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proteasome pathway in rat spinal cord neurons
Neuroscience Letters 527 (2012) 126–131
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cAMP stimulates the ubiquitin/proteasome pathway in rat spinal cord neurons Natura Myeku, Hu Wang, Maria E. Figueiredo-Pereira ∗ Department of Biological Sciences, Hunter College and Graduate Center, CUNY, New York, NY 10065, United States
h i g h l i g h t s Elevating cAMP stimulates proteasomes in rat spinal cord neurons. Elevating cAMP raises the levels of components of the UPP. These include p62/sequestosome1, CHIP, p97 and ubB. Targeting cAMP/PKA to prevent neurodegeneration linked to Ub–protein aggregation.
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Article history: Received 4 June 2012 Received in revised form 24 August 2012 Accepted 26 August 2012 Keywords: cAMP Proteasome p62/sequestosome1 CHIP Ubiquitin Prostaglandin J2
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SPINAL CORD NEURONS cAMP PKA UPP 26S proteasome p62/sqstm1; ubB CHIP; p97/VCP
a b s t r a c t Proteasome impairment and accumulation of ubiquitinated proteins are implicated in neurodegeneration associated with different forms of spinal cord injury. We show herein that elevating cAMP in rat spinal cord neurons increases 26S proteasome activity in a protein kinase A-dependent manner. Treating spinal cord neurons with dibutyryl-cAMP (db-cAMP) also raised the levels of various components of the UPP including proteasome subunits Rpt6 and 5, polyubiquitin shuttling factor p62/sequestosome1, E3 ligase CHIP, AAA-ATPase p97 and the ubiquitin gene ubB. Finally, db-cAMP reduced the accumulation of ubiquitinated proteins, proteasome inhibition, and neurotoxicity triggered by the endogenous product of inflammation prostaglandin J2. We propose that optimizing the effects of cAMP/PKA-signaling on the UPP could offer an effective therapeutic approach to prevent UPP-related proteotoxicity in spinal cord neurons. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The accumulation of ubiquitinated proteins is a pathological feature of spinal cord neurons detected upon injury [1] and in patients with amyotrophic lateral sclerosis (ALS) [5], implicating impairment of protein turnover by the ubiquitin/proteasome pathway
Abbreviations: ALS, amyotrophic lateral sclerosis; CHIP, C-terminus of Hsc70 interacting protein; db-cAMP, dibutyryl-cAMP; DMSO, dimethyl sulfoxide; p62/sqstm1, sequestosome1; PKA, cAMP-dependent protein kinase; PGJ2, prostaglandin J2; Rp-cAMPS, adenosine 3 ,5 -cyclic monophosphorothioate, Rp-Isomer, triethylammonium salt; Rpt, regulatory particle tripleA-ATPase; SucLLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; UPP, ubiquitin/ proteasome pathway. ∗ Corresponding author at: 695 Park Avenue, New York, NY 10065, United States. Tel.: +1 212 650 3565; fax: +1 212 772 5227. E-mail address: [email protected] (M.E. Figueiredo-Pereira). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.08.051
(UPP). From a therapeutic point of view, the UPP is an ideal early target for preventing failures in protein degradation prior to formation of protein aggregates and the onset of cell death pathways (reviewed in [12]). In the studies described herein, we demonstrate that elevating cAMP in rat spinal cord neurons stimulates different components of the UPP and diminishes neuronal damage induced by the endogenous product of inflammation prostaglandin J2. Enhancing protein turnover by the UPP via the cAMP/PKA pathway thus has potential as a neuroprotective strategy to prevent neuronal damage associated with proteasome impairment and accumulation/aggregation of ubiquitinated proteins in spinal cord neurons.
2. Materials and methods Animal procedures were approved by the Hunter College IACUC.
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2.1. Reagents Adenosine 3 ,5 -cyclic monophosphate dibutyryl sodium salt (db-cAMP) from Calbiochem/EMD Bioscience. Rolipram and prostaglandin J2 (PGJ2) from Cayman Chemical. Forskolin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) from Sigma–Aldrich. Substrate Suc-LLVY-AMC from BACHEM Bioscience Inc. Primary antibodies: anti-5 (1:1000, #PW8895) and antiRpt6 (1:1000, #PW9265) from BIOMOL; anti-p62/sqstm1 (1:1000, #PM045, MBL International Corp.); anti-PKA C-␣ (1:1000, #4782), anti-CHIP (1:1000, #C386), and anti-Parkin (1:1000, #2132) from Cell Signaling; anti-p47 (1:1000, #365215, Santa Cruz Biotech), anti-p97/VCP (1:1000, #612182, BD Biosciences); antiubiquitinated proteins (1:1500, cat# Z0458, Dako North America); anti-III-tubulin (1:5000, #MMS-435P, Covance). 2.2. Spinal cord neurons Dissociated cultures of Sprague Dawley rat embryonic (E18) spinal cords were prepared as in [11] with modifications. Isolated spinal cords free of meninges and dorsal root ganglia were digested with papain (2 mg/ml, Worthington Biochemical Corp.) in Hibernate E without calcium (BrainBits LLC.) at 37 ◦ C for 30 min in a humidified atmosphere containing 5% CO2 . After removal of the enzymatic solution, tissues were gently dissociated in NbActiv4 media (BrainBits LLC.) and centrifuged at 300 × g for 5 min. Pellets were resuspended in NBActiv4 media without antibiotics, and neurons were plated onto Lab-TekII-CC2 chamber slides (Nalgene Nunc International) pre-coated with 100 g/l polyd-lysine (Sigma–Aldrich). Cultures were maintained in NbActiv4 medium and incubated at 37 ◦ C in a humidified atmosphere containing 5% CO2 . Experiments were run upon 7DIV. According to manufacturer’s specifications, NbActiv4 medium contains several proprietary factors that ensure a mostly pure (>95%) neuronal culture; glial growth is inhibited without a need for the anti-mitotic agent arabinofuranosyl cytidine [3,22]. 2.3. Culture treatments Neuronal cultures were treated for 24 h with water or DMSO (control) or with different drugs: db-cAMP in ultra pure filtered water, forskolin/rolipram or PGJ2 in DMSO, added directly to the NbActiv4 media. The final DMSO concentration in the medium was 0.5%. 2.4. Western blotting Cells were plated (density of 1.5 × 106 cells) on 1-well chamber slides. After treatment, cells were rinsed twice with PBS and harvested by scraping into ice-cold lysis buffer [20 mM Tris–HCl, pH 7.5, 137 mM NaCl, 1 mM EGTA, 2.5 mM Na4 P2 O7 , 1 mM -glycerophosphate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1% NP40, 1 mM Na3 VO4 , 1% glycerol and protease inhibitor cocktail (Sigma–Aldrich)]. Following lysis (at least 30 min, −80 ◦ C) and centrifugation (19,000 × g for 10 min) at 4 ◦ C, protein concentration was determined (BCA kit, Pierce). Normalized samples were boiled for 5 min in Laemmli buffer and loaded onto 10% gels (40 g of protein/lane). Following electrophoresis, proteins were transferred onto Immobilon-P membranes (Millipore). After blocking for 30 min at 37 ◦ C [10 mM Tris–HCl, pH 7.3, 5% (w/v) non-fat dry milk, 10 mM NaCl, and 0.1% (v/v) Tween 20], membranes were probed with primary antibodies followed by the respective secondary antibodies with HRP conjugate (1:10,000, Bio-Rad Laboratories). Antigens were visualized by a chemiluminescent horseradish peroxidase method with the ECL reagent. Semi-quantitative analysis
