Defining the potential of HDAC5 and HDAC9 as therapeutic targets for Parkinson’s disease

Degeneration of midbrain nigrostriatal dopaminergic (DA) neurons is a pathological hallmark of Parkinson’s disease (PD). DA neuronal degeneration is driven by toxic aggregates of alpha-synuclein (α-syn), a key pathological hallmark of PD. Identification of neuroprotective and disease altering factors are essential for the development of new therapies. The targeted inhibition of histone deacetylases (HDACs), enzymes which regulate histone acetylation, have shown some promise in this regard. For example, pan-HDAC inhibition by pharmacological compounds in PD models have been reported to provide some neuroprotection in PD models. However, given the number of different classes of HDACs (ClassI-IV) and their varied biological functions, it is important to evaluate the therapeutic efficacy of each different class, to ensure target specificity and prevent any off-target negative effects, of which there have been some previous reports. The targeted inhibition of Class IIa HDACs, HDAC5 and HDAC9 have demonstrated some positive preliminary findings in PD models. Therefore, this thesis aims to better understand the therapeutic potential of the inhibition of these HDACs in the PD context. To do this, we utilised both pharmacological selective class IIa HDAC inhibitors, which have the therapeutic advantage of being able to be administered peripherally, as well as targeted short hairpin RNA (shRNA)-mediated inhibition. Furthermore, we assessed the effect of HDAC5 and HDAC9 inhibition on the neurotrophic factors (NTF), Bone morphogenetic protein (BMP)2 and Growth differentiation factor (GDF)5, respectively, given that they have been identified as being regulated by HDAC5 and HDAC9. Both BMP2 and GDF5 have been identified as promising NTFs involved in DA neuronal development and capable of protecting against α-syn-induced degeneration. Therefore, we hypothesised that mechanistically, the inhibition of HDAC5 and HDAC9 might offer protection in PD models, at least, in part through BMP2 and GDF5 upregulation and explored this throughout. In addition, given these reported benefits of BMP2 and GDF5, we also attempted to better understand the mechanisms underlying their neuroprotective effects. In the first experimental chapter we examined the neuroprotective potential of the Class-IIa HDAC inhibitor, TMP269. We showed that TMP269 protects against 6-hydroxydopamine (6-OHDA)-induced neurite injury in SH-SY5Y cells and cultured rat ventral mesencephalic primary DA neurons. We found that TMP269 upregulates the neurotrophic factor BMP2 and BMP-Smad dependent transcription signalling in SH-SY5Y cells, the activation of which has been previously demonstrated to be neuroprotective in PD models. Finally, we determined that this activation of BMP-Smad signalling was necessary for the neuroprotective effects of TMP269 against 6-OHDA-induced injury, with addition of dorsomorphin, a BMP receptor agonist, negating the protective effects of TMP269 administration in SH-SY5Y cells. In the second experimental chapter we investigated the protective effects of TMP269 in an in vivo 6-OHDA Sprague-Dawley (SD) rat model of PD. Using a mini-osmotic pump, we administered 0.5mg/kg of TMP269 for 7 days post-surgery through peripheral continuous infusion. We found TMP269 treatment reduced forelimb impairments induced by striatal 6-OHDA administration. TMP269 also protected DA neurons in the SN and their striatal terminals from striatal 6-OHDA-induced neurodegeneration and prevented the 6-OHDA-induced increases in the numbers of IBA1-positive microglia in the striatum and SN in vivo. Finally, we found that TMP269 treatment protects against 6-OHDA-induced reductions in BMP2 and Smad1 levels in DA cells in the SN. In the third experimental chapter we examined the neuroprotective potential of the selective HDAC4/5 inhibitor, LMK235. We showed that LMK235 promotes neurite outgrowth and protects against 6-OHDA-induced neurite injury in SH-SY5Y cells and promotes increases in neuronal length and branching in cultured rat ventral mesencephalic DA neurons and also protects them against 6-OHDA-induced degeneration. We found that LMK235 upregulates BMP2 transcription in SH-SY5Y cells. Furthermore, we found that peripheral continuous