Defining the potential of class-IIa histone deacetylases as a therapeutic target for Parkinson’s disease

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Mazzocchi, Martina
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University College Cork
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Parkinson’s disease (PD) is a neurodegenerative disease characterized by early dopaminergic (DA) neuron degeneration, coupled with intracellular aggregation of α-synuclein (α-syn) in Lewy Bodies (LB). Given the lack of a disease-modifying therapy for PD, histone deacetylases (HDACs) have emerged as potential druggable therapeutic targets. The neuroprotective effects of pan-HDAC inhibitors (HDIs) and of some specific HDIs have been tested in both in vitro and in vivo models of PD, showing various outcomes, however which is the best class of HDACs to be targeted for neuroprotection in PD is unclear. In the first experimental chapter, we used gene co-expression analysis to show that HDAC5 and HDAC9 were positively co-expressed with the DA neuronal markers TH, GIRK2 and ALDH1A1 in the human substantia nigra (SN). We then demonstrated that both siRNA against HDAC5 and HDAC9, and the HDI MC1568 which inhibits both HDAC5 and HDAC9, promoted neurite outgrowth in SH-SY5Y cells and DA neurons. Neurite growth was used as a single cell readout of neurotrophic action. Furthermore, MC1568 treatment increased the expression of the neurotrophic factor BMP2, and of its downstream effector SMAD1. A reporter assay revealed that these HDAC5 and HDAC9 siRNAs, or treatment with MC1568, increased BMP-Smad dependent transcription. Furthermore, inhibition of BMP signalling either pharmacologically with dorsomorphin, or by overexpression of Smad4 dominant negative or inhibitory Smad7, abolished the neurite outgrowth-promoting effects of BMP2. Moreover, siRNAs against HDAC5 or HDAC9, but not against HDAC4 or HDAC7, promoted neurite growth in the presence of WT and A53T α-syn. Treatment with MC1568 protected DA neurons from MPP+-induced neurodegeneration in vitro. These findings confirmed HDAC5 and HDAC9 as novel regulators of BMP-Smad neurotrophic factor signalling pathway, making them potential therapeutic targets for PD. In the second experimental chapter, we expanded our gene co-expression analysis and found that HDAC3, HDAC5, HDAC6 and HDAC9 were co-expressed with the DA neuronal markers SLC6A3 and NR4A2 in the human SN. A RT-qPCR confirmed that mRNA of all four HDACs exhibited similar temporal expression profiles during DA neuronal development. We next investigated whether class-specific pharmacological inhibition of these four HDACs could promote neurite growth. We found that inhibition of HDAC1 and HDAC3, or of HDAC3 alone, using RGFP109 or RGFP966 respectively, and inhibition of HDAC6 using ACY1215, did not promote neurite growth or affect survival in SH-SY5Y cells. Conversely, LMK235, which is a class-IIa-specific HDI which mainly inhibits HDAC4 and HDAC5, significantly increased both histone acetylation levels and neurite outgrowth in SH-SY5Y cells. Using a GFP reporter assay, we confirmed that administration of LMK235 increased Smad-dependent transcription. We then showed that inhibition of BMPR1B receptor in both SH-SY5Y cells and primary DA neurons prevented the neurite growth-promoting effects of LMK235. Furthermore, LMK235 was neuroprotective against MPP+ neurotoxic insult, and against wild type (WT) and A53T α-syn overexpression, in both SH-SY5Y cells and DA neurons. These data confirmed the neuroprotective effects of class-IIa HDAC inhibition in vitro. In the third experimental chapter, we investigated the cellular localisation of HDAC5 and the therapeutic potential of the classIIa inhibitor MC1568 in vivo, in 6-hydroxydopamine (6-OHDA) treated in vitro models of PD. We found that 6-OHDA treatment of both SH-SY5Y cells and DA neurons led to nuclear accumulation of HDAC5. This was prevented by PMA, which activates the canonical PKC pathway that is known to cause HDAC5 shuttling out of the nucleus. Having confirmed that MC1568 treatment protected SH-SY5Y cells from 6-OHDA-induced neurodegenration in vitro, we next examined whether peripheral administration (7 daily i.p. injections) of MC1568 was neuroprotective in the intrastriatal 6-OHDA lesion rat model of PD in vivo. Behavioural analysis at 8 and 12 days after lesion surgery showed that MC1568 treatment partially ameliorated 6-OHDA-induced forelimb akinesia. Post-mortem analysis of nigrostriatal integrity showed that MC1568 partially protected striatal dopaminergic terminals and DA neurons in the SN and prevented the increase in microglial numbers in both the striatum (ST) and SN. Finally, we confirmed that 6-OHDA lesion induced HDAC5 nuclear accumulation in DA neurons in the SN, which was prevented by MC1568 treatment. Collectively, these data rationalise the strategy of peripheral administration of MC1568 for neuroprotection in PD. In the fourth and final experimental chapter, we sought to determine whether HDAC5 nuclear accummulation occurred in other in vitro and in vivo models of PD . We verified that there are increased nuclear levels of HDAC5 in SH-SY5Y cells and in DA neurons treated with the neurotoxin MPP+, or in those overexpressing of α-syn or LRRK2 G2019S. We found that combined overexpression of nuclear-restricted HDAC5 and α-syn further increased neurite degeneration. Furthermore, activation of the canonical CaMK/PKC pathway prevented HDAC5 nuclear accumulation and the consequent neurite degeneration. Finally, we demonstrated that HDAC5 nuclear accumulation occurred in DA neurons in the SN, in both the MPTP mouse model and the AAV-αSyn rat model of PD. Collectively, the data presented in this thesis rationalise the future development of strategies focused on HDAC5 inhibition as a potential neuroprotective strategy for PD.
Parkinson's disease , HDACs , Epigenetic , Axons , Alpha-synuclein , Regeneration
Mazzocchi, M. 2021. Defining the potential of class-IIa histone deacetylases as a therapeutic target for Parkinson’s disease. PhD Thesis, University College Cork.