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of protein detection was done by image analysis (ImageJ program, Rasband, W.S., ImageJ, U.S. NIH, MD). 2.5. In-gel proteasome activity and detection Cells were plated (density of 1.5 × 106 cells) on 1-well chamber slides. Upon treatment cells were washed twice with PBS and harvested for the in-gel assay [20] with 80 g protein/lane loaded for proteasome activity and 40 g protein/lane loaded for western blotting. Proteasome activity was assessed by incubating the native gel for 10 min at 37 ◦ C with 15 ml of 400 M Suc-LLVY-AMC, followed by exposure to UV light (360 nm), and photographing with a NIKON Cool Pix 8700 camera with a 3-4219 fluorescent green filter (Peca Products, Inc.). Proteins on native gels were transferred onto PVDF membranes. Immunoblotting detected 20S and 26S proteasomes with anti-5 and anti-Rpt6 antibodies. The anti-5 antibody reacts with a core particle subunit detecting assembled 26S and 20S proteasomes. The anti-Rpt6 antibody reacts with a regulatory particle subunit detecting only assembled 26S proteasomes. The values reflect the semi-quantification obtained: with the Rtp6 antibody for 26S proteasome levels (single and double capped), as it generated the strongest signal; with the 5 antibody for 20S proteasomes. For loading control, aliquots of the samples were boiled for 5 min in Laemmli buffer and loaded onto 10% gels (40 g of protein/lane) for immunoblotting with anti-III-tubulin. 2.6. Quantitative reverse transcription-PCR analysis Cells were plated (density of 1.5 × 106 ) on 1-well chamber slides. Total RNA isolated with the RNAeasy Mini Kit from Qiagen, Inc., was evaluated for quantity and integrity (OD at 260/280 nm) and agarose gel electrophoresis. 10 ng of RNA was reverse transcribed with the DyNAmo cDNA Synthesis kit (Finnzymes Inc.). PCR primers (Applied Biosystems) to amplify rat cDNAs were: proteasome subunits Rpt6 [PSMC5 (Rn00579821 m1)] and 5 [PSMB5 (Rn01488741 m1)], PKA subunit C␣ [PRKACA (Rn01432302 m1)], p62/sqstm1 [SQSTM1 (Rn00709977 m1)], ubiquitin B [UBB (Rn03062801 gH)] and ubiquitin C [UBC (Rn01789812 g1)], and GAPDH [GAPDH (Rn01775763 g1)] to normalize. Quantitative realtime PCR in 384-well plates: each well contained 5 l of TaqMan Universal PCR Master Mix (Applied Biosystems), 0.5 l of each primer, 2.5 l of RNase-free water and 2 l of the reverse transcribed reaction in a LightCycler 480 (Roche Diagnostics Corp.). Thermal cycling conditions: initial denaturation step (10 min at 95 ◦ C) followed by 45 cycles of: 10 s at 95 ◦ C, 30 s at 60 ◦ C and a cooling step of 10 s at 40 ◦ C. Cycle threshold (CT ) values for each gene were obtained using a LightCycler 480 Software. Differences in CT values between each gene and the reference gene (CT ) were calculated: 2−[Ct(treated) − Ct(DMSO)] , where CT = CT (gene of interest) − CT (gapdh, reference gene as its mRNA level was not altered by db-cAMP or forskolin + rolipram). 2.7. PKA assay Cells were plated (density of 1.5 × 106 ) on 1-well chamber slides. After treatment cells were rinsed twice with PBS and harvested as for western blotting including the centrifugation step, but without boiling. PKA activity was determined with 0.15 g of protein per well. Absorbance (450 nm) was measured with a PowerWave HT Spectrophotometer. Relative kinase activity was determined as described in the nonradioactive assay kit (Assay Designs). 2.8. Cell viability Cells were plated (density of 2.5 × 105 cells/well) on 4well chamber slides. Cell viability was assessed with the
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [19] and expressed as percentages which represent the ratio between the data for each condition and control (DMSO) considered to be 100%. 2.9. Cell toxicity Cells were plated (density of 2.5 × 105 cells/well) on 4-well chamber slides. Cell toxicity was assessed with a bioluminescent cytotoxicity assay (aCella-TOX kit, Cell Technology Inc.) based on the release of GAPDH from dying cells. Cytotoxicity is expressed as RU (relative units) determined as follows: [(each drug treated sample) − (average solvent treated sample)]/[(maximum GAPDH release) − (average solvent treated sample)]. 2.10. Statistical analysis Statistical significance was estimated using one-way ANOVA (Tukey–Kramer multiple comparison test) with the Prism 5 program (GraphPad Software). 3. Results 3.1. Elevating cAMP stimulates 26S proteasomes via PKA In vitro and in vivo studies with non-neuronal cells reported that forskolin (an activator of adenylate cyclase that increases cAMP) or isoproterenol (a -adrenergic receptor agonist) activate PKA which in turn modulates proteasome activity [30]. We tested if db-cAMP (1 mM) stimulated proteasome activity in rat spinal cord neurons. db-cAMP is a cell permeable analog of cAMP that is significantly less susceptible to hydrolysis by phosphodiesterases [17]. Proteasomal chymotrypsin-like activity was assessed by a native in-gel assay with the substrate Suc-LLVY-AMC. This assay detects three forms of the proteasome: two-cap (2) and one-cap (1) 26S as well as the 20S. The one-cap 26S has the strongest activity, followed by the two-cap 26S, and lastly the 20S proteasome (Fig. 1A, left panel, control). db-cAMP (1 mM) approximately doubled 26S and 20S proteasome activities compared to control conditions. The positive effect of db-cAMP on proteasome activity was significant upon 24 h treatment, but not at earlier time points (4 h and 8 h, not shown). The adenylyl cyclase activator forskolin (100 M, Fk) in conjunction with rolipram (10 M, Rlp), a cell permeable phosphodiesterase (PDE4) inhibitor, also significantly enhanced proteasome activity. Immunoblot analyses of the native gels with anti-Rpt6 and anti5 antibodies assessed changes in 26S and 20S proteasome levels (Fig. 1A, middle and right panels). Treatment with db-cAMP but not with forskolin/rolipram, significantly increased 26S and 20S proteasome levels. These findings established that stimulation of proteasome activity with db-cAMP was due in part to a parallel elevation in proteasome levels. The PKA specific inhibitor Rp-cAMPS (Calbiochem/EMD Bioscience) abolished the increase in proteasome activity and levels induced by db-cAMP (Fig. 1B, compare lanes 2 and 3). These data demonstrate that proteasome stimulation by db-cAMP is mediated by PKA. 3.2. Elevating cAMP increases proteasome subunits Rpt6 and ˇ5 as well as p62/sqstm1 levels We previously demonstrated that 20S proteasome subunits are phosphorylated by a proteasome-associated PKA [23]. Moreover, PKA-dependent phosphorylation of proteasome subunits, in particular of Rpt6, enhances 26S proteasome assembly [24,34]. We now established that, besides phosphorylation, cAMP enhances proteasome activity by modulating proteasome subunit levels.
