal maturation and increased lysosomal degradation of AMPA-containing vesicles [78]; however, in a condition of DA hyperactivity, the sorting in the late endosome or the fusion in phagosomes are likely to be impaired, as suggested by reduced Rab9 and LC3-II. In addition, we found that DA overactivity reduces the transand cis-Golgi markers, Galt and GM130, respectively, together with a significant reduction of Rab9, a protein that mediates endosome-to-trans-Golgi network transport [79]. Taken together, these results suggest that an altered DA transmission may also affect the recycling pathway that retrogradely targets AMPA receptors back to Golgi outposts [21]. Since depletion of cisand trans-Golgi proteins may affect the Golgi ribbon and induce central Golgi fragmentation [46], we can speculate that the defective morphology of the Golgi apparatus, observed at the molecular level, likely contributes to the impaired endosomal traffic in DAT −/− rats. Taken together, such abnormalities in endosomal trafficking may render the glutamate synapse more vulnerable and less responsive to stimuli by promoting the intracellular accumulation of AMPA receptors, a mechanism that closely resembles the impaired endosomal-induced aberrant intracellular accumulation of neurofibrillary tangles of hyper-phosphorylated tau and amyloid-b plaques in Alzheimer's disease [80] and a-synuclein in Parkinson's disease [81].
We are aware that our manuscript has some limitations. Although we did not demonstrate a direct causal link between the alteration of AMPA receptor trafficking and vesicular sorting, the changes observed in proteins regulating endosomal sorting sustain our hypothesis. Further, we focused our attention on AMPA receptors, and, therefore, we do not know whether our findings generalize to other types of glutamate receptors (NMDA, kainate, or metabotropic receptors). We are also aware that the norepinephrine transporter may come into play as a functional DAT replacement in the prefrontal cortex of DAT −/− rats [82,83]. However, evidence exists that extracellular levels of DA are significantly enhanced in the PFC of DAT −/− mice [36], potentially ruling out the possibility that NET may have prevented cortical DA hyperactivity. In addition, since DAT functions may also be regulated by protein-protein interactions [84,85], we cannot rule out the possibility that a change in DAT interactor functions may contribute to the alteration of AMPA receptor trafficking at the PSD.

Conclusions
Taken together, our findings show that DA overactivity alters the homeostatic plasticity of the glutamate synapse in the mPFC through profound dysregulation of AMPA receptor trafficking, an effect likely involving increased internalization of such receptors and alterations in endosomal trafficking and degradation. Our data add further details to the interaction between DA and glutamate neurotransmission, thus reinforcing the hypothesis that an altered communication between these systems likely plays significant roles in shaping maladaptive endophenotypes underlying pathological conditions characterized by enhanced dopaminergic tone.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.