(T1530-03-20) Evaluation of Histone Deacetylase-6 (HDAC-6) Inhibitor Tubacin on 3D Midbrain Neurospheres: A New Therapeutic Avenue for Parkinson’s Disease

Purpose: Isoform specific inhibition of Histone Deacetylase-6 (HDAC-6) has been a long contested therapeutic strategy for Parkinson’s Disease (PD). Previous in-vitro studies report that HDAC-6 inhibition causes nuclear localization of HDAC-6, proteasomal system dysfunction, increased protein aggregation, and apoptosis. [1] On the contrary, most in-vivo studies show that HDAC-6 inhibition can have a protective effect against toxin-induced PD models via countering neuroinflammation, oxidative stress and microglial activation. [2][3] The likely reason postulated for this gap in translational research is the use of monolayer cultures of neuroblastoma (cancer) cell lines in most in-vitro PD models. We report a simple method to generate and midbrain-differentiate spheroids from Neural Progenitor Cells (NPCs) derived from healthy human and PD patients. We further characterized effect of HDAC-6 inhibition on α-synuclein levels and aggresome formation, two pathological hallmarks of PD, on the NPC cell lines using quantitative ELISA and confocal microscopy. Alpha-synuclein-based pre-formed fibrils (PFFs) were used as a positive control for disease induction. Our findings report that treatment with HDAC-6 inhibitor Tubacin can counter PFF-induced α-synuclein overexpression and aggresome-related pathology in NPCs.

Methods: Cell culture: The NPC cell lines ACS 5001 and ACS 5006 were cultured on NPC expansion media in standard culture conditions. The HDAC-6 inhibitor, Tubacin was dissolved in 10 mg/ml DMSO stock solution. Spheroids were generated by seeding NPCs in 96-well plates. Spheroid Generation: Spheroids were generated by seeding NPCs at a seeding density of 12,800 cells/well in Ultra-low attachment (ULA), round bottom 96-well plates.  Media was changed at 70% change rate. Midbrain Differentiation: The NPC spheroids were cultured in Dopaminergic differentiation media for 21 days, and their differentiation was confirmed by immunostaining for markers like FOXA2 (midbrain-specific marker), DAT (a specific dopaminergic neuron maker), and GIRK2 (A9 dopaminergic neuron-specific marker). Alamar Blue Assay: cytotoxicity of Tubacin was examined on NPC spheroids and monolayer cultures. Cells/ Spheroids were treated with 0-80 µM Tubacin, for a 48-hour incubation period. On test day, the media was changed and 10 µl Alamar blue HS viability agent was added. Absorbance was recorded at 570 & 600 nm to further compute viability. Sandwich ELISA: Differentiated NPCs from both the cell lines kept under different conditions were lysed and quantified using sandwich ELISA Samples were then incubated with detector-antibody co-incubation for 1 hour in microplate strips. Absorbance was measured at 450 nm and correlated with α-synuclein levels. Aggresome assay: The aggresome assay involved fixing the cells, permeabilizing them, and dual-staining them with Hoechst blue and aggresome-detector reagent. Images were acquired via confocal fluorescence microscopy and analyzed for mean fluorescence intensity from aggresome-positive cells.

Results: Spheroids from both cell lines were successfully generated using the ULA-microplate seeding method. Differentiation was confirmed by immunocytochemistry (ICC). The spheroids immunostained positively for FOXA2, DAT, & GIRK2 (validating the spheroid model for PD). The Resazurin assay yielded IC50 values of 4 µM and 35 µM for the 2D & 3D cultures of ACS 5006, & the IC50 values of 4 µM and 40 µM for the 2D & 3D cultures of ACS 5001. The sandwich ELISA quantified α -synuclein levels in cell lysates. PFF treatment increased α-synuclein levels, likely due to endogenous synucleopathy.[4] Tubacin treatment mitigated α-synuclein levels in most groups. The increased production of α-synuclein is a compensatory measure by the cell to replace the misfolded amount. Aggresome experiment was used to detect aggresome occurrence and quantity. Quantity of aggresomes was estimated by quantifying mean fluorescence intensities using Fiji (ImageJ). PFF treatment raised the MFI of aggresomes from NPCs which was expected as misfolded α-synuclein is known to deposit in aggresomes, and aggresome formation is upregulated in during PD & most neurodegenerative diseases.[5] Tubacin treatment reduced aggresome formation in PFF-treated cells and restored it to normal levels which is expected as HDAC-6 is one of the proteins responsible for aggresome formation.