Fig. 1. Elevating cAMP stimulates the 26S proteasome via PKA. Rat E18 spinal cord neurons were treated for 24 h with: (A) 0.5% DMSO (control), 1 mM db-cAMP, or 100 M forskolin/10 M rolipram (Fk + Rlp); (B) 0.5% DMSO (control), pretreated for 1 h with 100 M Rp-cAMPS followed by 1 mM db-cAMP (Rp-cAMPS + db-cAMP), or 1 mM db-cAMP. Clear lysates (80 g/sample) were subjected to non-denaturing gel electrophoresis. Proteasomal chymotrypsin-like activity was assessed with SucLLVY-AMC by the in-gel assay (left panels). 26S and 20S proteasome levels (indicated by arrows) were detected by immunoblotting with anti-Rpt6 and anti-5 (middle and right panels). III-Tubulin (loading control). Activity and immunoblot bands were semi-quantified by densitometry. Percentages represent the ratio between data for each condition and control (DMSO) considered to be 100%. Values indicate means from at least three experiments. Asterisks identify values that are significantly different from control (*p < 0.05; **p < 0.01; ***p < 0.001).
Accordingly, db-cAMP or forskolin/rolipram increased the mRNA and protein levels of Rpt6 and 5, the latter to a lesser extent (Fig. 2A and C). P62/sqstm1 shuttles polyubiquitinated proteins to the proteasome for degradation and is being considered as a therapeutic target and/or biomarker for neurodegenerative diseases including ALS [33]. We show that db-cAMP or forskolin/rolipram enhance the mRNA and protein levels of p62/sqstm1 (Fig. 2A and C). Treatment with forskolin/rolipram was significantly less effective than db-cAMP on elevating the levels of most proteins tested including PKA subunit C␣ (Fig. 2A and C). This was not surprising as forskolin/rolipram increased PKA activity to a lesser extent than db-cAMP (Fig. 2D).
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Fig. 2. Effect of elevating cAMP on components of the UPP. Rat E18 spinal cord neurons were treated with 0.5% DMSO (control), 1 mM db-cAMP, or 100 M forskolin/10 M rolipram (Fk + Rlp) for 24 h. Western blot analyses (40 g of protein/lane; 10% gels) detected in: (A) Proteasome subunits (Rpt6 and 5), p62/sqstm1, and PKA catalytic subunit C␣ (PKA sub C␣). (B) CHIP, p97/VCP, Parkin, and p47/Ubx1; III-tubulin in A and B (loading control). Molecular mass markers in kDa are on the left. (C) Expression of the listed genes (normalized to gapdh) by quantitative RT-PCR. Percentages represent the ratio between the relative intensities for each condition and control (DMSO) considered to be 100%. (D) PKA activity determined with a nonradioactive assay kit. Values indicate means (and SE in C and D) from at least three experiments. Asterisks identify values that are significantly different from control (*p < 0.05; **p < 0.01; ***p < 0.001).
3.3. db-cAMP increases the levels of the E3 ligase CHIP, the AAA-ATPase p97, and the ubiquitin gene ubB
3.4. db-cAMP reduces the accumulation of ubiquitinated proteins, proteasome inhibition and neurotoxicity induced by PGJ2
Since db-cAMP enhanced 26S proteasome activity we reasoned that the drug could also raise the levels of other components of the UPP that work in concert with the proteasome to promote protein degradation. We only tested the effect of db-cAMP, since it was the most effective drug stimulating the proteasome. dbcAMP (Fig. 2B) increased the levels of two enzymes that facilitate the degradation of misfolded proteins by the 26S proteasome: (1) the C-terminus of Hsc70 interacting protein (CHIP), which is a homodimeric ubiquitin ligase that links chaperone function with the degradation of misfolded or unstable proteins [7]; and (2) the homohexameric AAA-ATPase p97 (VCP/cdc48) that can associate with proteasomes, bind to polyubiquitinated proteins and promote their degradation [15]. The levels of CHIP and p97 were significantly elevated by 1.3-fold upon treatment with db-cAMP. db-cAMP also up-regulated the mRNA of the ubiquitin gene ubB by almost 2-fold but not ubC (Fig. 2C). Expression of both genes ubB and ubC, which encode tandemly repeated multiple ubiquitins with no spacer, are essential for stress tolerance [26,27]. Two other proteins relevant to protein degradation by the 26S proteasome, i.e. the E3 ligase Parkin and p47/Ubx1, a substrate-recruiting co-factor for p97, were not significantly altered by db-cAMP (Fig. 2B). We conclude that cAMP-signaling has a positive effect on specific components of the UPP.
