Contributive Role of TNF-α to L-DOPA-Induced Dyskinesia in a Unilateral 6-OHDA Lesion Model of Parkinson’s Disease.

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. 2021 Jan 11;11:617085.

doi: 10.3389/fphar.2020.617085. eCollection 2020.

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Maurício Dos Santos Pereira et al. Front Pharmacol. .

Abstract

Our present objective was to better characterize the mechanisms that regulate striatal neuroinflammation in mice developing L-DOPA-induced dyskinesia (LID). For that, we used 6-hydroxydopamine (6-OHDA)-lesioned mice rendered dyskinetic by repeated intraperitoneal injections of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) and quantified ensuing neuroinflammatory changes in the dopamine-denervated dorsal striatum. LID development was associated with a prominent astrocytic response, and a more moderate microglial cell reaction restricted to this striatal area. The glial response was associated with elevations in two pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β. Treatment with the phytocannabinoid cannabidiol and the transient receptor potential vanilloid-1 (TRPV-1) channel antagonist capsazepine diminished LID intensity and decreased TNF-α levels without impacting other inflammation markers. To possibly reproduce the neuroinflammatory component of LID, we exposed astrocyte and microglial cells in culture to candidate molecules that might operate as inflammatory cues during LID development, i.e., L-DOPA, dopamine, or glutamate. Neither L-DOPA nor dopamine produced an inflammatory response in glial cell cultures. However, glutamate enhanced TNF-α secretion and GFAP expression in astrocyte cultures and promoted Iba-1 expression in microglial cultures. Of interest, the antidyskinetic treatment with cannabidiol + capsazepine reduced TNF-α release in glutamate-activated astrocytes. TNF-α, on its own, promoted the synaptic release of glutamate in cortical neuronal cultures, whereas cannabidiol + capsazepine prevented this effect. Therefore, we may assume that the release of TNF-α by glutamate-activated astrocytes may contribute to LID by exacerbating corticostriatal glutamatergic inputs excitability and maintaining astrocytes in an activated state through a self-reinforcing mechanism.

Keywords: IL-1β; astrocyte; cannabidiol; dopamine; glutamate; microglia; neuroinflammation, TNF-α.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1

FIGURE 1

Schematic drawing summarizing the experimental design of in vivo experiments. Two groups of sham-operated and 6-OHDA-lesioned mice were sacrificed 21 days after stereotaxic surgery to assess the extent of nigrostriatal dopaminergic deficits and intensity of glial inflammatory changes in the striatum. Abnormal involuntary movements scores were also evaluated in the same groups before sacrifice. Two other groups of 6-OHDA-lesioned mice received daily i.p. injections of Veh or L-DOPA (25 mg/kg) + benserazide (10 mg/kg) from day 21 until day 42 to assess the same parameters as before and perform cytokine measurements. The two last groups of 6-OHDA-lesioned mice received L-DOPA in combination or not with CPZ (5 mg/kg i.p.) + CBD (30 mg/kg i.p.) from day 42 until day 45 to assess abnormal involuntary movements score and inflammation parameters. CPZ (5 mg/kg i.p.) and CBD (30 mg/kg i.p.) or corresponding Veh were given 30 and 15 min before L-DOPA, respectively or corresponding Veh. S: sacrifice.

FIGURE 2

FIGURE 2

Abnormal involuntary movements after L-DOPA treatment in mice with partial unilateral lesions of the nigrostriatal pathway. (A) Sum of axial, limb and orofacial (ALO) abnormal involuntary movements (AIMs) of sham-operated and 6-OHDA-lesioned mice, 21 or 42 days after surgery. Between days 21–42, 6-OHDA-lesioned mice received daily injections of Veh or L-DOPA. (B) Sum of locomotor activity in the same groups of mice as in (A). *p < 0.05 vs. sham, n = 7; Kruskal-Wallis analysis on ranks followed by Dunn’s test. (C) Density of TH+ cell bodies per 0.5 mm2 in the substantia nigra pars compacta and (D) Relative TH optical density (OD) in the striatum of the same groups of mice as in (A). *p < 0.05 vs. sham, n = 7; One-way ANOVA followed by Bonferroni test. Data are presented as both median (horizontal black lines) and individual values (circles) in (A) and (B) and as mean ± SEM in (C) and (D).

