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. 2023 Aug 1;13(1):140.
doi: 10.1186/s13578-023-01068-6.

Calcineurin inhibition protects against dopamine toxicity and attenuates behavioral decline in a Parkinson's disease model

Rupsha Mondal #  1   2 , Chayan Banerjee #  1   2 , Sumangal Nandy  1 , Moumita Roy  1   2 , Joy Chakraborty  3   4
Affiliations

Calcineurin inhibition protects against dopamine toxicity and attenuates behavioral decline in a Parkinson's disease model

Rupsha Mondal et al. Cell Biosci. .

Abstract

Background: Parkinson's disease (PD), a highly prevalent neuro-motor disorder is caused due to progressive loss of dopaminergic (DAergic) neurons at substantia nigra region of brain. This leads to depleted dopamine (DA) content at striatum, thus affecting the fine tuning of basal ganglia. In patients, this imbalance is manifested by akinesia, catalepsy and tremor. PD associated behavioral dysfunctions are frequently mitigated by l-DOPA (LD) therapy, a precursor for DA synthesis. Due to progressive neurodegeneration, LD eventually loses applicability in PD. Although DA is cytotoxic, it is unclear whether LD therapy can accelerate PD progression or not. LD itself does not lead to neurodegeneration in vivo, but previous reports demonstrate that LD treatment mediated excess DA can potentiate neurotoxicity when PD associated genetic or epigenetic aberrations are involved. So, minimizing DA toxicity during the therapy is an absolute necessity to halt or slowdown PD progression. The two major contributing factors associated with DA toxicity are: degradation by Monoamine oxidase and DAquinone (DAQ) formation.

Results: Here, we report that apoptotic mitochondrial fragmentation via Calcineurin (CaN)-DRP1 axis is a common downstream event for both these initial cues, inhibiting which can protect cells from DA toxicity comprehensively. No protective effect is observed, in terms of cell survival when only PxIxIT domain of CaN is obstructed, demonstrating the importance to block DRP1-CaN axis specifically. Further, evaluation of the impact of DA exposure on PD progression in a mice model reveal that LD mediated behavioral recovery diminishes with time, mostly because of continued DAergic cell death and dendritic spine loss at striatum. CaN inhibition, alone or in combination with LD, offer long term behavioral protection. This protective effect is mediated specifically by hindering CaN-DRP1 axis, whereas inhibiting interaction between CaN and other substrates, including proteins involved in neuro-inflammation, remained ineffective when LD is co-administered.

Conclusions: In this study, we conclude that DA toxicity can be circumvented by CaN inhibition and it can mitigate PD related behavioral aberrations by protecting neuronal architecture at striatum. We propose that CaN inhibitors might extend the therapeutic efficacy of LD treatment.