Inflammation is associated with neuronal damage following spinal cord injury [10] and ALS [4]. Prostaglandins of the J2 series are endogenous products of inflammation reported to be neurotoxic [31]. We and others previously showed that PGJ2 inhibits the 26S proteasome by causing its disassembly [32] and/or oxidative modification [14]. We now show that PGJ2-treatment of spinal cord neurons induces significant (a) accumulation (∼2.4-fold above control) of ubiquitinated proteins (Fig. 3A); (b) decrease in the activity and levels of both forms of the 26S proteasome (two cap and one cap), while slightly increasing the activity of the 20S proteasome (Fig. 3B); (c) loss of viability determined with the MTT assay (Fig. 3C); (d) cytotoxicity assessed by the release of GAPDH from dying cells (Fig. 3D). Notably, db-cAMP and forskolin/rolipram reduced the damaging effects of PGJ2 listed above. Prevention of PGJ2-induced proteasome inhibition by forskolin/rolipram was minor (not shown). 4. Discussion Means to increase proteasome activity in spinal cord neurons are currently lacking. We show herein that db-cAMP enhances proteasome activity and levels in a PKA-dependent manner in spinal cord neurons (to our knowledge for the first time). This effect was
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Fig. 3. Elevating cAMP diminishes (A) accumulation of ubiquitinated proteins, (B) 26S proteasome inhibition, (C) loss of cell viability, and (D) cytotoxicity induced by PGJ2. Rat E18 spinal cord neurons were treated separately with 0.5% DMSO (control), 1 mM db-cAMP, 100 M forskolin/10 M rolipram (Fk + Rlp) or 20 M PGJ2 for 24 h. Parallel cultures were pretreated for 1 h with 1 mM db-cAMP or 100 M forskolin/10 M rolipram (Fk + Rlp) followed by 20 M PGJ2 for 24 h (db-cAMP + J2 or Fk + Rlp + J2). (A) Western blot analysis (40 g of protein/lane; 10% gels) detecting ubiquitinated proteins (Ub–proteins) and III-tubulin (loading control). (B) Proteasomal chymotrypsin-like activity (left panel) and levels (middle and right panels) were assessed as described in Fig. 1. 26S and 20S proteasomes are indicated by arrows. III-Tubulin (loading control). (C) Cell viability measured with the MTT assay. In (A–C) percentages represent the ratio between the data for each condition and control (DMSO) considered to be 100%. (D) Cell toxicity was assessed with the bioluminescent cytotoxicity assay and expressed as RU (relative units). Values indicate means and SE from at least three experiments. Asterisks identify values that are significantly different from control (*p < 0.05; **p < 0.01; ***p < 0.001). Furthermore, in (A) there is no significant difference between control and db-cAMP or Fk + Rlp within the DMSO- or J2-treatment groups. In (C) and (D) there is no significant difference between control and db-cAMP or Fk + Rlp within the DMSO-treatment group. Within the J2-treatment group, J2 alone is significantly different (p < 0.001) from J2 + db-cAMP or J2 + (Fk + Rlp).
mimicked albeit to a lesser extent, by forskolin/rolipram. At the subunit level, db-cAMP raised the mRNA and protein levels of Rpt6, a subunit of the proteasome 19S regulatory particle, and to a lesser extent 5, which is a constitutive subunit of the 20S proteasome. These results suggest an independent regulation of proteasome 19S and 20S subunit pools in the primary neuronal cultures. A similar PKA-mediated regulation of proteasome 19S and 20S subunits was observed in isoproterenol-induced cardiac hyperthrophy in mice [8]. In contrast, PKA-mediated stimulation of 26S proteasome activity did not cause an increase in proteasome subunit levels following the intracoronary administration of isoproterenol, or forskolin, or ischemic pre-conditioning in the canine heart [2]. These opposing results indicate variation in 26S proteasome regulation under different conditions. In conclusion, in vitro and in vivo studies demonstrate that PKA regulates proteasome activity at the transcriptional subunit level, as well as by subunit phosphorylation which promotes proteasome assembly [16,30,34]. db-cAMP treatment also elevated the protein levels of p62/sqstm1. We propose that the rise in p62/sqstm1 is mediated by increased mRNA levels, and not by inhibition of its turnover. The latter premise is based on p62/sqstm1 being degraded by autophagy [13], in conjunction with the finding in yeast, that activation of the TOR or the PKA pathways, inhibit autophagy via
phosphorylation of the Atg1/Atg13 complex [29]. To our knowledge, ours is the first report showing that p62/sqstm1 levels are regulated by cAMP. This is important because, due to oxidative damage to its promoter, there is an aging-correlated decrease in p62/sqstm1 levels, which is also apparent in neurodegenerative disorders such as Alzheimer and Parkinson diseases [9]. Moreover, absence of p62/sqstm1 induces neuronal cell death [21,25]. The overall impact of p62/sqstm1 on neuronal survival is not surprising because it is a scaffold protein that integrates kinase-activated and ubiquitin-mediated survival signals [18]. The protein levels of the E3 ubiquitin ligase CHIP and the AAA-ATPase p97/VCP were also elevated by cAMP. These two components of the UPP are associated with turnover of abnormal proteins. Together with the observed increase in 26S proteasome activity, our results support the notion that activation of cAMP/PKA signaling in spinal cord neurons could be a promising strategy to promote protein degradation via the UPP and avoid Ub–protein accumulation/aggregation in spinal cord neurodegenerative conditions associated with proteasome impairment, such as those induced by inflammation. This would be beneficial to neurons because, compared to astrocytes, neurons are particularly sensitive to the toxic effects of proteasome inhibition [6]. Recent developments in cyclic nucleotide-related biochemical and pharmacological