infusion of 0.5mg/kg of LMK235 for 7 days post-surgery via a mini-osmotic pump reduced forelimb impairments induced by striatal 6-OHDA administration. LMK235 also protected DA neurons in the SN and their striatal terminals from striatal 6-OHDA-induced neurodegeneration and protected against 6-OHDA-induced increases in the numbers of IBA1-positive microglia in the striatum and SN in vivo. In the fourth experimental chapter we examine the neuroprotective potential of the selective inhibition of the Class IIa HDAC, HDAC9. We showed that HDAC9 inhibition via siRNA promotes neurite outgrowth and protected against α-syn-induced neurite injury in SH-SY5Y cells. Additionally, we found that HDAC9 inhibition in cultured rat ventral mesencephalic DA neurons promotes increases in neuronal length and branching and also protects against α-syn-induced degeneration. We found that inhibition of HDAC9 upregulates neuroprotective BMP-Smad dependent transcription in SH-SY5Y cells and increases in Smad1/5 phosphorylation. Finally, nigral administration of an AAV-shRNA to inhibit HDAC9 resulted in protection against DA neuronal degeneration induced by nigrally administered α-syn overexpression. HDAC9 protected both DA neurons in the SN and their striatal terminals from degeneration induced by α-syn overexpression and prevented α-syn-induced increases in the numbers of IBA1-positive microglia in both the striatum and SN in vivo. In the fifth experimental chapter we utilised transcriptomic analysis of the SH-SY5Y cell line in response to treatment with either BMP2 or GDF5 in order to attempt to better understand the mechanism underlying their protective effects. Both BMP2 and GDF5 showed a common upregulation of the T-box 3 transcription factor (TBX3). Gene expression analysis found high expression of TBX3 in the adult SN relative to other brain regions, and gene co-expression analysis found a positive correlation of TBX3 with genes associated with a number of biological pathways and processes associated with axonal and glial development along with potentially neuroprotective processes in the PD brain. TBX3 inhibition was found to inhibit the growth promoting effects of BMP2 and GDF5 in both SH-SY5Y cells and rat primary midbrain cultures. Gene expression analysis of the PD SN found a significant reduction in TBX3 levels in early (Braak 1/2) stages of PD relative to age matched controls, and gene co-expression analysis revealed a disruption of the co-expression patterns of TBX3 and α-syn in the PD brain throughout the progression of the disease. Finally, TBX3 showed trends towards reduction in primary midbrain cultures in response to treatment with α-syn pre-formed-fibrils. These data highlight TBX3 as a potential regulator of the neurotrophic effects of BMP2 and GDF5 in neurite outgrowth and potentially in DA neuronal development and highlight its potential dysregulation in PD which could make it a potential therapeutic target. Collectively, the data presented in this thesis adds to the growing body of evidence which suggests that targeted inhibition of the Class-IIa HDACs, in particular HDAC5 and HDAC9, may prove to be effective neuroprotective therapies for the treatment of PD, and justifies their continued investigation. It also provides some insight into the mechanisms underlying the protective effects resulting from the inhibition of these HDACs, highlighting the activation and upregulation of components of the neuroprotective BMP-Smad signalling pathway. Finally, it demonstrates that the neurotrophic/neuroprotective effects of this BMP-Smad signalling may require the activity of TBX3, which also identifies TBX3 as a potentially new and novel therapeutic target for the treatment of PD which should be explored further.

Grantor dc:publisher

University College Cork

Year dc:date.issued

2025

Author dc:creator

• O'Mahony, Adam

Advisors dc:contributor.advisor

• O'Keeffe, Gerard W.
• Sullivan, Aideen M.
• Collins, Louise

• Parkinson's disease
• Histone deacetylase
• Neuroprotection
• Histone deacetylase inhibitor
• HDAC5
• HDAC9

Statement dc:rights

• © 2025, Adam O'Mahony.

Licence dc:rights.uri

• https://creativecommons.org/licenses/by-nc-nd/4.0/

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10468/18945

OAI identifier oai:identifier

oai:cora.ucc.ie:10468/18945