Conclusion: NPC spheroids where successfully generated and differentiated into midbrain spheroids. The significant upregulation of FOXA2 (a transcription factor responsible for midbrain differentiation and catecholaminergic neuron differentiation), as well as expression of dopaminergic neuron markers (DAT & GIRK2) validates the spheroid model for in-vitro studies on PD. Tubacin treatment was able to reduce PFF-induced α-synuclein overexpression and aggresome formation in NPCs. We aim to characterize more PD-related biomarkers like Neuromelanin in midbrain spheroids.

References: [1] Su, M.; Shi, J.-J.; Yang, Y.-P.; Li, J.; Zhang, Y.-L.; Chen, J.; Hu, L.-F.; Liu, C.-F. HDAC6 Regulates Aggresome-Autophagy Degradation Pathway of α-Synuclein in Response to MPP+-Induced Stress. J. Neurochem. 2011, 117 (1), 112–120.
[2] Cao, Z.; Gu, Z.; Lin, S.; Chen, D.; Wang, J.; Zhao, Y.; Li, Y.; Liu, T.; Li, Y.; Wang, Y.; Lin, H.; He, B. Attenuation of NLRP3 Inflammasome Activation by Indirubin-Derived PROTAC Targeting HDAC6. ACS Chem. Biol. 2021, 16 (12), 2746–2751. https://doi.org/10.1021/acschembio.1c00681.
[3] Li, B.; Yang, Y.; Wang, Y.; Zhang, J.; Ding, J.; Liu, X.; Jin, Y.; Lian, B.; Ling, Y.; Sun, C. Acetylation of NDUFV1 Induced by a Newly Synthesized HDAC6 Inhibitor HGC Rescues Dopaminergic Neuron Loss in Parkinson Models. iScience 2021, 24 (4), 102302. https://doi.org/10.1016/j.isci.2021.102302.
[4] Volpicelli-Daley, L. A.; Luk, K. C.; Patel, T. P.; Tanik, S. A.; Riddle, D. M.; Stieber, A.; Meany, D. F.; Trojanowski, J. Q.; Lee, V. M.-Y. Exogenous α-Synuclein Fibrils Induce Lewy Body Pathology Leading to Synaptic Dysfunction and Neuron Death. Neuron 2011, 72 (1), 57–71. https://doi.org/10.1016/j.neuron.2011.08.033.
[5] Olzmann JA, Li L, Chin LS. Aggresome formation and neurodegenerative diseases: therapeutic implications. Curr Med Chem. 2008;15(1):47-60. doi: 10.2174/092986708783330692. PMID: 18220762; PMCID: PMC4403008.

Acknowledgements: Financial support from Health Science Strategic Investment Fund and School of Pharmacy and Health Professions, Creighton University. Conflice of Interest Statement: We confirm that there are no conflicts of interest associated with this work.


Figure 1: (a) Flowchart of ULA microplate method, (b) process of seeding a ULA microplate, & (c) brightfield image of spheroid generated using ULA plate method.


Figure 2: (a) Scheme of differentiation experiment. (b) FOXA2 stained & (c) DAT-GIRK2 double-stained images of NPC spheroids of both cell lines.


Figure 3: (a) ELISA standard curve for recombinant alpha-synuclein (provided by vendor). (b) Effect of differentiation on alpha-synuclein levels. (c) effect of PFF-treatment on alpha-synuclein levels; Effect of HDAC-6 inhibitor Tubacin on alpha-synuclein levels in (d) Control cell line (ACS 5006) & (e) Patient cell line (ACS 5001); (f) Images of NPCs dual stained with DAPI & Aggresome detector reagent. Comparison of Mean fluorescence Intensities (MFI)s of aggresome detector agent from various groups in (g) Control cell line (ACS 5006) & (h) Patient cell line (ACS 5001).