Carbon catabolite repression in A. nidulans is a regulatory system which allows the organism to utilize the most preferable carbon source by repressing the expression of genes encoding enzymes utilizing alternative carbon sources. A ubiquitination pathway was shown to be one of the key mechanisms which regulate carbon source utilization, when creB was found to encode a deubiquitinating enzyme. Strains containing mutations in creB show loss of repression for some metabolic pathways in carbon catabolite repressing conditions, and also grow very poorly on several sole carbon sources such as quinate and proline, suggesting CreB plays multiple roles in the cell. This work describes the analysis of the interaction of CreB with CreA, and with PrnB and QutD. Various epitope-tagged versions of CreA were expressed in A. nidulans, and an internally located HA-epitope tag was found to allow detection of CreA using Western analysis. A diploid strain was constructed between strains containing HA-tagged CreA and FLAG-tagged CreB. When CreB was immunoprecipitated, HA-tagged CreA was also precipitated in the diploid, indicating that CreA and CreB are present in a complex in vivo. To determine whether CreA is a ubiquitinated protein, a version of CreA that was tagged with both an HA epitope and a His-tag was expressed in A. nidulans, and protein extracts were precipitated with an UbiQapture™-Q matrix. Western analysis was used to show that CreA was present in the precipitate. These findings suggest that CreA is a ubiquitinated protein, and a target of the CreB deubiquitination enzyme. To determine whether the proline permease (PrnB) is a direct substrate of CreB, plasmids to express epitope-tagged versions of PrnB were constructed and introduced into the prnB mutant strain. No tagged protein could be detected by Western analysis, even when these constructs were over-expressed from the gpdA promoter. However, a construct to express an HA epitope tagged version of quinate permease (QutD) fully complemented the qutD mutant strain, and HA-tagged QutD could be easily detected in Western analysis when probed with the anti-HA monoclonal antibody. A diploid strain was made between a complementing transformant and a strain expressing a FLAG-tagged CreB construct. When QutDHA was immunoprecipitated, CreBFLAG was detected in the immunoprecipitate of the diploid. A proportion of QutDHA was also co-precipitated in the diploid when CreBFLAG was immunoprecipitated. Thus, CreB is present in a complex with QutD in vivo. Further results showed that the concentration of QutD in the cell is lower in a creB null mutant background than in the wild-type background, indicating that deubiquitination is required to prevent protein turnover. Northern analysis of mRNA showed that the failure of creB mutant strains to grow on quinate medium was not due to a failure of transcriptional induction of qutD, as the amount of mRNA was not lower in a creB1937 mutant background compared to the wild-type. Furthermore, experiments were undertaken that showed that QutD is a ubiquitinated protein. These findings suggest that quinate permease is regulated through deubiquitination involving the CreB deubiquitination protein in A. nidulans. In addition to the candidate protein approach asking whether CreA is a substrate of CreB, a proteomics approach was also used to identify proteins that interact with CreA. However, no clear interacting proteins were identified using this approach. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Nicotine Addiction is a chronic relapsing disorder. Relapse risk persists despite years of abstinence. Drug-associated cues have been demonstrated to induce craving and provoke relapse. Surprisingly, in human smokers, craving for nicotine increases or “incubates” with longer abstinence durations, a phenomenon that may explain persistent relapse liability. This incubation phenomenon also presents in animals trained to intravenously self-administer nicotine though the underlying mechanisms are unclear. Two proteins, ERK (Extra-cellular signal Regulated Kinase) and CREB (cAMP Response Element Binding protein) play important roles in learning, memory, and numerous aspects of drug addiction. We therefore examined whether changes in these proteins are associated with incubation of craving for nicotine in rats. We found increased nicotine-seeking behaviour after 14 days of abstinence (compared to 1 day) along with elevated ERK and CREB activity in the Accumbens brain region suggesting that these proteins may be involved in the incubation phenomenon.

