Sodium Phenylbutyrate Controls Neuroinflammatory and Antioxidant Activities and Protects Dopaminergic Neurons in Mouse Models of Parkinson’s Disease

Neuroinflammation and oxidative stress underlie the pathogenesis of various neurodegenerative disorders. Here we demonstrate that sodium phenylbutyrate (NaPB), an FDA-approved therapy for reducing plasma ammonia and glutamine in urea cycle disorders, can suppress both proinflammatory molecules and reactive oxygen species (ROS) in activated glial cells. Interestingly, NaPB also decreased the level of cholesterol but involved only intermediates, not the end product of cholesterol biosynthesis pathway for these functions. While inhibitors of both geranylgeranyl transferase (GGTI) and farnesyl transferase (FTI) inhibited the activation of NF-κB, inhibitor of GGTI, but not FTI, suppressed the production of ROS. Accordingly, a dominant-negative mutant of p21rac, but not p21ras, attenuated the production of ROS from activated microglia. Inhibition of both p21ras and p21rac activation by NaPB in microglial cells suggests that NaPB exerts anti-inflammatory and antioxidative effects via inhibition of these small G proteins. Consistently, we found activation of both p21ras and p21rac in vivo in the substantia nigra of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease. Oral administration of NaPB reduced nigral activation of p21ras and p21rac, protected nigral reduced glutathione, attenuated nigral activation of NF-κB, inhibited nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Consistently, FTI and GGTI also protected nigrostriata in MPTP-intoxicated mice. Furthermore, NaPB also halted the disease progression in a chronic MPTP mouse model. These results identify novel mode of action of NaPB and suggest that NaPB may be of therapeutic benefit for neurodegenerative disorders

NaPB inhibits the production of ROS in mouse microglial cells.

<p>Cells were treated with 500 µM NaPB for 6 h followed by stimulation with 1 µM MPP⁺. At 15 min of stimulation, the generation of ROS was monitored by carboxy-H₂DCFDA (A). At different intervals (measured in minutes), superoxide production was assayed in whole cells (B). Cells preincubated with 500 µM NaPB for 6 h were stimulated with LPS (1 µg/ml), TNF-α (50 ng/ml), IL-1β (20 ng/ml), gp120 (200 pg/ml), and fibrillar Aβ1-42 (1 µM). At 10 min of stimulation, superoxide production was assayed in whole cells (C). Results are mean <u>+</u> SD of three different experiments. <i>^ap<0.001</i> vs control; <i>^bp<0.001</i> vs stimuli. D) Cells were incubated with NaPB in the presence or absence of HMG-CoA, mevalonate, GGPP, FPP, cholesterol, and coenzyme Q. After 6 h of incubation, cells were stimulated with LPS for 10 min followed by assay of superoxide. Results are mean <u>+</u> SD of three different experiments. <i>^ap<0.001</i> vs control; <i>^bp<0.001</i> vs LPS; <i>^cp<0.001</i> vs LPS+NaPB.</p

NaPB protects dopaminergic neurons in MPTP-intoxicated mice.

<p>Mice receiving NaPB (200 mg/kg body wt/day) from 3 h after the last injection of MPTP were sacrificed 7 d after the last injection of MPTP followed by TH immunostaining of SNpc (A) and striatum (B), counting of TH-positive neurons in SNpc (C), quantification of TH-positive fibers in striatum (D), assay of neurotransmitters in striatum (E), and quantification of GSH in nigra (F). Data are means <u>+</u> SEM of eight mice per group. <i>^ap<0.0001</i> vs saline group; <i>^bp<0.0001</i> vs the MPTP group.</p

NaPB inhibits the activation of NF-κB and the production of ROS via modulation of p21^ras and p21^rac.

