ABSTRACT
Title
The γ-secretase modulator CHF5074 elicits neuroprotection and increases histone acetylation in primary cortical neurons exposed to oxygen glucose deprivation
Authors
V. Porrini1, I.Sarnico1, A. Lanzillotta1, C. Branca1, M. Benarese1, PF. Spano1, B.P. Imbimbo2, M. Pizzi1
1Department of Biomedical Sciences and Biotechnologies, School of Medicine, University of Brescia and Istituto Nazionale di Neuroscienze Brescia, Italy; 2Research & Development, Chiesi Farmaceutici, Via Palermo 26/A, 43100 Parma, Italy.
1Department of Biomedical Sciences and Biotechnologies, School of Medicine, University of Brescia and Istituto Nazionale di Neuroscienze Brescia, Italy; 2Research & Development, Chiesi Farmaceutici, Via Palermo 26/A, 43100 Parma, Italy.
Abstract
Background:CHF5074 is a new γ -secretase modulator in clinical development for the treatment of Alzheimer’s disease (AD). Recent studies have shown that CHF5074 can induce astrocyte stellation independently from gamma-secretase modulation (JAD 2010; 22: 1135-55). These results prompt us to investigate on neuroprotective activity of CHF5074 in experimental models unrelated to Aβaccumulation. Much research is actually focused on epigenetic mechanism regulating histone acetylation and involved in process of chromatin remodeling, microtubule dynamics, metabolism and ageing with relevance in different complex, late-onset neurodegenerative diseases. Histone deacetylase inhibitors have proved to be neuroprotective in animal models of AD and brain ischemia. The aim of our study was to investigate the effect of CHF5074 in mouse primary cortical neurons exposed to oxygen glucose deprivation (OGD), a cell-based model of brain ischemia. The effect of CHF5074 was compared to that elicited by ibuprofen and other neuroprotective agents, the sirtuin activator resveratrol and the HDAC inhibitor MS-275.
Methods: Fifteen days embryonic C57BL/6 mice were harvested with caesarean section from anaesthetized pregnant dams. Cerebral cortices were isolated and dissociated by manual dispersion. The cells were plated in Neurobasal medium supplemented with 2% B27, 0.5 mM L-glutamine and 50 U/mL penicillin/streptomicin. At 10 days in vitro near pure cortical neurons were exposed to OGD at 37° C for 3 hours. At the end of the OGD period, cortical neurons were allowed to recover for 24 hours in Neurobasal medium containing 0.4% B27 supplement, under normoxic conditions and with or without the test drugs. We analyzed the effect of CHF5074 in the concentration range eliciting anti-amyloidogenic activity, from 1-30 µM, ibuprofen at 500 µM, resveratrol at 30 µM and MS275 at 1 µM. Neuronal injury and neuroprotection were evaluated by measuring the amount of lactate dehydrogenase (LDH) released into the culture medium. To test histone acetylation, nuclear extracts were prepared 6 hours after the OGD exposure and nuclear proteins were processed for Western blot analysis by using antibody specific for histone H3 acetylated on K9/18 residues.
Results:Near pure cortical neurons were exposed to CHF5074 from 1 to 30 μM during the OGD period and the following 24 hour recovery. In another set of experiments, CHF5074 was added only after the OGD exposure. When compared to neurons exposed to OGD, cells treated with CHF5074 displayed higher survival at all the concentrations tested, i.e 1, 3, 10, 30 μM. Maximal neuroprotection (75 % reduction of LDH release) was evident at 10 μM concentration. If added only after the OGD period, CHF5074 was still neuroprotective, though with lower potency. Likewise 3 μM CHF5074, 30 μM resveratrol and 1 μM MS-275 added in the post-OGD period also elicited neuroprotection while 500 μM ibuprofen exacerbated the neuronal cell death. Analysis of histone acetylation, showed a decreased H3 acetylation on the K9/18 residues at 6 hours after OGD. The acetylation state of histone H3 recovered in cells treated in the post-OGD period with the HDAC inhibitor MS-275 or with CHF5074, but not with the sirtuin activator resveratrol.
Conclusions:These data indicate that CHF5074, but not ibuprofen, elicits neuroprotection in cortical neurons previously exposed to OGD. CHF5074 effects were associated with the increase of histone H3 acetylation. In spite of observed similar neuroprotection, CHF5074 effect on histone acetylation was reproduced by MS-275 but not by resveratrol. This leaves open the possibility that CHF5074 may directly or indirectly modulate the HDAC activity. The possible role of HDAC activity modulation in the beneficial effects of CHF5074 in AD deserves further investigations.
Methods: Fifteen days embryonic C57BL/6 mice were harvested with caesarean section from anaesthetized pregnant dams. Cerebral cortices were isolated and dissociated by manual dispersion. The cells were plated in Neurobasal medium supplemented with 2% B27, 0.5 mM L-glutamine and 50 U/mL penicillin/streptomicin. At 10 days in vitro near pure cortical neurons were exposed to OGD at 37° C for 3 hours. At the end of the OGD period, cortical neurons were allowed to recover for 24 hours in Neurobasal medium containing 0.4% B27 supplement, under normoxic conditions and with or without the test drugs. We analyzed the effect of CHF5074 in the concentration range eliciting anti-amyloidogenic activity, from 1-30 µM, ibuprofen at 500 µM, resveratrol at 30 µM and MS275 at 1 µM. Neuronal injury and neuroprotection were evaluated by measuring the amount of lactate dehydrogenase (LDH) released into the culture medium. To test histone acetylation, nuclear extracts were prepared 6 hours after the OGD exposure and nuclear proteins were processed for Western blot analysis by using antibody specific for histone H3 acetylated on K9/18 residues.
Results:Near pure cortical neurons were exposed to CHF5074 from 1 to 30 μM during the OGD period and the following 24 hour recovery. In another set of experiments, CHF5074 was added only after the OGD exposure. When compared to neurons exposed to OGD, cells treated with CHF5074 displayed higher survival at all the concentrations tested, i.e 1, 3, 10, 30 μM. Maximal neuroprotection (75 % reduction of LDH release) was evident at 10 μM concentration. If added only after the OGD period, CHF5074 was still neuroprotective, though with lower potency. Likewise 3 μM CHF5074, 30 μM resveratrol and 1 μM MS-275 added in the post-OGD period also elicited neuroprotection while 500 μM ibuprofen exacerbated the neuronal cell death. Analysis of histone acetylation, showed a decreased H3 acetylation on the K9/18 residues at 6 hours after OGD. The acetylation state of histone H3 recovered in cells treated in the post-OGD period with the HDAC inhibitor MS-275 or with CHF5074, but not with the sirtuin activator resveratrol.
Conclusions:These data indicate that CHF5074, but not ibuprofen, elicits neuroprotection in cortical neurons previously exposed to OGD. CHF5074 effects were associated with the increase of histone H3 acetylation. In spite of observed similar neuroprotection, CHF5074 effect on histone acetylation was reproduced by MS-275 but not by resveratrol. This leaves open the possibility that CHF5074 may directly or indirectly modulate the HDAC activity. The possible role of HDAC activity modulation in the beneficial effects of CHF5074 in AD deserves further investigations.