FIGURE 3

FIGURE 3

Quantification of the glial reaction in the dopamine-denervated dorsal striatum of mice receiving L-DOPA. (A) Intensity of the astroglial reaction evaluated by quantification of the GFAP immunosignal in the dorsal striatum of sham-operated and 6-OHDA-lesioned mice sacrificed at day 21 or at day 42 after stereotaxic surgery. Mice sacrificed at day 42 received Veh or L-DOPA treatment between days 21–42. *p < 0.05 vs. sham; #p < 0.05 vs. Veh, n = 7; One-way ANOVA followed by Bonferroni test. (B) Micrographs depicting GFAP+ cells in the dorsal area of the striatum of the same groups as before. Note profound changes in the number of GFAP+ astrocytes and in their morphology 21 days after 6-OHDA injection. At day 42, the astrocyte reaction is reduced but still intense in 6-OHDA-lesioned mice treated with L-DOPA. (C) Intensity of the microglial reaction was evaluated by quantifying the density of Iba-1+ cells in the dorsal striatum of the same groups as before. *p < 0.05 vs. sham; #p < 0.05 vs. Veh, n = 7; One-way ANOVA followed by Bonferroni test. (D) Micrographs depicting Iba-1+ cells in the dorsal area of the striatum of the same groups as before. Note that the microglial reaction is prominent in 6-OHDA-lesioned mice 21 days after stereotaxic surgery. At day 42 the microglial reaction is reduced but still present in L-DOPA-treated mice. (E) Immunocytochemical detection of TH, GFAP and Iba-1 in the dorsal striatum of 6-OHDA-lesioned mice treated with L-DOPA between day 21 and 42. Illustrations show that the glial reaction is restricted to the dorsal striatum where TH immunostaining is reduced. Dash lines represent the virtual boundary between the dorsal and ventral striatum. Micrographs for GFAP and Iba-1 are from slides at level +0.1 mm and +0.18 mm from Bregma, respectively. Scale bar: 100 µm.

FIGURE 4

FIGURE 4

Cytokine levels in the dorsal striatum of L-DOPA dyskinetic mice. (A) TNF-α levels in sham-operated (21 days) and 6-OHDA-treated mice (42 days) receiving or not L-DOPA. (B) IL1-β levels in sham-operated (21 days) and 6-OHDA-treated mice (42 days) receiving or not L-DOPA. *p < 0.05 vs. sham, n = 6; One-way ANOVA followed by Bonferroni test. (C) Correlation between TNF-α values and abnormal involuntary movements scores in mice treated with L-DOPA. (D) Correlation between IL1-β values and abnormal involuntary movements scores in mice treated with L-DOPA. In (C) and (D) filled circles represent data values from individual animals. Spearman’s correlation coefficient and p-values are presented for each cytokine in the figure above.

FIGURE 5

FIGURE 5

Impact of CPZ + CBD on LID manifestation and striatal inflammation parameters. (A) Impact of a treatment with CPZ (5 mg/kg i.p.) + CBD (30 mg/kg i.p.) on the abnormal involuntary movements (AIMs) score of dyskinetic mice receiving L-DOPA. Data are presented as median (horizontal lines) and individual values (filled circles). *p < 0.05 vs. Veh, n = 6; Mann-Whitney’s test. (B) Impact of CPZ + CBD on TNF-α levels within the dorsal injured striatum of dyskinetic mice. *p < 0.05 vs. Veh, n = 6; Mann-Whitney’s test. (C) Correlation plot between TNF-α values and AIMs scores in mice treated with Veh + L-DOPA or CPZ + CBD + L-DOPA. The Spearman correlation coefficient r and the p value are given above in the plot. (D) Absence of impact of CPZ + CBD on IL1-β levels within the dorsal injured striatum of dyskinetic mice. (E) Absence of impact of the CPZ + CBD treatment on the intensity of the GFAP immunosignal in the dorsal striatum of dyskinetic mice. (F) Absence of impact of the CPZ + CBD treatment on the number of Iba-1+ microglial cells in the dorsal striatum of dyskinetic mice. (G) Representative illustrations showing that the treatment with CPZ + CBD had no impact on the intensity of the GFAP immunosignal from astrocytes, the density of Iba-1+ microglial cells and the TH immunosignal of dopaminergic nerve endings in the dorsal striatal area of dyskinetic mice receiving L-DOPA. Scale bar: 50 µm.

FIGURE 6

FIGURE 6

Neither L-DOPA nor dopamine promotes activation of astrocytes or microglial cells in culture. (A) Western immunoblotting characterization of GFAP expression in astrocyte cultures treated with L-DOPA (3 and 10 µM) or (B) dopamine (3 and 10 μM). (C) Western immunoblotting characterization of Iba-1 expression in microglial cultures treated with L-DOPA (3 and 10 µM) or (D) dopamine (3 and 10 μM). Note that 10 µM dopamine caused a significant decrease of the Iba-1 signal. *p < 0.05 vs. control, n = 5; One-way ANOVA followed by Bonferroni test. Data are expressed in arbitrary units (AU).

