Preclinical Evidence Supporting Early Initiation of Citalopram Treatment in Machado-Joseph Disease

Spinocerebellar ataxias are dominantly inherited neurodegenerative disorders with no disease-modifying treatment. We previously identified the selective serotonin reuptake inhibitor citalopram as a safe and effective drug to be repurposed for Machado-Joseph disease. Pre-symptomatic treatment of transgenic (CMVMJD135) mice strikingly ameliorated mutant ataxin-3 (ATXN3) pathogenesis. Here, we asked whether citalopram treatment initiated at a post-symptomatic age would still show efficacy. We used a cohort of CMVMJD135 mice that shows increased phenotypic severity and faster disease progression (CMVMJD135hi) compared to the mice used in the first trial. Groups of hemizygous CMVMJD135hi mice were orally treated with citalopram. Behavior, protein analysis, and pathology assessment were performed blindly to treatment. Our results show that even when initiated after symptom onset, treatment of CMVMJD135hi mice with citalopram ameliorated motor coordination and balance, attenuating disease progression, albeit to a lesser extent than that seen with pre-symptomatic treatment initiation. There was no impact on ATXN3 aggregation, which contrasts with the robust reduction in ATXN3-positive inclusions observed in CMVMJD135 mice, when treated pre-symptomatically. Post-symptomatic treatment of CMVMJD135hi mice revealed, however, a limited neuroprotective effect by showing a tendency to repair cerebellar calbindin staining, and to increase the number of motor neurons and of NeuN-positive cells in certain brain regions. While supporting that early initiation of treatment with citalopram leads to a marked increase in efficacy, these results strengthen our previous observation that modulation of serotonergic signaling by citalopram is a promising therapeutic approach for Machado-Joseph disease even after symptom onset.European Regional Development Funds (FEDER), through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038. This article has been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the FEDER. This work was also supported by FCT and COMPETE through the projects [PTDC/SAU-GMG/112617/2009] (to PM) and [EXPL/BIM-MEC/0239/2012] (to AT-C), by FCT through the project [POCI-01-0145-FEDER-016818 (PTDC/NEU-NMC/3648/2014)] (to PM), by National Ataxia foundation (to PM and to AT-C), and by Ataxia UK (to PM). SE, SD-S, SO, and AT-C were supported by the FCT individual fellowships, SFRH/BD/78554/2011, SFRH/BD/78388/2011, PD/BD/127818/2016, and SFRH/BPD/102317/2014, respectively. FCT fellowships are co-financed by POPH, QREN, Governo da República Portuguesa, and EU/FSEinfo:eu-repo/semantics/publishedVersio

Antidepressant stimulation of CDP-diacylglycerol synthesis does not require monoamine reuptake inhibition

<p>Abstract</p> <p>Background</p> <p>Recent studies demonstrate that diverse antidepressant agents increase the cellular production of the nucleolipid CDP-diacylglycerol and its synthetic derivative, phosphatidylinositol, in depression-relevant brain regions. Pharmacological blockade of downstream phosphatidylinositide signaling disrupted the behavioral antidepressant effects in rats. However, the nucleolipid responses were resistant to inhibition by serotonin receptor antagonists, even though antidepressant-facilitated inositol phosphate accumulation was blocked. Could the neurochemical effects be additional to the known effects of the drugs on monoamine transmitter transporters? To examine this question, we tested selected agents in serotonin-depleted brain tissues, in PC12 cells devoid of serotonin transporters, and on the enzymatic activity of brain CDP-diacylglycerol synthase - the enzyme that catalyzes the physiological synthesis of CDP-diacylglycerol.</p> <p>Results</p> <p>Imipramine, paroxetine, and maprotiline concentration-dependently increased the levels of CDP-diacylglycerol and phosphatidylinositides in PC12 cells. Rat forebrain tissues depleted of serotonin by pretreatment with <it>p</it>-chlorophenylalanine showed responses to imipramine or maprotiline that were comparable to respective responses from saline-injected controls. With fluoxetine, nucleolipid responses in the serotonin-depleted cortex or hippocampus were significantly reduced, but not abolished. Each drug significantly increased the enzymatic activity of CDP-diacylglycerol synthase following incubations with cortical or hippocampal brain tissues.</p> <p>Conclusion</p> <p>Antidepressants probably induce the activity of CDP-diacylglycerol synthase leading to increased production of CDP-diacylglycerol and facilitation of downstream phosphatidylinositol synthesis. Phosphatidylinositol-dependent signaling cascades exert diverse salutary effects in neural cells, including facilitation of BDNF signaling and neurogenesis. Hence, the present findings should strengthen the notion that modulation of brain phosphatidylinositide signaling probably contributes to the molecular mechanism of diverse antidepressant medications.</p

Scaffold dependent histone deacetylase (HDAC) inhibitor induced re-equilibration of the subcellular localization and post-translational modification state of class I HDACs

