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. 2019 Jun;14(2):173-187.
doi: 10.1007/s11481-018-9808-3. Epub 2018 Sep 5.

Low-Dose Aspirin Upregulates Tyrosine Hydroxylase and Increases Dopamine Production in Dopaminergic Neurons: Implications for Parkinson's Disease

Suresh B Rangasamy  1   2 , Sridevi Dasarathi  2 , Priyanka Pahan  2 , Malabendu Jana  1   2 , Kalipada Pahan  3   4
Affiliations

Low-Dose Aspirin Upregulates Tyrosine Hydroxylase and Increases Dopamine Production in Dopaminergic Neurons: Implications for Parkinson's Disease

Suresh B Rangasamy et al. J Neuroimmune Pharmacol. 2019 Jun.

Erratum in

Abstract

Increasing the function of residual dopaminergic neurons in the nigra of PD patients is an important area of research as it may eventually compensate the loss. Although tyrosine hydroxylase (TH) is the rate-limiting enzyme in the dopamine (DA) biosynthesis pathway, there are no effective drugs/molecules to upregulate TH and increase the production of DA in nigral dopaminergic neurons. This study underlines the importance of aspirin in stimulating the expression of TH and increasing the level of DA in dopaminergic neurons. At low doses, aspirin increased the expression of TH and the production of DA in mouse MN9D dopaminergic neuronal cells. Accordingly, oral administration of aspirin increased the expression of TH in the nigra and upregulated the level of DA in striatum of normal C57/BL6 mice and aged A53T α-syn transgenic mice. Oral aspirin also improved locomotor activities of normal mice and A53T transgenic mice. While investigating mechanisms, we found the presence of cAMP response element (CRE) in the promoter of TH gene and the rapid induction of cAMP response element binding (CREB) activation by aspirin in dopaminergic neuronal cells. Aspirin treatment also increased the level of phospho-CREB in the nigra of C57/BL6 mice. The abrogation of aspirin-induced expression of TH by siRNA knockdown of CREB and the recruitment of CREB to the TH gene promoter by aspirin suggest that aspirin stimulates the transcription of TH in dopaminergic neurons via CREB. These results highlight a new property of aspirin in stimulating the TH-DA pathway, which may be beneficial in PD patients. Graphical Abstract ᅟ.

Keywords: Aspirin; CREB; Dopamine; Striatum; Substantia nigra pars compacta; Tyrosine hydroxylase.

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Conflict of interest statement

