Figure 1.
Reduction of CREB expression in HPRT-deficiency.
Reduction of CREB expression in HPRT-deficient human SH-SY5Y (A & B) and mouse MN9D (D & F) cells lines. Cells were then stimulated with DMSO (as control) or Forskolin (see methods). Immuno-blot as well as the quantification of protein through densitometry analysis show impaired expression of CREB in response to forskolin. The asterisks (*) represent statistical significance between forskolin treated cells (p<0.05, t-test n = 3). Reduced agonist-induced cAMP accumulation in HPRT-deficient SH-SY5Y (C) and MN9D (F) cells. Cells were stimulated with DMSO (CTL) or forskolin. Cyclic AMP level was evaluated as described in material and methods. The data are expressed as level of cAMP normalized to protein content. Error bars represent mean ± SEM of triplicate measurements of two experiments (n = 6). The asterisks (*p<0.05) represent statistical significance between forskolin treated cells (t-test). (G & H) Altered-CREB-mediated transcriptional activity in HPRT-deficiency; HEK293 cells lines were infected with lentivirus vector encoding small hairpin against luciferase (HPRT+) and HPRT gene (HPRT−). Cells were subsequently transfected with pCRE-DD-Zs-Green1 (CREB probe) and then stimulated with DMSO (CTL) or 50 µM Forskolin for 30 min. Figure shows microscopy images of DAPI staining and green fluorescence which is a measure of the overall CREB-related transcriptional activity. There is diminished green fluorescence in HPRT-deficient cells relative to control cells after stimulation with forskolin. (Bar scale, 100 µm). This is confirmed by the quantification of the mean fluorescent intensity illustrated in Figure H. Error bars represent mean ± SEM of duplicate measurements of two independent experiments. The asterisks (*) represent statistical significance between forskolin treated cells (p<0.05, t-test).
Figure 2.
Reduction of Synapsin I mRNA level in HPRT-deficient cells.
Synapsin I mRNA expression is reduced in HPRT-Deficient (mutant) MN9D cells. The asterisks (*) represent statistical significance between control (open bar) and mutant cells (closed bar) (n = 4, p<0.05, t-test).
Figure 3.
Blunted PKA-mediated signaling in HPRT-deficient MN9D cells.
(A), Immuno-blot analysis of phospho-PKA-substrate expression and p-Syn I (Ser9) in response to forskolin exposure. (B), quantification phospho-synapsin relative to the total amount of protein measured as beta-actin. Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisk (*) represents statistical significance between forskolin treated cells (p<0.05 t-test). (C&D), Effect of 6-bnz-cAMP on phospho-PKA-substrate and p-Syn I (Ser9) expression in HPRT-deficient MN9D cells. Data show reduced expression of phospho-PKA-substrates including p-Syn I (Ser9) in HPRT-deficient MN9D cells, after 6-bnz-cAMP treatment. The quantification of p-Syn relative to beta-actin is seen in figure D. Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisk (*) represents statistical significance between 6-bnz-cAMP treated cells (p<0.05 t-test).
Figure 4.
Increased PDE10A protein expression in HPRT-deficient MN9D cells.
(A&B) Immuno-blot and quantification analysis for various PDEs that can affect cAMP/PKA signaling. Data show that the expression of PDE1C, PDE4B, and PDE7B are similar between control (parent) and HPRT-deficient (mutant) MN9D cell lines. Expression of PDE10A protein is significantly increased in HPRT-deficient cells. Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisk (*) represents the statistical significant between the control (open bars) and HPRT-deficient (closed bars) MND9 cells (p<0.05 t-test).
Figure 5.
PDE10 inhibition restores PKA-mediated expression.
(A & B), immuno-blot and quantification analysis of p-Syn (Ser9), data show that the lower expression of phospho-PKA substrate and p-Syn (Ser9) in response to forskolin treatment in HPRT-deficient MN9D cells is restored in the presence of Papaverine (200 µM). Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisks (*) represent statistical significance between forskolin treated cells without papaverine treatment (p<0.05, t-test). (C & D), immuno-blot and quantification analysis of PDE10A expression after transfection of HPRT-deficient MN9D cells with siRNA directed to the mouse PDE10 gene. The data show lower expression PDE10A protein in cells transfected with siRNA-PDE10A relative to cells transfected with scramble control siRNA (siRNA-CTL). Error bars represent mean ± SEM of duplicate measurements carried out independently twice (n = 4). The asterisks (*) represent the statistical significant (p<0.05, t-test) between the control (open bar, siRNA-CTL) and siRNA-PDE10A transfected cells (closed bar). This significant reduction of PDE10A protein contributes to the enhanced expression of phospho-PKA-substrates including p-Syn observed in (E & F). Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisk (*) represents the statistical significant (p<0.05, t-test) between the siRNA-CTL and siRNA-PDE10A HPRT-deficient transfected MND9 cells treated with forskolin.
Figure 6.
Effect of HPRT-rescue on cAMP/PKA signaling.
Immuno-blot and densitometry quantification analysis of Syn 1and phospho-Syn I (A, B & C). Data show that the lower expression of Synapsin I protein in response to forskolin treatment in HPRT-deficient MN9D cells is abrogated in HPRT-reconstituted MN9D cells (B). p-Syn I (Ser9) expression in HPRT-reconstituted cells is partially restored (C). Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisk (*) represents statistical significance (p<0.05) between forskolin treated cells control versus HPRT-deficient cells, the double asterisks (**), between forskolin-treated HPRT-deficient cells versus forskolin-treated HPRT-reconstituted cells (ANOVA and Tukey, post-hoc test).
Figure 7.
Schematic of the possible mechanisms by which HPRT-deficiency via blunting of cAMP/PKA signaling leads to the neural-related LND phenotype.
HPRT-deficiency is primarily a purine metabolism deficiency that affects the cellular purine pools. The deficit in purine pools causes changes in expression and activity of PDE and/or AC reduces cAMP production. We propose that the cAMP deficit in HPRT-deficient causes a reduction in CREB and thereby expression of neural genes such as Synapsin I. The decrease in PKA activity blunts phosphorylation of substrates, such as p-Syn I (Ser9), which is known to affect neurotransmitter release. We conclude HPRT-deficiency via inhibiting cAMP/PKA signaling could affect neurotransmission and neuro-modulation that underlie the neurological phenotype in LND.