<p>A) Mouse BV-2 microglial cells preincubated with different concentrations of FTI and GGTI were stimulated with LPS for 60 min followed by monitoring the activation of NF-κB by EMSA. B) Cells preincubated with different concentrations of FTI and GGTI were stimulated with LPS for 10 min followed by monitoring the production of superoxide. Results are mean <u>+</u> SD of three different experiments. <i>^ap<0.001</i> vs control; <i>^bp<0.001</i> vs LPS. C) Cells were transfected with Δp21^ras, Δp21^rac or an empty vector using Lipofectamine Plus. Twenty-four hour after transfection, cells were stimulated with LPS followed by monitoring superoxide production at 15 and 30 min of stimulation. <i>^ap<0.001</i> vs control; <i>^bp<0.001</i> vs LPS-15 min; <i>^cp<0.001</i> vs LPS-30 min. D) Cells preincubated with 500 µM NaPB were stimulated with LPS. At different time points, activation of p21^ras and p21^rac was monitored. Results represent three independent experiments.</p

Activation of small G proteins (p21^ras and p21^rac) and NF-κB in ventral midbrain of MPTP-intoxicated mice is NaPB-sensitive.

<p>A) Mice were treated with NaPB (200 mg/kg body wt/d) via gavage from 1 d prior to MPTP injection. Six h after the last injection of MPTP, activation of p21^ras and p21^rac was monitored in ventral midbrain tissues. Experiment was repeated three times each time using two animal in each group. B) Bands from three different mice were quantified and activation of p21^ras and p21^rac is shown as percent of control. C) Mice were treated with NaPB (200 mg/kg body wt/d) from 3 h after the last injection of MPTP. Twenty-four h after the last injection of MPTP, ventral midbrain sections were immunostained for p65 (low magnification). Midbrain sections of MPTP-intoxicated mice were also double-labeled for p65 and glial cell markers (GFAP for astrocytes and CD11b for microglia). Results represent three independent experiments. D) NF-κB p65 positive cells counted in four nigral sections (two images per slide) from each of four mice in an Olympus IX81 fluorescence microscope using the MicroSuite™ imaging software are mentioned as cells/mm². <i>^ap<0.0001</i> vs saline-control; <i>^bp<0.0001</i> vs MPTP.</p

NaPB attenuates activation of NF-κB in mouse microglial cells.

<p>(A) BV-2 microglial cells preincubated with 0.5 mM NaPB for 6 h were stimulated with 1 µg/ml LPS. At different minute of stimulation, the level of phospho-IκBα was monitored by Western blot. B) Cells preincubated with different concentrations of NaPB for 6 h were stimulated with LPS for 1 h followed by monitoring the DNA-binding activity of NF-κB by EMSA. (C) Cells plated in 12-well plates were co-transfected with 0.25 µg of PBIIX-Luc (an NF-κB-dependent reporter construct) and 12.5 ng of pRL-TK. Twenty-four h after transfection, cells received different concentrations of NaPB. After 6 h of incubation, cells were stimulated with LPS for 4 h. Firefly (ff-Luc) and Renilla (r-Luc) luciferase activities were obtained by analyzing the total cell extract. Results are mean <u>+</u> S.D. of three different experiments. <i>^ap<0.001</i> vs control; <i>^bp<0.05</i> vs LPS; <i>^cp<0.001</i> vs LPS. D) Cells preincubated with 0.5 mM NaPB for 6 h were stimulated with LPS for 2 h followed by monitoring the recruitment of RelA p65 to the mouse iNOS promoter by ChIP assay. E) Cells were co-transfected with 0.25 µg of PBIIX-Luc and 12.5 ng of pRL-TK. Twenty-four hours after transfection, cells were incubated with NaPB in the presence or absence of HMG-CoA, mevalonate, FPP, GGPP, cholesterol, and coenzyme Q. After 6 h of incubation, cells were stimulated with LPS for 4 h followed by assay of firefly (ff-Luc) and Renilla (r-Luc) luciferase activities. Results are mean <u>+</u> S.D. of three different experiments. <i>^ap<0.001</i> vs control; <i>^bp<0.001</i> vs LPS; <i>^cp<0.001</i> vs LPS+NaPB.</p