Conflict of interests: None

Figures

Figure 1.
Figure 1.. Dose- and time-dependent induction of tyrosine hydroxylase (TH) in mouse MN9D neuronal cells by aspirin.
(A) Mouse MN9D neuronal cells were treated with different concentrations of aspirin under serum-free conditions for 2 h followed by monitoring the mRNA expression of TH by semi-quantitative RT-PCR (A) and real-time PCR (B). Cells were treated with 2 µM aspirin for different time periods under serum-free condition followed by monitoring the mRNA expression of TH by semi-quantitative RT-PCR (C) and real-time PCR (D). Results are mean ± SD of at least three independent experiments. ***p < 0.001 vs control; ns, not significant. One-way ANOVA with Tukey’s multiple comparison test shows that both dose- (B) or time- (C) dependent increase in TH mRNA by aspirin is significant (p < 0.0001). Cells were treated with different concentrations of aspirin under serum-free conditions for 2 h followed by monitoring the protein level of TH by Western blot (E). Bands were scanned and values (TH/actin) presented as relative to control (F). Results are mean ± SD of three different experiments. *p < 0.05 & **p < 0.01 vs control; ns, not significant. One-way ANOVA with Tukey’s multiple comparison test shows that dose-dependent increase in TH protein by aspirin is significant (p < 0.01). Cells were treated with 2 µM aspirin for 2 h followed by double-label immunofluorescence with antibodies against TH and actin (G). Mean fluorescence intensity (MFI) of TH and actin was calculated in 20 different cells (H) and presented as MFI-TH/MFI-actin. Results are mean ± SEM of 20 different cells per group. *p < 0.05 & ***p < 0.001 vs control. I) Cells were treated with 2 µM aspirin for different time periods followed by measuring the level of DA, DOPAC and HVA in supernatants by HPLC. Results are mean ± SD of at least three independent experiments. ***p < 0.001 vs control; ns, not significant. One-way ANOVA with Tukey’s multiple comparison test shows that time-dependent increase in DA by aspirin is significant (p < 0.0001).
Figure 2.
Figure 2.. Increase in TH and dopamine in differentiated mouse MN9D neuronal cells by aspirin.
(A) Mouse MN9D neuronal cells were allowed to differentiate in neurobasal media containing B27 for 2 d following treatment with 2 μM aspirin for different time periods in neurobasal media without B27. The protein level of TH was monitored by Western blot (A). Bands were scanned and values (TH/actin) presented as relative to control (B). Results are mean SD of different experiments. *p < 0.05 & ***p < 0.001 vs control; ns, not significant. Cells were treated with 2 µM aspirin for 2 h followed by double-label immunofluorescence with antibodies against TH and actin (C). Mean fluorescence intensity (MFI) of TH and actin was calculated in 20 different cells (D) and presented as MFI-TH/MFI-actin. Results are mean ± SEM of 20 different cells per group. ***p < 0.001 vs control; ns, not significant. E) Cells were treated with 2 µM aspirin for different time periods followed by measuring the level of DA, DOPAC and HVA in supernatants by HPLC. Results are mean ± SD of at least three independent experiments. *p < 0.05 & ***p < 0.001 vs control; ns, not significant. One-way ANOVA with Tukey’s multiple comparison test shows that time-dependent increase in DA by aspirin is significant (p < 0.0001).
Figure 3.
Figure 3.. Oral administration of aspirin increases the level of TH in vivo in the nigra of C57/BL6 mice.
Male C57/BL6 mice (n=5 per group) were treated with aspirin (2 mg/kg body wt/d) mixed in 0.5% methylcellulose orally via gavage. Control mice received 0.5% methylcellulose as vehicle. After 30 d of treatment, the level of TH was monitored in the SNpc by Western blot (A). Actin was run as loading control. Bands were scanned and values (TH/actin) presented as relative to control (B). Results are mean + SEM of five mice per group. ***p<0.001 (= 5.28 × 10−4) vs control by two-tailed paired t-tests. The level of TH was monitored in ventral midbrain sections by immunofluorescence (C). MFI of TH (D) was calculated in two nigral sections of each of five mice per group. Results are mean ± SEM of five mice per group. ***p<0.001 (=1 × 10−7) vs control by two-tailed paired t-tests. The level of TH was monitored in ventral midbrain sections by DAB immunostaining (E). Optical density of TH (F) was calculated in two nigral sections of each of five mice per group. Results are mean ± SEM of five mice per group. *p<0.05 (= 0.012632) vs control by two-tailed paired t-tests.
Figure 4.
Figure 4.. Oral administration of aspirin increases the level of TH in vivo in the nigra of A53T-Tg mice.
Male A53T-Tg mice (n=5 per group) were treated with aspirin (2 mg/kg body wt/d) mixed in 0.5% methylcellulose orally via gavage. Control mice received 0.5% methylcellulose as vehicle. After 30 d of treatment, the level of TH was monitored in the SNpc by Western blot (A). Actin was run as loading control. Bands were scanned and values (TH/actin) presented as relative to control (B). Results are mean + SEM of five mice per group. ***p<0.001 (= 1.865 × 10−4) vs control by two-tailed paired t-tests. The level of TH was monitored in ventral midbrain sections by immunofluorescence (C). MFI of TH (D) was calculated in two nigral sections of each of five mice per group. ***p<0.001 (= 4.69 × 10−9) vs control by two-tailed paired t-tests. The level of TH was monitored in ventral midbrain sections by DAB immunostaining (E). Optical density of TH (F) was calculated in two nigral sections of each of five A53T-Tg mice per group. Results are mean ± SEM of five mice per group. **p<0.01 (= 0.00245) vs control by two-tailed paired t-tests.