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research, renders this field potentially very attractive for future therapeutic targeting [28]. Competing interests None. Authors’ contributions N.M. and H.W. performed the experiments. N.M. and M.E.F.P. designed the study and wrote the manuscript. All authors read and approved the final manuscript. Acknowledgments We thank Dr. Charlotte D‘Hulst and Dr. Lisa Abrams for assistance with the RT-PCR and statistical analysis, respectively. This work was supported by NIH (R01 AG028847, U54 NS041073, UL1 RR024996, G12 RR003037). References [1] S. Ahlgren, G.L. Li, Y. Olsson, Accumulation of beta-amyloid precursor protein and ubiquitin in axons after spinal cord trauma in humans: immunohistochemical observations on autopsy material, Acta Neuropathologica 92 (1996) 49–55. [2] M. Asai, O. Tsukamoto, T. Minamino, H. Asanuma, M. Fujita, Y. Asano, H. Takahama, H. Sasaki, S. Higo, M. Asakura, S. Takashima, M. Hori, M. Kitakaze, PKA rapidly enhances proteasome assembly and activity in in vivo canine hearts, Journal of Molecular and Cellular Cardiology 46 (2009) 452–462. [3] G.J. Brewer, J.R. Torricelli, E.K. Evege, P.J. Price, Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination, Journal of Neuroscience Research 35 (1993) 567–576. [4] L.I. Bruijn, T.M. Miller, D.W. Cleveland, Unraveling the mechanisms involved in motor neuron degeneration in ALS, Annual Review of Neuroscience 27 (2004) 723–749. [5] J. Costa, C. Gomes, M. de Carvalho, Diagnosis, pathogenesis and therapeutic targets in amyotrophic lateral sclerosis, CNS Neurological Disorders Drug Targets 9 (2010) 764–778. [6] K. Dasuri, P.J. Ebenezer, L. Zhang, S.O. Fernandez-Kim, R.M. Uranga, E. Gavilan, B.A. Di, J.N. Keller, Selective vulnerability of neurons to acute toxicity after proteasome inhibitor treatment: implications for oxidative stress and insolubility of newly synthesized proteins, Free Radical Biology and Medicine 49 (2010) 1290–1297. [7] C.A. Dickey, C. Patterson, D. Dickson, L. Petrucelli, Brain CHIP: removing the culprits in neurodegenerative disease, Trends in Molecular Medicine 13 (2007) 32–38. [8] O. Drews, O. Tsukamoto, D. Liem, J. Streicher, Y. Wang, P. Ping, Differential regulation of proteasome function in isoproterenol-induced cardiac hypertrophy, Circulation Research 107 (2010) 1094–1101. [9] Y. Du, M.C. Wooten, M.W. Wooten, Oxidative damage to the promoter region of SQSTM1/p62 is common to neurodegenerative disease, Neurobiology of Disease 35 (2009) 302–310. [10] O.N. Hausmann, Post-traumatic inflammation following spinal cord injury, Spinal Cord 41 (2003) 369–378. [11] C.E. Henderson, E. Bloch-Gallego, W. Camu, Purified embryonic motoneurons, in: J. Cohen, G.P. Wilkin (Eds.), Neural Cell culture, Oxford University Press, Oxford, 2002, pp. 69–81. [12] Q. Huang, M.E. Figueiredo-Pereira, Ubiquitin/proteasome pathway impairment in neurodegeneration: therapeutic implications, Apoptosis 15 (2010) 1292–1311. [13] Y. Ichimura, M. Komatsu, Selective degradation of p62 by autophagy, Seminars in Immunopathology 32 (2010) 431–436.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
cAMP stimulates the ubiquitin/proteasome pathway in rat spinal cord neurons Natura Myeku, Hu Wang, Maria E. Figueiredo-Pereira ∗ Department of Biological Sciences, Hunter College and Graduate Center, CUNY, New York, NY 10065, United States
h i g h l i g h t s Elevating cAMP stimulates proteasomes in rat spinal cord neurons. Elevating cAMP raises the levels of components of the UPP. These include p62/sequestosome1, CHIP, p97 and ubB. Targeting cAMP/PKA to prevent neurodegeneration linked to Ub–protein aggregation.
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Article history: Received 4 June 2012 Received in revised form 24 August 2012 Accepted 26 August 2012 Keywords: cAMP Proteasome p62/sequestosome1 CHIP Ubiquitin Prostaglandin J2
g r a p h i c a l
a b s t r a c t
SPINAL CORD NEURONS cAMP PKA UPP 26S proteasome p62/sqstm1; ubB CHIP; p97/VCP
a b s t r a c t Proteasome impairment and accumulation of ubiquitinated proteins are implicated in neurodegeneration associated with different forms of spinal cord injury. We show herein that elevating cAMP in rat spinal cord neurons increases 26S proteasome activity in a protein kinase A-dependent manner. Treating spinal cord neurons with dibutyryl-cAMP (db-cAMP) also raised the levels of various components of the UPP including proteasome subunits Rpt6 and 5, polyubiquitin shuttling factor p62/sequestosome1, E3 ligase CHIP, AAA-ATPase p97 and the ubiquitin gene ubB. Finally, db-cAMP reduced the accumulation of ubiquitinated proteins, proteasome inhibition, and neurotoxicity triggered by the endogenous product of inflammation prostaglandin J2. We propose that optimizing the effects of cAMP/PKA-signaling on the UPP could offer an effective therapeutic approach to prevent UPP-related proteotoxicity in spinal cord neurons. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The accumulation of ubiquitinated proteins is a pathological feature of spinal cord neurons detected upon injury [1] and in patients with amyotrophic lateral sclerosis (ALS) [5], implicating impairment of protein turnover by the ubiquitin/proteasome pathway
Abbreviations: ALS, amyotrophic lateral sclerosis; CHIP, C-terminus of Hsc70 interacting protein; db-cAMP, dibutyryl-cAMP; DMSO, dimethyl sulfoxide; p62/sqstm1, sequestosome1; PKA, cAMP-dependent protein kinase; PGJ2, prostaglandin J2; Rp-cAMPS, adenosine 3 ,5 -cyclic monophosphorothioate, Rp-Isomer, triethylammonium salt; Rpt, regulatory particle tripleA-ATPase; SucLLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; UPP, ubiquitin/ proteasome pathway. ∗ Corresponding author at: 695 Park Avenue, New York, NY 10065, United States. Tel.: +1 212 650 3565; fax: +1 212 772 5227. E-mail address: [email protected] (M.E. Figueiredo-Pereira). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.08.051
(UPP). From a therapeutic point of view, the UPP is an ideal early target for preventing failures in protein degradation prior to formation of protein aggregates and the onset of cell death pathways (reviewed in [12]). In the studies described herein, we demonstrate that elevating cAMP in rat spinal cord neurons stimulates different components of the UPP and diminishes neuronal damage induced by the endogenous product of inflammation prostaglandin J2. Enhancing protein turnover by the UPP via the cAMP/PKA pathway thus has potential as a neuroprotective strategy to prevent neuronal damage associated with proteasome impairment and accumulation/aggregation of ubiquitinated proteins in spinal cord neurons.