Keywords: Calcineurin; Dendritic spine; Dopamine toxicity; L-DOPA therapy; Mitochondrial fragmentation; Parkinson’s disease.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Dopamine, tyramine or DAquinone leads to cell death. A, B Bar graphs show cell death after 24 h, as quantified by trypan blue or propidium iodide (PI) positive cells. SH-SY5Y cells are treated with different concentrations of dopamine (DA). N-acetyl cysteine (NAC)/mitoTEMPO/pargyline are administered 30 min before DA treatment. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 when compared to control group. #P ≤ 0.05, ###P ≤ 0.001, ####P ≤ 0.0001 when compared to the 300 µM DA treated group. C Cell death is measured in SH-SY5Y cells, as mentioned in A, after 24 h of tyramine (Tyr) treatment. **P ≤ 0.01 when compared to control group. D Monoamine oxidase A is overexpressed in SH-SY5Y cells and tyramine (Tyr) in mentioned concentration is treated for 24 h. Bar graphs represent cell as in A. ***P ≤ 0.001, ****P ≤ 0.0001 as compared to control. E SH-SY5Y cells are treated with tyramine (Tyr) for 48 h. NAC or mitoTEMPO is treated 30 min before Tyr treatment. Cell death is measured as mentioned above. **P ≤ 0.01, ***P ≤ 0.001 when compared to control group. #P ≤ 0.05 when compared to the 1000 µM Tyr treated group. F Cells are treated with DA (300 µM) and Tyrosinase (co: co treated) for 24 h. Alternately DA and Tyrosinase is incubated beforehand (pre: pre-incubation) and then administered to the cells. In the relevant groups mitoTEMPO is administered 30 min before the treatment. Cell death is measured as mentioned in A. ****P ≤ 0.0001 when compared to control group., ##P ≤ 0.01, ###P ≤ 0.0001 when compared to the respective 300 µM DA + Tyrosinase treated group. Bar graphs represent mean ± SEM. N = at least 3 for each group. P values are calculated by one way ANOVA followed by Tukey’s/Dunnett’s multiple comparison test, when compared between mean of each group or mean of control group, respectively
Fig. 2
Fig. 2
Dopamine, Tyramine, or DA-quinone treatment induce mitochondrial fragmentation, decrease p-Ser637 DRP1 and increase DRP1 mitochondrial localization. A SH-SY5Y cells are treated with DA (200 µM), Tyramine (1 mM) or DAQ (200 µM co-incubation with Tyrosinase) for 16 h and mitochondria is visualized by immunostaining for ATP5a. Scale bar 10 µm. B Mitochondrial morphology is analyzed after the treatment as mentioned above, and mean proportion of filamentous, rod shaped or punctate mitochondria is represented in the graphs. At least 30 cells are analyzed from 3 independent experiments for each group. C SH-SY5Y cells are treated as mentioned in A and total cell lysate is subjected to SDS PAGE followed by immunoblotting for the mentioned proteins. Immunoblots are representative of at least 3 different experiments. D Mean normalized band intensities of the mentioned proteins in C are represented as bar graphs. E SH-SY5Y cells are treated as mentioned in A and protein lysate from mitochondria is subjected to SDS PAGE followed by immunoblotting for the mentioned proteins. Immunoblots are representative of at least 3 different experiments. F Mean normalized band intensities of the mentioned proteins in E are represented as bar graphs. Bar graphs represent mean ± SEM. N = at least 3 for each group. P values are calculated by Student’s t test. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 compared to control group
Fig. 3
Fig. 3
Dopamine induced p-Ser637DRP1 depletion, mitochondrial fragmentation and apoptotic Cytochrome c re-localization is attenuated by Calcineurin inhibition. A SH-SY5Y cells are treated with DA (200 µM) or as mentioned for 16 h and total cell lysate is subjected to SDS PAGE followed by immunoblotting for the mentioned proteins. Bar graphs represent mean intensity value of p-Ser637DRP1 normalized by total DRP1. Immunoblots are representative of at least 3 different experiments. **P ≤ 0.05 as compared to control, one way ANOVA followed by Tukey’s multiple comparison test. B After the mentioned treatment for 16 h, Calcineurin (CaN) activity from the SH-SY5Y cell lysate is measured and the mean activity is represented. *P ≤ 0.05. C SH-SY5Y cells are treated for 16 h and cytosolic free Ca2+ is measured by Fluo-4-AM intensity. Mean intensity of each group is represented as bar graph. *P ≤ 0.05 as compared to control, #P ≤ 0.05 when compared to DA treated group. D After treatment, as mentioned, mitochondrial morphology of SH-SY5Y cells is analyzed from the captured images. Images are representative of 3 different experiments and at least 30 cells were analyzed. Scale bar 10 µm. Mitochondrial morphology is classified as mentioned earlier and mean proportion of filamentous, rod shaped or punctate mitochondria is represented in the bar graphs. *P ≤ 0.05, **P ≤ 0.01 as compared to control. E Control or MEF cells expressing DRP1K38A/dominant negative Calcineurin (ΔCnAH151Q) are treated as mentioned, for 16 h and mitochondria are imaged. Mitochondrial