Pharmacological modulation of HDAC1 and HDAC6 in vivo in a zebrafish model: Therapeutic implications for Parkinson’s disease

Abstract

Histone deacetylases (HDACs) are key epigenetic enzymes and emerging drug targets in cancer and neurodegeneration. Pan-HDAC inhibitors provided neuroprotection in Parkinson's Disease (PD) models, however, the HDAC isoforms with highest neuroprotective potential remain unknown. Zebrafish larvae (powerful pharmacological testing tools bridging cellular and in vivo studies) have thus far been used in PD modelling with limited phenotypic characterization. Here we characterize the behavioural and metabolic phenotypes of a zebrafish PD model induced with MPP(+), assess the feasibility of targeting zebrafish HDAC1 and HDAC6 isoforms, and test the in vivo effects of their selective inhibitors MS-275 and tubastatin A, respectively. MPP(+) induced a concentration-dependent decrease in metabolic activity and sensorimotor reflexes, and induced locomotor impairments rescuable by the dopaminergic agonist apomorphine. Zebrafish HDAC1 and HDAC6 isoforms show high sequence identity with mammalian homologues at the deacetylase active sites, and pharmacological inhibition increased acetylation of their respective histone and tubulin targets. MS-275 and tubastatin rescued the MPP(+)-induced decrease in diencephalic tyrosine hydroxylase immunofluorescence and in whole-larvae metabolic activity, without modifying mitochondrial complex activity or biogenesis. MS-275 or tubastatin alone modulated spontaneous locomotion. When combined with MPP(+), however, neither MS-275 nor tubastatin rescued locomotor impairments, although tubastatin did ameliorate the head-reflex impairment. This study demonstrates the feasibility of pharmacologically targeting the zebrafish HDAC1 and HDAC6 isoforms, and indicates that their inhibition can rescue cellular metabolism in a PD model. Absence of improvement in locomotion, however, suggests that monotherapy with either HDAC1 or HDAC6 inhibitors is unlikely to provide strong benefits in PD. This study highlights parameters dependent on the integrity of zebrafish neuronal circuits as a valuable complement to cell-based studies. Also, the demonstrated feasibility of pharmacologically targeting HDAC1 and HDAC6 in this organism paves the way for future studies investigating HDAC inhibitors in other diseases modelled in zebrafish.

HDAC6 inhibition by tubastatin A is protective against oxidative stress in a photoreceptor cell line and restores visual function in a zebrafish model of inherited blindness

“… 34 The data are deposited in the NCBI Gene Expression Omnibus (GEO) database, under accession number GSE86155. Furthermore, in a recent study Pinho et al 35 have demonstrated that zebrafish HDAC6 shows high sequence identity with the mammalian homolog at its deacetylase active site. In their study the effects of HDAC inhibitors on a zebrafish model of Parkinson’s disease were evaluated.…”

Histone Deacetylases Inhibitors in Neurodegenerative Diseases, Neuroprotection and Neuronal Differentiation

“…K560, a HDAC 1 and 2 isoform specific inhibitor, attenuated MPTP induced cell death in SH-SY5Y cells in vitro and prevented MPTP-induced loss of dopaminergic neurons in substantia nigra in mice in vivo (Choong et al, 2016). Similarly, M275 (HDAC 1 inhibitor) and tubastatin A (HDAC 6 inhibitor) improved sensorimotor reflexes and locomotor impairments (Pinho et al, 2016). The fibroblasts from PD patients with G2019S mutation in leucine rich repeat kinase 2 showed increased mitophagy and activation of class III HDACs (Yakhine-Diop et al, 2018).…”

Chemical Genetics Screen Identifies Epigenetic Mechanisms Involved in Dopaminergic and Noradrenergic Neurogenesis in Zebrafish

“…An increase in the levels of histone acetylation has been reported in zebrafish tissues after treatment with the HDAC inhibitors Entinostat, Mocetinostat and Vorinostat. Treatments of larval zebrafish from 72 until 120 hpf with Entinostat causes hyperacetylation of H3K9 as evident from Western Blot analysis of whole larval extracts as well as from acetylated H3K9 immunostain (1 µM Entinostat/MS-275; Pinho et al, 2016). Mocetinostat caused a similar hyperacetylation of histones H3 and H4 in lysates from adult zebrafish fin regenerates, as evident from Western Blot analysis (5 µM Mocetinostat/MGCD0103; Pfefferli et al, 2014).…”

“…In this study, we did not experimentally validate, whether the identified HDAC and Bromodomain inhibitors alter histone acetylation or recognition in zebrafish larvae. However, for most of these compounds, the specificity and activity on histone acetylation or acetylated histone recognition, respectively, have recently been reported for larval and adult zebrafish tissues (Pfefferli et al, 2014;Vleeshouwer-Neumann et al, 2015;Pinho et al, 2016;Chan et al, 2019;Sato et al, 2019). An increase in the levels of histone acetylation has been reported in zebrafish tissues after treatment with the HDAC inhibitors Entinostat, Mocetinostat and Vorinostat.…”

“…Together, these data suggest that the bromodomain inhibitor JQ1 is active in larval zebrafish tissues and specifically blocks recognition of acetylated histones. Considering the high sequence conservation of histones as well as of active sites in epigenetic factors (for example, see Pinho et al, 2016), we assume that for many epigenetic compounds active concentration range and specificity may be conserved.…”