2. Materials and methods Animal procedures were approved by the Hunter College IACUC.
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2.1. Reagents Adenosine 3 ,5 -cyclic monophosphate dibutyryl sodium salt (db-cAMP) from Calbiochem/EMD Bioscience. Rolipram and prostaglandin J2 (PGJ2) from Cayman Chemical. Forskolin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) from Sigma–Aldrich. Substrate Suc-LLVY-AMC from BACHEM Bioscience Inc. Primary antibodies: anti-5 (1:1000, #PW8895) and antiRpt6 (1:1000, #PW9265) from BIOMOL; anti-p62/sqstm1 (1:1000, #PM045, MBL International Corp.); anti-PKA C-␣ (1:1000, #4782), anti-CHIP (1:1000, #C386), and anti-Parkin (1:1000, #2132) from Cell Signaling; anti-p47 (1:1000, #365215, Santa Cruz Biotech), anti-p97/VCP (1:1000, #612182, BD Biosciences); antiubiquitinated proteins (1:1500, cat# Z0458, Dako North America); anti-III-tubulin (1:5000, #MMS-435P, Covance). 2.2. Spinal cord neurons Dissociated cultures of Sprague Dawley rat embryonic (E18) spinal cords were prepared as in [11] with modifications. Isolated spinal cords free of meninges and dorsal root ganglia were digested with papain (2 mg/ml, Worthington Biochemical Corp.) in Hibernate E without calcium (BrainBits LLC.) at 37 ◦ C for 30 min in a humidified atmosphere containing 5% CO2 . After removal of the enzymatic solution, tissues were gently dissociated in NbActiv4 media (BrainBits LLC.) and centrifuged at 300 × g for 5 min. Pellets were resuspended in NBActiv4 media without antibiotics, and neurons were plated onto Lab-TekII-CC2 chamber slides (Nalgene Nunc International) pre-coated with 100 g/l polyd-lysine (Sigma–Aldrich). Cultures were maintained in NbActiv4 medium and incubated at 37 ◦ C in a humidified atmosphere containing 5% CO2 . Experiments were run upon 7DIV. According to manufacturer’s specifications, NbActiv4 medium contains several proprietary factors that ensure a mostly pure (>95%) neuronal culture; glial growth is inhibited without a need for the anti-mitotic agent arabinofuranosyl cytidine [3,22]. 2.3. Culture treatments Neuronal cultures were treated for 24 h with water or DMSO (control) or with different drugs: db-cAMP in ultra pure filtered water, forskolin/rolipram or PGJ2 in DMSO, added directly to the NbActiv4 media. The final DMSO concentration in the medium was 0.5%. 2.4. Western blotting Cells were plated (density of 1.5 × 106 cells) on 1-well chamber slides. After treatment, cells were rinsed twice with PBS and harvested by scraping into ice-cold lysis buffer [20 mM Tris–HCl, pH 7.5, 137 mM NaCl, 1 mM EGTA, 2.5 mM Na4 P2 O7 , 1 mM -glycerophosphate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1% NP40, 1 mM Na3 VO4 , 1% glycerol and protease inhibitor cocktail (Sigma–Aldrich)]. Following lysis (at least 30 min, −80 ◦ C) and centrifugation (19,000 × g for 10 min) at 4 ◦ C, protein concentration was determined (BCA kit, Pierce). Normalized samples were boiled for 5 min in Laemmli buffer and loaded onto 10% gels (40 g of protein/lane). Following electrophoresis, proteins were transferred onto Immobilon-P membranes (Millipore). After blocking for 30 min at 37 ◦ C [10 mM Tris–HCl, pH 7.3, 5% (w/v) non-fat dry milk, 10 mM NaCl, and 0.1% (v/v) Tween 20], membranes were probed with primary antibodies followed by the respective secondary antibodies with HRP conjugate (1:10,000, Bio-Rad Laboratories). Antigens were visualized by a chemiluminescent horseradish peroxidase method with the ECL reagent. Semi-quantitative analysis
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of protein detection was done by image analysis (ImageJ program, Rasband, W.S., ImageJ, U.S. NIH, MD). 2.5. In-gel proteasome activity and detection Cells were plated (density of 1.5 × 106 cells) on 1-well chamber slides. Upon treatment cells were washed twice with PBS and harvested for the in-gel assay [20] with 80 g protein/lane loaded for proteasome activity and 40 g protein/lane loaded for western blotting. Proteasome activity was assessed by incubating the native gel for 10 min at 37 ◦ C with 15 ml of 400 M Suc-LLVY-AMC, followed by exposure to UV light (360 nm), and photographing with a NIKON Cool Pix 8700 camera with a 3-4219 fluorescent green filter (Peca Products, Inc.). Proteins on native gels were transferred onto PVDF membranes. Immunoblotting detected 20S and 26S proteasomes with anti-5 and anti-Rpt6 antibodies. The anti-5 antibody reacts with a core particle subunit detecting assembled 26S and 20S proteasomes. The anti-Rpt6 antibody reacts with a regulatory particle subunit detecting only assembled 26S proteasomes. The values reflect the semi-quantification obtained: with the Rtp6 antibody for 26S proteasome levels (single and double capped), as it generated the strongest signal; with the 5 antibody for 20S proteasomes. For loading control, aliquots of the samples were boiled for 5 min in Laemmli buffer and loaded onto 10% gels (40 g of protein/lane) for immunoblotting with anti-III-tubulin. 2.6. Quantitative reverse transcription-PCR analysis Cells were plated (density of 1.5 × 106 ) on 1-well chamber slides. Total RNA isolated with the RNAeasy Mini Kit from Qiagen, Inc., was evaluated for quantity and integrity (OD at 260/280 nm) and agarose gel electrophoresis. 10 ng of RNA was reverse transcribed with the DyNAmo cDNA Synthesis kit (Finnzymes Inc.). PCR primers (Applied Biosystems) to amplify rat cDNAs were: proteasome subunits Rpt6 [PSMC5 (Rn00579821 m1)] and 5 [PSMB5 (Rn01488741 m1)], PKA subunit C␣ [PRKACA (Rn01432302 m1)], p62/sqstm1 [SQSTM1 (Rn00709977 m1)], ubiquitin B [UBB (Rn03062801 gH)] and ubiquitin C [UBC (Rn01789812 g1)], and GAPDH [GAPDH (Rn01775763 g1)] to normalize. Quantitative realtime PCR in 384-well plates: each well contained 5 l of TaqMan Universal PCR Master Mix (Applied Biosystems), 0.5 l of each primer, 2.5 l of RNase-free water and 2 l of the reverse transcribed reaction in a LightCycler 480 (Roche Diagnostics Corp.). Thermal cycling conditions: initial denaturation step (10 min at 95 ◦ C) followed by 45 cycles of: 10 s at 95 ◦ C, 30 s at 60 ◦ C and a cooling step of 10 s at 40 ◦ C. Cycle threshold (CT ) values for each gene were obtained using a LightCycler 480 Software. Differences in CT values between each gene and the reference gene (CT ) were calculated: 2−[Ct(treated) − Ct(DMSO)] , where CT = CT (gene of interest) − CT (gapdh, reference gene as its mRNA level was not altered by db-cAMP or forskolin + rolipram). 2.7. PKA assay Cells were plated (density of 1.5 × 106 ) on 1-well chamber slides. After treatment cells were rinsed twice with PBS and harvested as for western blotting including the centrifugation step, but without boiling. PKA activity was determined with 0.15 g of protein per well. Absorbance (450 nm) was measured with a PowerWave HT Spectrophotometer. Relative kinase activity was determined as described in the nonradioactive assay kit (Assay Designs). 2.8. Cell viability Cells were plated (density of 2.5 × 105 cells/well) on 4well chamber slides. Cell viability was assessed with the