classification and quantification are done as mentioned above. *P ≤ 0.05 as compared to control. Scale bar 10 µm. F SH-SY5Y cells are treated with DA or DA + FK-506 for 16 h. Fixed cells are stained for ATP5a and Cytochrome c. Co-localization is quantified by measuring Mander’s coefficient and mean values are represented as bar graph. At least 30 cells were analyzed from 3 independent experiments. *P ≤ 0.05, **P ≤ 0.01 as compared to control, #P ≤ 0.05 as compared to DA treated group. G SH-SY5Y cell death is analyzed by propidium iodide (PI) or trypan blue staining, after DA or DA + FK-506 treatment for 24 h. *P ≤ 0.05, ***P ≤ 0.001when compared to control group; #P ≤ 0.05 and ##P ≤ 0.01 when compared to DA treated group. Bar graphs represent mean ± SEM. N = at least 3 for each group. P values are calculated by one way ANOVA followed by Tukey’s multiple comparison test unless mentioned otherwise
Fig. 4
Fig. 4
Calcineurin inhibition protects against MPTP induced behavioral deficiency in presence/absence of L-DOPA while VIVIT peptide fails to offer any. A Treatment paradigm for MPTP induced Parkinson’s disease model generation. MPTP (30 mg/kg) is treated on day 1 and 2 (16 h apart). From 3rd day the mentioned treatments are initiated, alone or in combination. Behavior is evaluated on 7th, 8th, 14th and 15th day. On 8th and 15th day, behavioral assays are done after 16 h of the last treatment. Neurotransmitter level analysis and histochemistry are done on either 8th or 15th day. [*] in red denotes the day in which animals are sacrificed. B Akinesia and catalepsy on the mentioned days for the treatment groups are evaluated. At least 4 animals are analyzed for each experiment. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001 when compared to control group; #P ≤ 0.05, ##P ≤ 0.01,###P ≤ 0.001,####P ≤ 0.0001 when compared to MPTP treated group. P values are calculated by two way ANOVA followed by Tukey’s multiple comparison test. C Trajectories from the mean swim score (± SEM) for the mentioned time is plotted and the cumulative score for 10 min, for each group on 8th and 15th day is represented. At least 4 animals are taken for the analysis. *P ≤ 0.05, **P ≤ 0.01,***P ≤ 0.001 when compared to the control group. P values are calculated by one way ANOVA followed by Tukey’s multiple comparison test. D Akinesia and catalepsy are evaluated on the mentioned days for the treatment groups. At least 4 animals are taken for the evaluation. *P ≤ 0.05, **P ≤ 0.01,***P ≤ 0.001, ****P ≤ 0.0001 when compared to control group; ##P ≤ 0.01 when compared to MPTP treated group. P values are calculated by two way ANOVA followed by Dunnetts multiple comparison test. E Trajectories from the average swim score (± SEM) for the mentioned time is plotted and the cumulative score for 10 min, for each group on 15th day is represented. At least 4 animals are taken for the analysis. **P ≤ 0.01,***P ≤ 0.001 and **** P ≤ 0.0001 when compared to the control group. P values are calculated by one way ANOVA followed by Dunnett’s multiple comparison test. Bar graphs represent mean ± SEM
Fig. 5
Fig. 5
Calcineurin inhibition protects against MPTP induced depletion of TH positive neurons at SN and dendritic spine density at striatum, while VIVIT peptide fails to safeguard the later. A Immunohistochemical staining for Tyrosine hydroxylase (TH) is performed after 14 days of treatment for substantia nigra (SN) and striatal cryosections (20 µm). Scale bar—100 µm. B TH positive neuronal cell bodies at SN or striatal projections are measured. N = 3 (C–E) After the treatment period striatal dopamine (DA) is quantified by HPLC method and represented as pmol/mg tissue. At least 4 animal striatum is utilized for the analysis. (C, D) denote changes in DA levels on 8th and 15th day respectively, while (E) shows the changes on 15th day. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 when compared to the control group, ##P ≤ 0.01 when compared to the MPTP treated group. @P ≤ 0.05 when compared to the MPTP + VIVIT treated group. P values are calculated by one way ANOVA followed by Tukey’s multiple comparison test. F Striatal sections (60 µm) are stained by Golgi-Cox method and representative images are provided. The full images of the neurons are presented in the inset. Scale bar 20 µm for inset image and 10 µm for the enlarged dendritic portion. G–I Spine number in entire length on individual dendritic projections from each neuron is counted and the mean number of spines are represented for each treatment group on the 8th (G) and 15th (H, I) day. At least 5 animal striatum and 4–5 neurons from each striatum are considered for the analysis. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 when compared to control group; #P ≤ 0.05, ##P ≤ 0.01, ###P ≤ 0.001, when compared to MPTP treated group. Bar graphs represent mean ± SEM. P values are calculated by one way ANOVA followed by Tukey’s multiple comparison test
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