FIGURE 7

FIGURE 7

Glutamate promotes activation of cultured astrocytes. (A) Impact of glutamate (50 and 500 μM) on GFAP expression as characterized by western immunoblotting in astrocyte cultures. (B) Impact of glutamate (50 and 500 μM) on Iba-1 expression as characterized by western immunoblotting in microglial cultures. *p < 0.05 vs. control, n = 6; One-way ANOVA followed by Bonferroni test. (C) Impact of glutamate (50 and 500 µM) on TNF-α levels in astrocyte and (D) microglial cultures. In both cases, LPS (0.5 ng/ml) was used as reference inflammogen. *p < 0.05 vs. control, n = 6–8; One-way ANOVA followed by Bonferroni test. ND: Non-detectable.

FIGURE 8

FIGURE 8

CPZ + CBD reduces TNF-α production in cultured astrocytes exposed to glutamate. Impact of CPZ (0.1 µM) + CBD (0.1 μM) on TNF-α elevation produced by glutamate (50, 500 µM) in astrocyte cultures. *p < 0.05 vs. control, #p < 0.05 vs. same glutamate concentration, n = 6; One-way ANOVA followed by Bonferroni test.

FIGURE 9

FIGURE 9

CPZ + CBD prevents the release of glutamate induced by TNF-α in cultures of cortical neurons. (A) Impact that a 4-days exposure to TNF-α (50 ng/ml) or the reference treatment 4AP (2.5 mM) + BIC (50 μM) exert on the release of glutamate in pure neuronal cortical cultures. A co-treatment with CPZ + CBD (both at 0.1 μM) reduced glutamate release induction in both paradigms. TNF-α failed to modulate glutamate levels in astrocyte (B) or microglial (C) cell cultures. *p < 0.05 vs. control. #p < 0.05 vs. TNF-α or 4-AP + BIC, n = 7; One-way ANOVA followed by Bonferroni test.

FIGURE 10

FIGURE 10

Hypothetical mechanisms through which astrocytes, glutamate, and TNF-α might contribute to LID onset and perpetuation. The dyskinesiogenic treatment with L-DOPA causes the stimulation of corticostriatal inputs, elevating glutamate levels at corticostriatal synapses. The elevation of glutamate elicits an inflammatory-type response in astrocytes. Reactive astrocytes release the pro-inflammatory cytokine TNF-α, which is capable of further enhancing the release of glutamate by cortical neurons in a self-reinforcing manner, thus creating a vicious circle that contributes to LID perpetuation. Note that glutamate might be also released by activated microglial cells through the activation of the Xc antiporter system (Farber and Kettenmann, 2005). The elevation of striatal IL1-β levels in dyskinetic mice appears unrelated to glutamate release. DA: dopamine; Glu: glutamate; SNpc: substantia nigra pars compacta.

References

    1. Barnum C. J., Eskow K. L., Dupre K., Blandino P., Jr., Deak T., Bishop C. (2008). Exogenous corticosterone reduces L-DOPA-induced dyskinesia in the hemi-parkinsonian rat: role for interleukin-1beta. Neuroscience 156, 30–41. 10.1016/j.neuroscience.2008.07.016 – DOI PMC PubMed
    1. Baufreton J., Milekovic T., Li Q., McGuire S., Moraud E. M., Porras G., et al. (2018). Inhaling xenon ameliorates l‐dopa‐induced dyskinesia in experimental parkinsonism. Mov. Disord 33, 1632–1642. 10.1002/mds.27404 – DOI PMC PubMed
    1. Bortolanza M., Cavalcanti-Kiwiatkoski R., Padovan-Neto F. E., da-Silva C. A., Mitkovski M., Raisman-Vozari R., et al. (2015a). Glial activation is associated with l-DOPA induced dyskinesia and blocked by a nitric oxide synthase inhibitor in a rat model of Parkinson’s disease. Neurobiol. Dis 73, 377–387. 10.1016/j.nbd.2014.10.017 – DOI PubMed
    1. Bortolanza M., Padovan-Neto F. E., Cavalcanti-Kiwiatkoski R., Dos Santos-Pereira M., Mitkovski M., Raisman-Vozari R., et al. (2015b). Are cyclooxygenase-2 and nitric oxide involved in the dyskinesia of Parkinson’s disease induced by L-DOPA?. Philos. Trans. R. Soc. Lond. B Biol. Sci 370 (1672), 20140190 10.1098/rstb.2014.0190 – DOI PMC PubMed
    1. Bortolanza M., Nascimento G., Raisman‐Vozari R., Del‐Bel E. (2020). Doxycycline, an anti‐inflammatory agent, alleviates dyskinesia induced by L‐DOPA in Parkinsonian rats. 10.22541/au.159285563.30385356 – DOI

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