cAMP stimulates the ubiquitin/proteasome pathway in rat spinal cord neurons

Abstract

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 β5, 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.

Figure 1. Elevating cAMP stimulates the 26S proteasome via PKA

Rat E18 spinal cord neurons were treated for 24h with: (A) 0.5% DMSO (control), 1 mM db-cAMP, or 100 μM forskolin/10 μM rolipram (Fk+Rlp); (B) 0.5% DMSO (control), pretreated for one hour with 100 μM Rp-cAMPS followed by 1 mM db-cAMP (Rp-cAMPS + Db-cAMP), or 1 mM db-cAMP. Clear lysates (80 μg/sample) were subjected to non-denaturing gel electrophoresis. Proteasomal chymotrypsin-like activity was assessed with Suc-LLVY-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-β5 (middle and right panels). βIII-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).

Figure 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 μM forskolin/10 μM rolipram (Fk+Rlp) for 24h. Western blot analyses (40 μg of protein/lane; 10% gels) detected in: (A) proteasome subunits (Rpt6 and β5), p62/sqstm1, and PKA catalytic subunit Cα (PKA sub Cα); (B) CHIP, p97/VCP, Parkin, and p47/Ubx1; βIII-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 S.E. 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).

Figure 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 μM forskolin/10 μM rolipram (Fk+Rlp) or 20 μM PGJ2 for 24h. Parallel cultures were pretreated for one hour with 1 mM db-cAMP or 100 μM forskolin/10 μM rolipram (Fk+Rlp) followed by 20 μM PGJ2 for 24h (Db-cAMP+J2 or Fk+Rlp+J2). (A) Western blot analysis (40 μg of protein/lane; 10% gels) detecting ubiquitinated proteins (Ub-proteins) and βIII-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. βIII-Tubulin (loading control). (C) Cell viability measured with the MTT assay. In A, B and 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 S.E. 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).