Figure 5.
Figure 5.. Oral administration of aspirin increases the density of TH fibers in vivo in the striatum of C57/BL6 and A53T-Tg mice.
Male C57/BL6 (A & B) and A53T-Tg (C & D) mice (n=5 per group) were treated with aspirin (2 mg/kg body wt/d) mixed in 0.5% methylcellulose orally via gavage. Control mice received 0.5% methylcellulose as vehicle. After 30 d of treatment, striatal sections were immunostained for TH (A, C57/BL6; C, A53T-Tg). Optical density of TH fibers was quantified in two striatal sections of each of five mice per group (B, C57/BL6; D, A53T-Tg). Results are mean ± SEM of five mice per group. *p<0.05 (= 0.03206) vs control (B); **p<0.01 (= 0.00124) vs control (D) by two-tailed paired t-tests.
Figure 6.
Figure 6.. Oral administration of aspirin increases the level of DA in vivo in the striatum of C57/BL6 and A53T-Tg mice.
Male C57/BL6 (A-C) and A53T-Tg (D-F) mice (n=5 per group) were treated with aspirin (2 mg/kg body wt/d) mixed in 0.5% methylcellulose orally via gavage. Control mice received 0.5% methylcellulose as vehicle. After 30 d of treatment, levels of DA (A D), DOPAC (B & E) and HVA (C & F) were measured in striatum by HPLC. Results are mean ± SEM of five mice per group. **p<0.01 (= 0.00102) vs control (A); **p<0.01 (= 0.00914) vs control (B); **p<0.01 (= 0.00744) vs control (D); *p<0.05 (= 0.04617) vs control (E); *p<0.05 (= 0.04054) vs control by two-tailed paired t-tests. NS, not significant.
Figure 7.
Figure 7.. Oral administration of aspirin improves locomotor activities in C57/BL6 and A53T-Tg mice.
Male C57/BL6 (A, C-E) and A53T-Tg (B, F-H) mice (n=5 per group) were treated with aspirin (2 mg/kg body wt/d) mixed in 0.5% methylcellulose orally via gavage. Control mice received 0.5% methylcellulose as vehicle. After 30 d of treatment, locomotor activities were monitored (A & B, track plot; C & F, distance traveled; D & G, velocity; E & H, rearing). Results are mean ± SEM of five mice per group. **p<0.01 (= 0.00518) vs control (C); **p<0.01 (= 0.00312) vs control (D); *p<0.05 (= 0.03509) vs control (E); *p<0.05 (= 0.01560) vs control (F); **p<0.01 (= 0.00197) vs control by two-tailed paired t-tests. NS, not significant.
Figure 8.
Figure 8.. Aspirin induces the activation of CREB in MN9D neuronal cells and in vivo in the nigra of C57/BL6 mice.
(A) Position of CRE in the mouse TH gene promoter. MN9D neuronal cells were treated with 2 µM aspirin for different min under serum-free conditions followed by monitoring the level of phospho-CREB (pCREB) by Western blot (B). Graph represents densitometric analysis of pCREB protein levels normalized to total CREB (loading control) (C). Results are mean ± SD of three independent experiments. **p < 0.01 vs control; *p < 0.05 vs control. D) Cells were incubated with 2 µM aspirin for different min under serum-free conditions followed by monitoring the DNS-binding activity of CREB by EMSA. E) Male C57/BL6 mice (n=5 per group) were treated with aspirin (2 mg/kg body wt/d) mixed in 0.5% methylcellulose orally via gavage. Control mice received 0.5% methylcellulose as vehicle. After 7 d of treatment, the level of phospho-CREB and total CREB was monitored in the SNpc by Western blot. F) Bands were scanned and values (P-CREB/CREB) presented as relative to control. Results are mean + SEM of five mice per group. ***p < 0.001 vs control. Nigral sections were double-labeled for either TH & P-CREB (G) or TH & CREB (I). Mean fluorescence intensities (MFI) of P-CREB (H) and CREB (J) were calculated in two sections (one image per section) of each of five mice per group. **p < 0.01 vs control (H); NS, not significant (J).
Figure 9.
Figure 9.. Aspirin increases the transcription of TH in MN9D neuronal cells via CREB.
A) Cells were transfected with either control or CREB siRNA. Forty-eight h after transfection, level of CREB was monitored by Western blot. B) Graph represents densitometric analysis of CREB protein levels normalized to β-Actin (loading control). Results are mean ± SD of three independent experiments. **p < 0.01 vs control (no siRNA). C) Cells were transfected with either control siRNA or CREB siRNA. Forty-eight h after transfection, cells were treated with 2 μM aspirin for 2 h under serum-free condition followed by monitoring the level of TH by Western Blot. D) Graph represents densitometric analysis of TH protein levels normalized to β-Actin (loading control). Results are mean ± SD of three independent experiments. **p < 0.01 vs control; *p < 0.05 vs control-siRNA. Cells were treated with 2 μM aspirin for 30 min in serum free media. Then immunoprecipitated chromatin fragments were amplified by semi-quantitative and quantitative (F) PCR as described under “Materials and Methods”. Results are the mean ± SD of three separate experiments. ***p < 0.001 vs control.
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