Aberrant chromatin landscape following loss of the H3.3 chaperone Daxx in haematopoietic precursors leads to Pu.1-mediated neutrophilia and inflammation.

Defective silencing of retrotransposable elements has been linked to inflammageing, cancer and autoimmune diseases. However, the underlying mechanisms are only partially understood. Here we implicate the histone H3.3 chaperone Daxx, a retrotransposable element repressor inactivated in myeloid leukaemia and other neoplasms, in protection from inflammatory disease. Loss of Daxx alters the chromatin landscape, H3.3 distribution and histone marks of haematopoietic progenitors, leading to engagement of a Pu.1-dependent transcriptional programme for myelopoiesis at the expense of B-cell differentiation. This causes neutrophilia and inflammation, predisposing mice to develop an autoinflammatory skin disease. While these molecular and phenotypic perturbations are in part reverted in animals lacking both Pu.1 and Daxx, haematopoietic progenitors in these mice show unique chromatin and transcriptome alterations, suggesting an interaction between these two pathways. Overall, our findings implicate retrotransposable element silencing in haematopoiesis and suggest a cross-talk between the H3.3 loading machinery and the pioneer transcription factor Pu.1.

Astrocyte Depletion Impairs Redox Homeostasis and Triggers Neuronal Loss in the Adult CNS.

Although the importance of reactive astrocytes during CNS pathology is well established, the function of astroglia in adult CNS homeostasis is less well understood. With the use of conditional, astrocyte-restricted protein synthesis termination, we found that selective paralysis of GFAP(+) astrocytes in vivo led to rapid neuronal cell loss and severe motor deficits. This occurred while structural astroglial support still persisted and in the absence of any major microvascular damage. Whereas loss of astrocyte function did lead to microglial activation, this had no impact on the neuronal loss and clinical decline. Neuronal injury was caused by oxidative stress resulting from the reduced redox scavenging capability of dysfunctional astrocytes and could be prevented by the in vivo treatment with scavengers of reactive oxygen and nitrogen species (ROS/RNS). Our results suggest that the subpopulation of GFAP(+) astrocytes maintain neuronal health by controlling redox homeostasis in the adult CNS.

Selective lentiviral-mediated suppression of microRNA124a in the hippocampus evokes antidepressants-like effects in rats.

Several lines of evidences suggest that the brain-derived neutrophic factor (BDNF) is implicated in the pathophysiology of depression. However, the molecular mechanisms are not fully understood. In the current study we aimed to investigate how genetic modulation of BDNF in the hippocampus using microRNa124a (miR124a)-expressing lentiviral vectors (LV) might affect depression-like behavior in adult rats. For this purpose, we assessed the expression level of miR124a and its direct target BDNF in the hippocampus and the cortex after 21-days exposure to social defeat stress. Results demonstrated that miR124a was up-regulated in the hippocampus but not in the cortex. In contrast, and as expected, BDNF transcripts were down-regulated. In a different set of experiments, male Wistar rats received bilateral intra-hippocampal or intra-cortical infusions of BDNF- and miR124a-expressing lentiviral vectors and depression-like behavior was assessed after 21-days social defeat stress using the novelty suppressed feeding, the sucrose preference and the forced swim tests. The results indicated that miR124a overexpression exacerbated depression-like behavior. However, an anti-depressant like effect was observed when BDNF or miR124a-silencers (siR124a) were injected into the hippocampus. Importantly, when expressed into the cortex, LV-miR124a, LV-siR124a and LV-BDNF had no effect on depression. Our findings indicate that hippocampal miR124a and its direct target BDNF play an important role in depression-like behavior. Taken together, the current results reveal, for the first time, a potential molecular regulation of miR124a on BDNF, and the pronounced behavioral consequences of this regulation shed light on the mechanisms underlying BDNF anti-depressant potential.

Regulation of MiR-124, Let-7d, and MiR-181a in the accumbens affects the expression, extinction, and reinstatement of cocaine-induced conditioned place preference.

