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Treating Parkinson’s Disease via Activation of BDNF/TrkB Signaling Pathways and Inhibition of Delta-Secretase

Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disease with motor disorders as the key clinical features. BDNF/TrkB neurotrophic signalings are progressively reduced, whereas δ-secretase, a protease that cleaves α-synuclein (α-Syn) at N103 and promotes its aggregation and neurotoxicity, is gradually escalated in PD patient brains, associated with dopaminergic neuronal loss in the Substantia Nigra. Here, we show that stimulation of deficient BDNF/TrkB signalings with its small molecular agonist CF3CN displays the promising therapeutic effect, and blockade of δ-secretase with an optimal specific inhibitor #11A exhibits marked therapeutic effect, and combination of both demonstrates additive restorative efficacy in MPTP-induced human SNCA transgenic PD mice. Upon oral administration, CF3CN robustly activates TrkB-mediated neurotrophic pathway in the brains of SNCA mice and decreases α-Syn N103 cleavage by δ-secretase, and #11A strongly blocks δ-secretase and reduces α-Syn N103 fragmentation, increasing TH-positive dopaminergic neurons. The mixture of CF3CN and #11A shows the maximal TH and dopamine levels with demonstrable BDNF as compared to negligible BDNF in vehicle-treated MPTP/SNCA mice, leading to the climaxed motor functions. Notably, both compounds possess the appropriate in vivo PK profiles. Hence, our findings support that CF3CN and #11A are promising therapeutic pharmaceutical agents for treating PD.

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Acknowledgements

This work was supported by the NIH (R01AG051538; R01NS 02338) to K. Y., the National Natural Science Foundation of China (no. 82071370) to Z.W., Shanghai Pujiang Program (no. 19PJ1409200) to Z.W. We thank the Emory Alzheimer’s Disease Research Center for postmortem human AD and healthy control samples. We thank the University of Washington Alzheimer’s Disease Research Center (NIH P50AG005136) and the Kaiser Permanente Adult Changes in Thought Study (NIH U01 AG006781) research subjects and their families for brain donation, a tremendous gift to science. This study was supported in part by the Rodent Behavioral Core (RBC) and Viral Vector Core, which are subsidized by the Emory University School of Medicine and are part of the Emory Integrated Core Facilities. Additional support was provided by the Emory Neuroscience NINDS Core Facilities (P30NS055077). Further support was provided by the Georgia Clinical & Translational Science Alliance of the National Institutes of Health under award number UL1TR002378.

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Disclosure

K.Y. is a shareholder of Shanghai Braegen Pharmaceuticals, Inc. and Wuhan Yuanzhen Pharmaceuticals, Inc.

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KY conceived the project, designed the experiments, analyzed the data, and wrote the manuscript. SSK and ZW designed and performed most of the experiments and analyzed the data. XL conducted genotype and breed the transgenic mice. LEM assisted with data analysis and interpretation and critically read the manuscript.

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Correspondence to Keqiang Ye.

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13311_2022_1248_MOESM1_ESM.tif

Supplementary file1 Compound #11 reduced AEP activity in SH-SY5Y cells treated with MPP+. (A). Immunoblotting demonstrated reduced AEP activity in the SH-SY5Y cells treated with #11. To provide a clear AEP cleavage, human wild-type α-Syn was overexpressed by transfection before the MPP+ treatment. AEP levels were increased with the treatment of MPP+ (1st panel), leading to the cleavage production, including α-Syn N103 (2nd panel), APP N585 (3rd panel) and Tau N368 (4th panel). Compound 11 (#11, 2nd lane, 1~4 panels) inhibited activation of AEP, resulting in diminished AEP substrates cleavage. Arrow indicates full-length AEP, arrowhead indicates active AEP. The intensities of the IB were quantified by densitometry. Data are shown as mean ± SEM. (n = 3 each group). (*p<0.05; **p<0.01, One-way ANOVA). (B). AEP activity assay demonstrated that #11 inhibited AEP activity induced by MPP+ treatment. (C). #11 treatment alleviated MPP+-caused cytotoxicity. Mean ± SEM, n = 3 in each group, one-way ANOVA with Tukey’s multiple-comparisons test, ** p < 0.01. (TIF 1149 KB)

13311_2022_1248_MOESM2_ESM.tif

Supplementary file2 Compound 11 oral gavage treatment rescued PD-related pathologies in human SNCA transgenic mice. (A) Immunoblotting of PD-characteristic markers TH and MAOB (1st ~ 2nd panels), AEP and its cleavage products α-Syn N103, Tau N368 (3rd ~ 5th panels). Arrow indicates full-length α-Syn, arrowhead indicates AEP-cleaved α-Syn (α-Syn N103). (B–E) Quantification analysis of IB intensity for TH, active AEP and α-Syn N103 (Mean ± SEM, one-way ANOVA with Tukey’s multiple-comparisons test, * p < 0.05, ** p < 0.01). (F) Immunostaining demonstrated the loss of TH+ neurons and generation of α-Syn N103 after treatment of MPTP. #11 oral gavage rescued TH neuron loss and reduced α-Syn N103 levels by inhibiting AEP activity in substantia nigra and striatum tissues. Scale bar: 50 μm. (G–H) Quantification of TH-positive and α-Syn N103-positive cells. Mean ± SEM, n = 6 in each group, one-way ANOVA with Tukey’s multiple-comparisons test, * p < 0.05, ** p < 0.01. (TIF 4055 KB)

13311_2022_1248_MOESM3_ESM.tif

Supplementary file3 Characterization of the AEP inhibitor effect of #11 in human SNCA mice. (A) Rotarod test evaluated locomotor impairment and restore with the treatment of MPTP and #11 in both SNCA and SNCA/AEP -/- mice. Mean ± SEM, n = 12 in each group, one-way ANOVA with Tukey’s multiple-comparisons test, * p < 0.05, ** p < 0.01. (B) Open field test evaluation for the treatment of MPTP and #11 in both SNCA and SNCA/AEP -/- mice. Mean ± SEM, n = 12 in each group, one-way ANOVA with Tukey’s multiple-comparisons test, * p < 0.05, ** p < 0.01. (C) HPLC assay examined the absolute amount of dopamine (left) and DOPAC (right). Mean ± SEM, n = 6 in each group, one-way ANOVA with Tukey’s multiple-comparisons test, * p < 0.01, ** p < 0.01. (D) Immunostaining demonstrated the AEP (left) and phosphorylated α-Syn S129 (right) levels with the treatment of MPTP and #11 in substantia nigra and striatum tissues. Scale bar: 50 μm. (E–F) Quantification of AEP-positive and p-α-Syn S129-positive cells. Mean ± SEM, n = 6 in each group, one-way ANOVA with Tukey’s multiple-comparisons test, * p < 0.05, ** p < 0.01. (G) #11A, CF3CN, or #11A+CF3CN were administered for 5 weeks into SNCA mice without MPTP injection. Substantia nigra (SN) tissues were lysed and assessed for the expression of TH, AEP, α-synuclein cleavage, and TrkB signaling by drugs using immunoblotting. (TIF 3522 KB)

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Kang, S.S., Wu, Z., Liu, X. et al. Treating Parkinson’s Disease via Activation of BDNF/TrkB Signaling Pathways and Inhibition of Delta-Secretase. Neurotherapeutics (2022). https://doi.org/10.1007/s13311-022-01248-1

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Keywords

  • TrkB agonist
  • AEP inhibitor
  • Motor dysfunctions
  • Lewy bodies
  • Dopaminergic neurons