PSMC5, a 19S Proteasomal ATPase, Regulates Cocaine Action in the Nucleus Accumbens

ΔFosB is a stable transcription factor which accumulates in the nucleus accumbens (NAc), a key part of the brain’s reward circuitry, in response to chronic exposure to cocaine or other drugs of abuse. While ΔFosB is known to heterodimerize with a Jun family member to form an active transcription factor complex, there has not to date been an open-ended exploration of other possible binding partners for ΔFosB in the brain. Here, by use of yeast two-hybrid assays, we identify PSMC5—also known as SUG1, an ATPase-containing subunit of the 19S proteasomal complex—as a novel interacting protein with ΔFosB. We verify such interactions between endogenous ΔFosB and PSMC5 in the NAc and demonstrate that both proteins also form complexes with other chromatin regulatory proteins associated with gene activation. We go on to show that chronic cocaine increases nuclear, but not cytoplasmic, levels of PSMC5 in the NAc and that overexpression of PSMC5 in this brain region promotes the locomotor responses to cocaine. Together, these findings describe a novel mechanism that contributes to the actions of ΔFosB and, for the first time, implicates PSMC5 in cocaine-induced molecular and behavioral plasticity.National Institutes of Health (U.S.)National Institute on Drug AbuseIshibashi FoundationJapan Society for the Promotion of Science (KAKENHI 24591735)Japan Society for the Promotion of Science (KAKENHI 26290064)Japan Society for the Promotion of Science (KAKENHI 25116010

PSMC5, a 19S Proteasomal ATPase, Regulates Cocaine Action in the Nucleus Accumbens.

ΔFosB is a stable transcription factor which accumulates in the nucleus accumbens (NAc), a key part of the brain's reward circuitry, in response to chronic exposure to cocaine or other drugs of abuse. While ΔFosB is known to heterodimerize with a Jun family member to form an active transcription factor complex, there has not to date been an open-ended exploration of other possible binding partners for ΔFosB in the brain. Here, by use of yeast two-hybrid assays, we identify PSMC5-also known as SUG1, an ATPase-containing subunit of the 19S proteasomal complex-as a novel interacting protein with ΔFosB. We verify such interactions between endogenous ΔFosB and PSMC5 in the NAc and demonstrate that both proteins also form complexes with other chromatin regulatory proteins associated with gene activation. We go on to show that chronic cocaine increases nuclear, but not cytoplasmic, levels of PSMC5 in the NAc and that overexpression of PSMC5 in this brain region promotes the locomotor responses to cocaine. Together, these findings describe a novel mechanism that contributes to the actions of ΔFosB and, for the first time, implicates PSMC5 in cocaine-induced molecular and behavioral plasticity

FOSB proteins in the orbitofrontal and dorsolateral prefrontal cortices of human alcoholics

The transcription factor DeltaFosB is accumulated in the addiction circuitry, including the orbitofrontal and medial prefrontal cortices of rodents chronically exposed to ethanol or other drugs of abuse, and has been suggested to play a direct role in addiction maintenance. To address this hypothesis in the context of substance dependence in humans, we compared the immunoreactivities of FOSB proteins in the orbitofrontal and dorsolateral prefrontal cortices (OFC and DLPFC respectively) between controls and alcoholics using semiquantitative immunoblotting. In both structures, we detected three forms of FOSB, one of which was DeltaFOSB, but in neither case did their immunoreactivities differ between the groups. Our results indicate that the DeltaFOSB immunoreactivity in the human brain is very low, and that it is not accumulated in the OFC and DLPFC of human alcoholics, suggesting that it may not be directly involved in addiction maintenance, at least not in ethanol dependence

FOSB proteins in the orbitofrontal and dorsolateral prefrontal cortices of human alcoholics

The transcription factor DeltaFosB is accumulated in the addiction circuitry, including the orbitofrontal and medial prefrontal cortices of rodents chronically exposed to ethanol or other drugs of abuse, and has been suggested to play a direct role in addiction maintenance. To address this hypothesis in the context of substance dependence in humans, we compared the immunoreactivities of FOSB proteins in the orbitofrontal and dorsolateral prefrontal cortices (OFC and DLPFC respectively) between controls and alcoholics using semiquantitative immunoblotting. In both structures, we detected three forms of FOSB, one of which was DeltaFOSB, but in neither case did their immunoreactivities differ between the groups. Our results indicate that the DeltaFOSB immunoreactivity in the human brain is very low, and that it is not accumulated in the OFC and DLPFC of human alcoholics, suggesting that it may not be directly involved in addiction maintenance, at least not in ethanol dependence

PSMC5 regulation of FosB/ΔFosB expression in Rat 1A cells.

<p>A. Rat 1A cells were transfected with 4 μg of PSMC5 or control DNA. PSMC5 overexpression had no effect on basal expression levels of FosB or ΔFosB protein as determined by Western blotting, but produced a small but significant increase in the induction of ΔFosB by serum stimulation (F(2,21) = 9.75, p = 0.001). B. Rat 1A cells were transfected with 5 pmol of either of two siRNAs or scrambled RNA (control). Both siRNAs effectively decreased PSMC5 protein levels compared to control conditions (siRNA #1, 23 ± 5% of control; siRNA #2, 18 ± 6%; p<0.05; n = 4). PSMC5 knockdown had no effect on basal levels of FosB or ΔFosB but attenulated the induction of both FosB and ΔFosB by serum stimulation (FosB: F(2,6) = 20.99, p = 0.002; ΔFosB: F(2,6) = 22.83, p = 0.002).</p

ΔFosB binds to PSMC5 in vitro.

<p>A. Schematic of ΔFosB, ΔFosB-LZM in which the leucine zipper domain is mutated to obliterate ΔFosB heterodimerization with Jun proteins, and Δ2ΔFosB which lacks the first 78 amino acids of the ΔFosB N-terminus. B. Schematic of PSMC5, PSMC5-NT which comprises the first 151 amino acids of PSMC5, PSMC5-CT which lacks the first 235 amino acids of PSMC5, and PSMC5-ΔCC which lacks the coiled-coil domain (amino acids 28–68). The AAA domain corresponds to a motif, ATPases Associated with diverse cellular Activities, present in many ATPases. C. 2.4 μg of pcDNA3.1-ΔFosB (lanes 1–4) or ΔFosB-LZM (lane 5) was co-transfected with 2.4 μg of FLAG-tagged PSMC5 or various deletion mutants into Neuro2a cells. Two days after transfection, cells were lysed and subjected to immunoprecipitation with an anti-FLAG antibody and then Western blotted with anti-ΔFosB or anti-FLAG antibody. Note that ΔFosB, but not ΔFosB-LZM, binds robustly to PSMC5 or PSMC5-NT, but not PSMC5-CT or PSMC5-ΔCC. The data shown in the figure were replicated in triplicate in each of three separate experiments.</p