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. 2013 Jan;10(1):143-53.
doi: 10.1007/s13311-012-0165-2.

Neuroprotective and Anti-Inflammatory Properties of a Coffee Component in the MPTP Model of Parkinson's Disease

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Free PMC article

Neuroprotective and Anti-Inflammatory Properties of a Coffee Component in the MPTP Model of Parkinson's Disease

Kang-Woo Lee et al. Neurotherapeutics. .
Free PMC article

Abstract

Consumption of coffee is associated with reduced risk of Parkinson's disease (PD), an effect that has largely been attributed to caffeine. However, coffee contains numerous components that may also be neuroprotective. One of these compounds is eicosanoyl-5-hydroxytryptamide (EHT), which ameliorates the phenotype of α-synuclein transgenic mice associated with decreased protein aggregation and phosphorylation, improved neuronal integrity and reduced neuroinflammation. Here, we sought to investigate if EHT has an effect in the MPTP model of PD. Mice fed a diet containing EHT for four weeks exhibited dose-dependent preservation of nigral dopaminergic neurons following MPTP challenge compared to animals given control feed. Reductions in striatal dopamine and tyrosine hydroxylase content were also less pronounced with EHT treatment. The neuroinflammatory response to MPTP was markedly attenuated, and indices of oxidative stress and JNK activation were significantly prevented with EHT. In cultured primary microglia and astrocytes, EHT had a direct anti-inflammatory effect demonstrated by repression of lipopolysaccharide-induced NFκB activation, iNOS induction, and nitric oxide production. EHT also exhibited a robust anti-oxidant activity in vitro. Additionally, in SH-SY5Y cells, MPP(+)-induced demethylation of phosphoprotein phosphatase 2A (PP2A), the master regulator of the cellular phosphoregulatory network, and cytotoxicity were ameliorated by EHT. These findings indicate that the neuroprotective effect of EHT against MPTP is through several mechanisms including its anti-inflammatory and antioxidant activities as well as its ability to modulate the methylation and hence activity of PP2A. Our data, therefore, reveal a strong beneficial effect of a novel component of coffee in multiple endpoints relevant to PD.

Figures

Fig. 1
Fig. 1
EHT treatment attenuates MPTP-induced nigrostriatal dopaminergic neuronal damage. Mice were treated with EHT for 4 weeks and challenged with MPTP (10 mg/kg, every 2 h X4). Brains were analyzed 7 days post MPTP. a, b Stereological counting of TH-positive and cresyl-violet positive neurons in sections of the substantia nigra from one hemisphere. Normal diet-saline injections (control), n = 4; normal diet-MPTP, n = 3; EHT in diet (0.01 %)-MPTP, n = 4; and EHT in diet (0.1 %)-MPTP, n = 3. c Representative images of TH immunohistochemistry of midbrain sections. Scale bar = 200 μm. d Striatal dopamine (DA) level measured by HPLC. Control (no EHT or MPTP), n = 6; normal diet-MPTP, n = 5; EHT (0.01 %)-MPTP, n = 6; and EHT (0.1 %)-MPTP, n = 5. e Dopamine turnover measured by HVA/dopamine ratio. f Striatal TH content measured by ELISA. Control, n = 6; normal diet-MPTP, n = 5; EHT (0.01 %)-MPTP, n = 6; and EHT (0.1 %)-MPTP, n = 5. g EHT treatment had no impact on MPTP metabolism in vivo. Mice were treated with two dose levels of EHT for 2 weeks prior to receiving a single dose of 30 mg/kg MPTP. Animals were sacrificed 90 minutes later (n = 4 per group). MPP+ levels were assessed by HPLC. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 2
Fig. 2
EHT treatment attenuates MPTP-induced glial activation in the striatum. a EHT attenuates MPTP-induced microglial activation. Three days post MPTP, the increased microglia detected by Iba-1 immunoreactivity is significantly diminished in mice pretreated with EHT. b EHT attenuates MPTP-induced astrocytosis. Sections are stained with GFAP. c Quantification of images in panel A. d Quantification of images in panel C. Normal diet-saline, n = 4; normal diet-MPTP, n = 5; EHT (0.01 %)-MPTP and EHT (0.1 %)-MPTP, n = 5. Scale bar = 100 μm. *p < 0.05, ***p < 0.001
Fig. 3
Fig. 3
EHT treatment prevents MPTP-induced depletion of reduced glutathione. a The decline in the ratio of reduced/oxidized glutathione (GSH/GSSG) 7 days following MPTP was completely prevented by EHT treatment in the substantia nigra (n = 5 per group). b No significant change was seen in the cortex (n = 3 per group). *p < 0.05, **p < 0.01
Fig. 4
Fig. 4
EHT treatment attenuates MPTP-induced activation/phosphorylation of JNK. a Western blot for phosphorylated and total JNK in the striatum 3 days post-MPTP. b Quantification of p-JNK band intensities. **p < 0.01, ***p < 0.001
Fig. 5
Fig. 5
EHT inhibits p65 activation, iNOS activation and nitric oxide production in primary microglia and astrocytes. a EHT inhibits LPS-induced p65 activation in primary microglia. Cells were pretreated with 10 μM EHT for 1 h and then stimulated with 1 μg/ml LPS for 15 min. Western blots were done for phospho-p65 (Ser536), p65, and β-actin. Bar graph depicts relative p-p65 level. ** p < 0.01 (n = 3). b EHT inhibits iNOS induction by LPS in primary microglia. Cells were pretreated with 10 μM EHT for 1 h and then stimulated with 1 μg/ml LPS for 24 h. c LPS induced nitric oxide production by microglia is significantly decreased by EHT. n = 6. **p < 0.001. d EHT suppresses iNOS expression in primary astrocytes. Cells were pretreated with 10 or 25 μM EHT for 1 h and then stimulated with 1 μg/ml LPS for 48 h. e EHT suppresses LPS induced nitric oxide production in astrocytes, n = 6. ** p < 0.01 relative to LPS treatment without EHT
Fig. 6
Fig. 6
EHT is a direct anti-oxidant. Compounds tested in the ABTS radical scavenging colorimetric assay: ascorbic acid (▼), EHT (○) and Trolox (●). Data shown are means ± SEM from three independent experiments
Fig. 7
Fig. 7
EHT prevents MPP+-induced demethylation of PP2A and cell death in SH-SY5Y cells. a EHT inhibits MPP+-induced PP2A demethylation. Cells were pretreated with 10 μM EHT for 6 h and then stimulated with 3 mM MPP+ for 18 h. Western blots were done for demethyl-PP2A, total PP2A, and β-actin. b Bar graph depicts relative demethyl-PP2A level. **p < 0.01 (n = 4). c EHT suppresses MPP+-induced cell death assessed by LDH release. Cells were pretreated with 10 μM EHT for 6 h and then stimulated with 3 mM MPP+ for 18 h, *p < 0.05 (n = 6). d EHT rescues MPP+-induced diminished cell survival assessed by MTT assay. Cells were pretreated with 10 μM EHT for 6 h and then stimulated with 3 mM MPP+ for 18 h, *p < 0.05 (n = 6)

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