<div><p>The mechanism of action of histone deacetylase inhibitors (HDACi) is mainly attributed to the inhibition of the deacetylase catalytic activity for their histone substrates. In this study, we analyzed the abundance of class I HDACs in the cytosolic, nuclear soluble and chromatin bound cellular fractions in breast cancer cells after HDACi treatment. We found that potent <i>N</i>-hydroxy propenamide-based HDACi induced a concentration dependent decrease in the HDAC1 associated with chromatin and a lasting concomitant increase in cytoplasmic HDAC1 while maintaining total protein expression. No such change occurred with HDAC2 or 8, however, an increase in cytoplasmic non-phosphorylated HDAC3 was also observed. The subcellular re-equilibration of HDAC1 was subsequent to the accumulation of acetylated histones and might be cell cycle dependent. This study suggests that the biological activity of a subset of <i>N</i>-hydroxy propenamide-based HDACi may stem from direct competition with histone substrates of HDACs as well as from spatial separation from their substrates in the nucleus and/or change in post-translational modification status of HDACs.</p></div

A model of the mechanism of action of propenamide-based HDACi including re-equilibration of the subcellular distribution and modulation of the post-translational modification of HDACs.

<p>Treatment of with <i>N</i>-hydroxy propenamide-based HDACi increase histone acetylation at 2 hours without affecting the subcellular localization or the phosphorylation status of HDACs. At 12 hours, re-equilibration of subcellular localization as well as a decrease in phosphorylation of HDACs is observed, along with a greater increase in histone acetylation. The induced change in subcellular localization and phosphorylation of HDACs is sustained 24 hours after removal of HDACi. This is in line with the time necessary to induce commitment to cell death with HDACi.</p

Increase in cytosolic HDAC1 is irreversible up to 24 hours.

<p>MCF-7 cells were treated with 10 μM panobinostat for 12 hours, the compound was then removed and cells allowed to recover for 24 hours. A) Western blot analysis of the abundance of HDAC1 in the cytosolic, nuclear soluble, and chromatin bound fractions. B) Densitometry analysis of the abundance of HDAC1 normalized to GAPDH (cytosolic fraction) or to TATA-binding protein (TBP, nuclear soluble and chromatin bound fractions). * Statistically significant difference compared with DMSO control (Student’s t-test, P<0.01). Western blots shown are representative of at least two independent experiments. HDAC1 fold change is presented as the mean of at least two independent experiments ± standard deviation.</p

Potent HDACi alter the subcellular localization of HDAC1.

<p>MCF-7 cells were treated with indicated concentrations of panobinostat or trichostatin A for 12 hours. A) Western blot analysis of the abundance of HDAC1 in the cytosolic, nuclear soluble, and chromatin bound fractions. B) Densitometry analysis of the abundance of HDAC1 normalized to GAPDH (cytosolic fraction) or to TATA-binding protein (TBP, nuclear soluble and chromatin bound fractions). C) Western blot analysis of the total abundance of class I HDACs and the loading controls TBP, GAPDH, and histone H3 after treatment with indicated concentrations of panobinostat for 12 hours. * Statistically significant difference compared with DMSO control (Student’s t-test, P<0.01). Western blots shown are representative of at least two independent experiments. HDAC1 fold change is presented as the mean of at least two independent experiments ± standard deviation.</p

Structures of a diverse selection of HDACi.

<p>Panobinostat, trichostatin A, and SAHA are pan-isoform inhibitors. Entinostat is a class I selective inhibitor. PCI-34051 is an HDAC8 selective inhibitor.</p

HDACi-induced re-equilibration of HDAC1 is confirmed by confocal microscopy.

<p>MCF-7 cells were treated with indicated concentrations of panobinostat (optical sections A-D, respectively) or trichostatin A (optical sections E-H, respectively) for 12 hours, fixed, permeabilized and optical sections were obtained by laser scanning confocal microscopy. Fluorescence signal for HDAC1 is shown in green (left panels), DAPI staining is shown in blue (middle panels), and merged optical sections are shown in the right panels. Colocalization analysis of HDAC1 fluorescence signal and the DAPI stain signal was performed with JACoP (ImageJ) and shown below. * Statistically significant difference compared with DMSO control (Student’s t-test, P<0.01). Pearson’s Coefficient is presented as the mean of at least two independent experiments ± standard deviation. Optical sections shown are representatives of at least two independent experiments.</p

HDAC1 re-equilibration induced by HDACi is subsequent to histone acetylation and is affected by mitogenic stimuli.

<p>A) Western blot analysis of cytosolic fractions of MCF-7 cells treated with indicated concentrations of trichostatin A for 2 hours. B) Densitometry analysis of western blots of chromatin fractions from MCF-7 cells treated with 10 μM trichostatin A for 2 and 12 hours; change in AcH3 was normalized to TBP. C) Western blot analysis of chromatin bound fractions from MCF-7 cells treated with indicated concentrations of HDACi for 12 hours. D) Laser scanning confocal microscopy of MCF-7 cells, grown with 10% serum and treated with DMSO for 12 hours. Fluorescence signal for HDAC1 is shown in green (left panels), DAPI staining is shown in blue (middle panels), and merged optical sections are shown in the right panels. Representative optical section from two independent experiments is shown. Arrows indicate mitotic cells where HDAC1 is dispersed off chromatin, as indicated by DAPI staining. * Statistically significant difference compared with DMSO control (Student’s t-test, P<0.01).</p