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [19] and expressed as percentages which represent the ratio between the data for each condition and control (DMSO) considered to be 100%. 2.9. Cell toxicity Cells were plated (density of 2.5 × 105 cells/well) on 4-well chamber slides. Cell toxicity was assessed with a bioluminescent cytotoxicity assay (aCella-TOX kit, Cell Technology Inc.) based on the release of GAPDH from dying cells. Cytotoxicity is expressed as RU (relative units) determined as follows: [(each drug treated sample) − (average solvent treated sample)]/[(maximum GAPDH release) − (average solvent treated sample)]. 2.10. Statistical analysis Statistical significance was estimated using one-way ANOVA (Tukey–Kramer multiple comparison test) with the Prism 5 program (GraphPad Software). 3. Results 3.1. Elevating cAMP stimulates 26S proteasomes via PKA In vitro and in vivo studies with non-neuronal cells reported that forskolin (an activator of adenylate cyclase that increases cAMP) or isoproterenol (a -adrenergic receptor agonist) activate PKA which in turn modulates proteasome activity [30]. We tested if db-cAMP (1 mM) stimulated proteasome activity in rat spinal cord neurons. db-cAMP is a cell permeable analog of cAMP that is significantly less susceptible to hydrolysis by phosphodiesterases [17]. Proteasomal chymotrypsin-like activity was assessed by a native in-gel assay with the substrate Suc-LLVY-AMC. This assay detects three forms of the proteasome: two-cap (2) and one-cap (1) 26S as well as the 20S. The one-cap 26S has the strongest activity, followed by the two-cap 26S, and lastly the 20S proteasome (Fig. 1A, left panel, control). db-cAMP (1 mM) approximately doubled 26S and 20S proteasome activities compared to control conditions. The positive effect of db-cAMP on proteasome activity was significant upon 24 h treatment, but not at earlier time points (4 h and 8 h, not shown). The adenylyl cyclase activator forskolin (100 M, Fk) in conjunction with rolipram (10 M, Rlp), a cell permeable phosphodiesterase (PDE4) inhibitor, also significantly enhanced proteasome activity. Immunoblot analyses of the native gels with anti-Rpt6 and anti5 antibodies assessed changes in 26S and 20S proteasome levels (Fig. 1A, middle and right panels). Treatment with db-cAMP but not with forskolin/rolipram, significantly increased 26S and 20S proteasome levels. These findings established that stimulation of proteasome activity with db-cAMP was due in part to a parallel elevation in proteasome levels. The PKA specific inhibitor Rp-cAMPS (Calbiochem/EMD Bioscience) abolished the increase in proteasome activity and levels induced by db-cAMP (Fig. 1B, compare lanes 2 and 3). These data demonstrate that proteasome stimulation by db-cAMP is mediated by PKA. 3.2. Elevating cAMP increases proteasome subunits Rpt6 and ˇ5 as well as p62/sqstm1 levels We previously demonstrated that 20S proteasome subunits are phosphorylated by a proteasome-associated PKA [23]. Moreover, PKA-dependent phosphorylation of proteasome subunits, in particular of Rpt6, enhances 26S proteasome assembly [24,34]. We now established that, besides phosphorylation, cAMP enhances proteasome activity by modulating proteasome subunit levels.
Fig. 1. Elevating cAMP stimulates the 26S proteasome via PKA. Rat E18 spinal cord neurons were treated for 24 h with: (A) 0.5% DMSO (control), 1 mM db-cAMP, or 100 M forskolin/10 M rolipram (Fk + Rlp); (B) 0.5% DMSO (control), pretreated for 1 h with 100 M Rp-cAMPS followed by 1 mM db-cAMP (Rp-cAMPS + db-cAMP), or 1 mM db-cAMP. Clear lysates (80 g/sample) were subjected to non-denaturing gel electrophoresis. Proteasomal chymotrypsin-like activity was assessed with SucLLVY-AMC by the in-gel assay (left panels). 26S and 20S proteasome levels (indicated by arrows) were detected by immunoblotting with anti-Rpt6 and anti-5 (middle and right panels). III-Tubulin (loading control). Activity and immunoblot bands were semi-quantified by densitometry. Percentages represent the ratio between data for each condition and control (DMSO) considered to be 100%. Values indicate means from at least three experiments. Asterisks identify values that are significantly different from control (*p < 0.05; **p < 0.01; ***p < 0.001).
Accordingly, db-cAMP or forskolin/rolipram increased the mRNA and protein levels of Rpt6 and 5, the latter to a lesser extent (Fig. 2A and C). P62/sqstm1 shuttles polyubiquitinated proteins to the proteasome for degradation and is being considered as a therapeutic target and/or biomarker for neurodegenerative diseases including ALS [33]. We show that db-cAMP or forskolin/rolipram enhance the mRNA and protein levels of p62/sqstm1 (Fig. 2A and C). Treatment with forskolin/rolipram was significantly less effective than db-cAMP on elevating the levels of most proteins tested including PKA subunit C␣ (Fig. 2A and C). This was not surprising as forskolin/rolipram increased PKA activity to a lesser extent than db-cAMP (Fig. 2D).
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Fig. 2. Effect of elevating cAMP on components of the UPP. Rat E18 spinal cord neurons were treated with 0.5% DMSO (control), 1 mM db-cAMP, or 100 M forskolin/10 M rolipram (Fk + Rlp) for 24 h. Western blot analyses (40 g of protein/lane; 10% gels) detected in: (A) Proteasome subunits (Rpt6 and 5), p62/sqstm1, and PKA catalytic subunit C␣ (PKA sub C␣). (B) CHIP, p97/VCP, Parkin, and p47/Ubx1; III-tubulin in A and B (loading control). Molecular mass markers in kDa are on the left. (C) Expression of the listed genes (normalized to gapdh) by quantitative RT-PCR. Percentages represent the ratio between the relative intensities for each condition and control (DMSO) considered to be 100%. (D) PKA activity determined with a nonradioactive assay kit. Values indicate means (and SE in C and D) from at least three experiments. Asterisks identify values that are significantly different from control (*p < 0.05; **p < 0.01; ***p < 0.001).