Memory stabilization following encoding (synaptic consolidation) or memory reactivation (reconsolidation) requires gene expression and protein synthesis. Although consolidation and reconsolidation may be mediated by distinct molecular mechanisms, disrupting the function of the transcription factor CREB (cAMP responsive element binding protein) impairs both processes. We use a gain-of-function approach to show that CREB (and CREB-coactivator CRTC1) can facilitate both synaptic and systems consolidation and reconsolidation. We first examine whether acutely increasing CREB levels in the dorsal hippocampus is sufficient to enhance spatial memory formation in the watermaze. Locally and acutely increasing CREB in the dorsal hippocampus using viral vectors is sufficient to induce robust spatial memory in two conditions which do not normally support consolidation, weakly-trained wild-type (WT) mice and strongly-trained mutant mice with brain-wide disrupted CREB function. CRTCs (CREB regulated transcription co-activators) are a powerful co-activator of CREB, but their role in memory is virtually unexplored. We show, for the first time, that the novel CREB co-activator CRTC1 enhances memory consolidation. Locally increasing CRTC1 (or CREB) in the dorsal hippocampus of WT mice prior to weak context fear conditioning facilitates consolidation of precise context memory. Last, we show that CREB or CRTC1 facilitates precise and enduring memory consolidation and reconsolidation. Acute enhancement of hippocampal CREB or CRTC1 during initial synaptic consolidation can maintain precision of remote context memory, while increasing CREB or CRTC1 just prior to reactivation of a weak remote context memory enhances context memory reconsolidation. These gain-of-function manipulations indicate that increasing CRTC1 or CREB function is sufficient to enhance the strength of new, as well as reactivated established, memories without compromising memory specificity. Together with previous results, these findings indicate that CREB is both necessary and sufficient for hippocampal-dependent memory formation, and underline its pivotal role in the hippocampal molecular machinery underlying long-term memory consolidation and reconsolidation.

Nicotine Addiction is a chronic relapsing disorder. Relapse risk persists despite years of abstinence. Drug-associated cues have been demonstrated to induce craving and provoke relapse. Surprisingly, in human smokers, craving for nicotine increases or “incubates” with longer abstinence durations, a phenomenon that may explain persistent relapse liability. This incubation phenomenon also presents in animals trained to intravenously self-administer nicotine though the underlying mechanisms are unclear. Two proteins, ERK (Extra-cellular signal Regulated Kinase) and CREB (cAMP Response Element Binding protein) play important roles in learning, memory, and numerous aspects of drug addiction. We therefore examined whether changes in these proteins are associated with incubation of craving for nicotine in rats. We found increased nicotine-seeking behaviour after 14 days of abstinence (compared to 1 day) along with elevated ERK and CREB activity in the Accumbens brain region suggesting that these proteins may be involved in the incubation phenomenon.

Memory stabilization following encoding (synaptic consolidation) or memory reactivation (reconsolidation) requires gene expression and protein synthesis. Although consolidation and reconsolidation may be mediated by distinct molecular mechanisms, disrupting the function of the transcription factor CREB (cAMP responsive element binding protein) impairs both processes. We use a gain-of-function approach to show that CREB (and CREB-coactivator CRTC1) can facilitate both synaptic and systems consolidation and reconsolidation. We first examine whether acutely increasing CREB levels in the dorsal hippocampus is sufficient to enhance spatial memory formation in the watermaze. Locally and acutely increasing CREB in the dorsal hippocampus using viral vectors is sufficient to induce robust spatial memory in two conditions which do not normally support consolidation, weakly-trained wild-type (WT) mice and strongly-trained mutant mice with brain-wide disrupted CREB function. CRTCs (CREB regulated transcription co-activators) are a powerful co-activator of CREB, but their role in memory is virtually unexplored. We show, for the first time, that the novel CREB co-activator CRTC1 enhances memory consolidation. Locally increasing CRTC1 (or CREB) in the dorsal hippocampus of WT mice prior to weak context fear conditioning facilitates consolidation of precise context memory. Last, we show that CREB or CRTC1 facilitates precise and enduring memory consolidation and reconsolidation. Acute enhancement of hippocampal CREB or CRTC1 during initial synaptic consolidation can maintain precision of remote context memory, while increasing CREB or CRTC1 just prior to reactivation of a weak remote context memory enhances context memory reconsolidation. These gain-of-function manipulations indicate that increasing CRTC1 or CREB function is sufficient to enhance the strength of new, as well as reactivated established, memories without compromising memory specificity. Together with previous results, these findings indicate that CREB is both necessary and sufficient for hippocampal-dependent memory formation, and underline its pivotal role in the hippocampal molecular machinery underlying long-term memory consolidation and reconsolidation.