3.3. db-cAMP increases the levels of the E3 ligase CHIP, the AAA-ATPase p97, and the ubiquitin gene ubB
3.4. db-cAMP reduces the accumulation of ubiquitinated proteins, proteasome inhibition and neurotoxicity induced by PGJ2
Since db-cAMP enhanced 26S proteasome activity we reasoned that the drug could also raise the levels of other components of the UPP that work in concert with the proteasome to promote protein degradation. We only tested the effect of db-cAMP, since it was the most effective drug stimulating the proteasome. dbcAMP (Fig. 2B) increased the levels of two enzymes that facilitate the degradation of misfolded proteins by the 26S proteasome: (1) the C-terminus of Hsc70 interacting protein (CHIP), which is a homodimeric ubiquitin ligase that links chaperone function with the degradation of misfolded or unstable proteins [7]; and (2) the homohexameric AAA-ATPase p97 (VCP/cdc48) that can associate with proteasomes, bind to polyubiquitinated proteins and promote their degradation [15]. The levels of CHIP and p97 were significantly elevated by 1.3-fold upon treatment with db-cAMP. db-cAMP also up-regulated the mRNA of the ubiquitin gene ubB by almost 2-fold but not ubC (Fig. 2C). Expression of both genes ubB and ubC, which encode tandemly repeated multiple ubiquitins with no spacer, are essential for stress tolerance [26,27]. Two other proteins relevant to protein degradation by the 26S proteasome, i.e. the E3 ligase Parkin and p47/Ubx1, a substrate-recruiting co-factor for p97, were not significantly altered by db-cAMP (Fig. 2B). We conclude that cAMP-signaling has a positive effect on specific components of the UPP.
Inflammation is associated with neuronal damage following spinal cord injury [10] and ALS [4]. Prostaglandins of the J2 series are endogenous products of inflammation reported to be neurotoxic [31]. We and others previously showed that PGJ2 inhibits the 26S proteasome by causing its disassembly [32] and/or oxidative modification [14]. We now show that PGJ2-treatment of spinal cord neurons induces significant (a) accumulation (∼2.4-fold above control) of ubiquitinated proteins (Fig. 3A); (b) decrease in the activity and levels of both forms of the 26S proteasome (two cap and one cap), while slightly increasing the activity of the 20S proteasome (Fig. 3B); (c) loss of viability determined with the MTT assay (Fig. 3C); (d) cytotoxicity assessed by the release of GAPDH from dying cells (Fig. 3D). Notably, db-cAMP and forskolin/rolipram reduced the damaging effects of PGJ2 listed above. Prevention of PGJ2-induced proteasome inhibition by forskolin/rolipram was minor (not shown). 4. Discussion Means to increase proteasome activity in spinal cord neurons are currently lacking. We show herein that db-cAMP enhances proteasome activity and levels in a PKA-dependent manner in spinal cord neurons (to our knowledge for the first time). This effect was
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Fig. 3. Elevating cAMP diminishes (A) accumulation of ubiquitinated proteins, (B) 26S proteasome inhibition, (C) loss of cell viability, and (D) cytotoxicity induced by PGJ2. Rat E18 spinal cord neurons were treated separately with 0.5% DMSO (control), 1 mM db-cAMP, 100 M forskolin/10 M rolipram (Fk + Rlp) or 20 M PGJ2 for 24 h. Parallel cultures were pretreated for 1 h with 1 mM db-cAMP or 100 M forskolin/10 M rolipram (Fk + Rlp) followed by 20 M PGJ2 for 24 h (db-cAMP + J2 or Fk + Rlp + J2). (A) Western blot analysis (40 g of protein/lane; 10% gels) detecting ubiquitinated proteins (Ub–proteins) and III-tubulin (loading control). (B) Proteasomal chymotrypsin-like activity (left panel) and levels (middle and right panels) were assessed as described in Fig. 1. 26S and 20S proteasomes are indicated by arrows. III-Tubulin (loading control). (C) Cell viability measured with the MTT assay. In (A–C) percentages represent the ratio between the data for each condition and control (DMSO) considered to be 100%. (D) Cell toxicity was assessed with the bioluminescent cytotoxicity assay and expressed as RU (relative units). Values indicate means and SE from at least three experiments. Asterisks identify values that are significantly different from control (*p < 0.05; **p < 0.01; ***p < 0.001). Furthermore, in (A) there is no significant difference between control and db-cAMP or Fk + Rlp within the DMSO- or J2-treatment groups. In (C) and (D) there is no significant difference between control and db-cAMP or Fk + Rlp within the DMSO-treatment group. Within the J2-treatment group, J2 alone is significantly different (p < 0.001) from J2 + db-cAMP or J2 + (Fk + Rlp).
mimicked albeit to a lesser extent, by forskolin/rolipram. At the subunit level, db-cAMP raised the mRNA and protein levels of Rpt6, a subunit of the proteasome 19S regulatory particle, and to a lesser extent 5, which is a constitutive subunit of the 20S proteasome. These results suggest an independent regulation of proteasome 19S and 20S subunit pools in the primary neuronal cultures. A similar PKA-mediated regulation of proteasome 19S and 20S subunits was observed in isoproterenol-induced cardiac hyperthrophy in mice [8]. In contrast, PKA-mediated stimulation of 26S proteasome activity did not cause an increase in proteasome subunit levels following the intracoronary administration of isoproterenol, or forskolin, or ischemic pre-conditioning in the canine heart [2]. These opposing results indicate variation in 26S proteasome regulation under different conditions. In conclusion, in vitro and in vivo studies demonstrate that PKA regulates proteasome activity at the transcriptional subunit level, as well as by subunit phosphorylation which promotes proteasome assembly [16,30,34]. db-cAMP treatment also elevated the protein levels of p62/sqstm1. We propose that the rise in p62/sqstm1 is mediated by increased mRNA levels, and not by inhibition of its turnover. The latter premise is based on p62/sqstm1 being degraded by autophagy [13], in conjunction with the finding in yeast, that activation of the TOR or the PKA pathways, inhibit autophagy via
phosphorylation of the Atg1/Atg13 complex [29]. To our knowledge, ours is the first report showing that p62/sqstm1 levels are regulated by cAMP. This is important because, due to oxidative damage to its promoter, there is an aging-correlated decrease in p62/sqstm1 levels, which is also apparent in neurodegenerative disorders such as Alzheimer and Parkinson diseases [9]. Moreover, absence of p62/sqstm1 induces neuronal cell death [21,25]. The overall impact of p62/sqstm1 on neuronal survival is not surprising because it is a scaffold protein that integrates kinase-activated and ubiquitin-mediated survival signals [18]. The protein levels of the E3 ubiquitin ligase CHIP and the AAA-ATPase p97/VCP were also elevated by cAMP. These two components of the UPP are associated with turnover of abnormal proteins. Together with the observed increase in 26S proteasome activity, our results support the notion that activation of cAMP/PKA signaling in spinal cord neurons could be a promising strategy to promote protein degradation via the UPP and avoid Ub–protein accumulation/aggregation in spinal cord neurodegenerative conditions associated with proteasome impairment, such as those induced by inflammation. This would be beneficial to neurons because, compared to astrocytes, neurons are particularly sensitive to the toxic effects of proteasome inhibition [6]. Recent developments in cyclic nucleotide-related biochemical and pharmacological
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