Carbon catabolite repression in A. nidulans is a regulatory system which allows the organism to utilize the most preferable carbon source by repressing the expression of genes encoding enzymes utilizing alternative carbon sources. A ubiquitination pathway was shown to be one of the key mechanisms which regulate carbon source utilization, when creB was found to encode a deubiquitinating enzyme. Strains containing mutations in creB show loss of repression for some metabolic pathways in carbon catabolite repressing conditions, and also grow very poorly on several sole carbon sources such as quinate and proline, suggesting CreB plays multiple roles in the cell. This work describes the analysis of the interaction of CreB with CreA, and with PrnB and QutD. Various epitope-tagged versions of CreA were expressed in A. nidulans, and an internally located HA-epitope tag was found to allow detection of CreA using Western analysis. A diploid strain was constructed between strains containing HA-tagged CreA and FLAG-tagged CreB. When CreB was immunoprecipitated, HA-tagged CreA was also precipitated in the diploid, indicating that CreA and CreB are present in a complex in vivo. To determine whether CreA is a ubiquitinated protein, a version of CreA that was tagged with both an HA epitope and a His-tag was expressed in A. nidulans, and protein extracts were precipitated with an UbiQapture™-Q matrix. Western analysis was used to show that CreA was present in the precipitate. These findings suggest that CreA is a ubiquitinated protein, and a target of the CreB deubiquitination enzyme. To determine whether the proline permease (PrnB) is a direct substrate of CreB, plasmids to express epitope-tagged versions of PrnB were constructed and introduced into the prnB mutant strain. No tagged protein could be detected by Western analysis, even when these constructs were over-expressed from the gpdA promoter. However, a construct to express an HA epitope tagged version of quinate permease (QutD) fully complemented the qutD mutant strain, and HA-tagged QutD could be easily detected in Western analysis when probed with the anti-HA monoclonal antibody. A diploid strain was made between a complementing transformant and a strain expressing a FLAG-tagged CreB construct. When QutDHA was immunoprecipitated, CreBFLAG was detected in the immunoprecipitate of the diploid. A proportion of QutDHA was also co-precipitated in the diploid when CreBFLAG was immunoprecipitated. Thus, CreB is present in a complex with QutD in vivo. Further results showed that the concentration of QutD in the cell is lower in a creB null mutant background than in the wild-type background, indicating that deubiquitination is required to prevent protein turnover. Northern analysis of mRNA showed that the failure of creB mutant strains to grow on quinate medium was not due to a failure of transcriptional induction of qutD, as the amount of mRNA was not lower in a creB1937 mutant background compared to the wild-type. Furthermore, experiments were undertaken that showed that QutD is a ubiquitinated protein. These findings suggest that quinate permease is regulated through deubiquitination involving the CreB deubiquitination protein in A. nidulans. In addition to the candidate protein approach asking whether CreA is a substrate of CreB, a proteomics approach was also used to identify proteins that interact with CreA. However, no clear interacting proteins were identified using this approach. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Rationale and objective: The adenosine A(2A) receptor forms a mutually inhibitory heteromer with the dopamine D2 receptor, and A(2A) agonists decrease D2 signaling. This study analyzed whether an adenosine A(2A) agonist would alleviate deficits in sensorimotor gating and increases in cyclic-AMP response element binding protein (CREB) in the nucleus accumbens (NAc) in the neonatal quinpirole model of schizophrenia (SZ). Methods: Male and female Sprague-Dawley rats were neonatally treated with saline (NS) or quinpirole HCl (NQ; 1 mg/kg) from postnatal days (P) 1–21. Animals were raised to P44 and behaviorally tested on auditory sensorimotor gating as measured through prepulse inhibition (PPI) from P44 to P48. Approximately 15 min before each session, animals were given an ip administration of saline or the adenosine A(2A) agonist CGS 21680 (0.03 or 0.09 mg/kg). One day after PPI was complete on P49, animals were administered a locomotor activity test in the open field after saline or CGS 21680 treatment, respectively. On P50, the nucleus accumbens (NAc) was evaluated for CREB protein. Results: NQ-treated rats demonstrated a deficit in PPI that was alleviated to control levels by either dose of CGS 21680. The 0.03 mg/kg dose of CGS 21680 increased startle amplitude in males. The 0.09 mg/kg dose of CGS 21680 resulted in an overall decrease in locomotor activity. NQ treatment significantly increased NAc CREB that was attenuated to control levels by either dose of CGS 21680. Conclusions: This study revealed that an adenosine A(2A) receptor agonist was effective to alleviate PPI deficits in the NQ model of SZ in both male and female rats.

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