Molecular adaptations underlying drug seeking and relapse remain largely unknown. Studies highlight post-transcriptional modifications mediated by microRNAs (miRNAs) in addiction and other neurological disorders. We have previously shown that chronic cocaine suppresses miR-124 and let-7d and induces the expression of miR-181a in mesolimbic pathway. To further address the role and target gene regulation network of these miRNAs in vivo in cocaine addiction, we developed lentiviral vector (LV)-expressing miRNAs and their corresponding silencers for stable and regulatable miRNA expression. We tested in vivo miRNA gain and loss of function on cocaine-induced conditioned place preference (CPP) by localized LV-miRNA regulation in the nucleus accumbens (NAc). LV-miR-124 and let-7d expression in the NAc attenuates cocaine CPP, whereas LV-miR-181a enhances it. Silencing miRNAs by corresponding LV-miRNA silencers expressing perfect miRNA target sequences inversed this effect on cocaine CPP. Doxycycline treatment for switching off silencer expression abolished the observed behavioral changes. Behavioral changes mediated by LV-miRNA regulation resulted in dynamic alterations in transcription factors, receptors, and other effector genes involved in cocaine-induced plasticity. Our results describe a complex regulatory pathway mediated by miRNAs in cocaine-mediated neuronal adaptations.

The Brain-Specific Neural Zinc Finger Transcription Factor 2b (NZF-2b/7ZFMyt1) Suppresses Cocaine Self-Administration in Rats.

Brain-specific neural-zinc-finger transcription factor-2b (NZF2b/7ZFMyt1) is induced in the mesolimbic dopaminergic region after chronic cocaine exposure and lentiviral-mediated expression of NZF2b/7ZFMyt1 in the nucleus accumbens results in decreased locomotor activity (Chandrasekar and Dreyer, 2010). In this study the role of NZF2b/7ZFMyt1 in active cocaine seeking and of its interaction with histone deacetylase on the altered behavior has been observed. Localized expression of NZF2b/7ZFMyt1 in the nucleus accumbens resulted in attenuated cocaine self-administration, whereas silencing this transcription factor with lentiviruses expressing siRNAs increased the animal's motivation to self-infuse cocaine. Low doses of sodium butyrate, a potent inhibitor of histone deacetylase, were sufficient to reverse the NZF2b/7ZFMyt1-mediated decrease in cocaine self-administration. NZF2b/7ZFMyt1 expression resulted in strong induction of transcription factors REST1 and NAC1 and of the dopamine D2 receptor, with concomitant inhibition of BDNF and its receptor TrkB. We show that NZF2b/7ZFMyt1 colocalizes with histone deacetylase-2 (HDAC2), probably overcoming the suppression of transcriptional activity caused by Lingo1. These findings show that molecular adaptations mediated by NZF2b/7ZFMyt1 expression possibly lead to decreased responsiveness to the reinforcing properties of cocaine and play a prominent role in affecting the behavioral changes induced by the drug.

The brain-specific Neural Zinc Finger transcription factor 2b (NZF-2b/7ZFMyt1) causes suppression of cocaine-induced locomotor activity.

Chronic cocaine induces high expression of the brain-specific Neural-Zinc-Finger transcription factor-2b (NZF-2b/7ZFMyt1), particularly in the mesolimbic dopaminergic pathway, resulting in a 11-fold increase in NZF-2b/7ZFMyt1 expression in the Nucleus Accumbens (NAc). Overexpression of this gene in the NAc with a NZF-2b/7ZFMyt1-expressing lentivirus resulted in >55% decrease in locomotor activity upon chronic cocaine administration, compared to control animals. In contrast knocking-down the gene in the NAc with lentiviruses expressing shRNAs against NZF-2b/7ZFMyt1 induced strong hyperlocomotor activity upon cocaine. Strong inhibition of BDNF is observed upon NZF-2b/7ZFMyt1 expression, concomitant with strong induction of transcription factors REST1 (RE silencing transcription factor-1) and NAC1, probably leading to regulation of gene expression by interaction with histone deacetylases. These changes lead to decreased responsiveness of the animal to the locomotor-activating effects of cocaine, indicating that NZF-2b/7ZFMyt1 expression plays an important role in phenotypic changes induced by the drug.

microRNAs miR-124, let-7d and miR-181a regulate cocaine-induced plasticity.

MicroRNAs play key regulatory roles in cellular processes including neurogenesis, synapse development and plasticity in the brain. Psychostimulants induce strong neuroadaptive changes through a surfeit of gene regulatory mechanisms leading to addiction. MicroRNA profiling for identifying miRNAs regulating cocaine-induced, plasticity-related genes revealed significant regulation of a set of miRNAs upon cocaine administration, especially let-7d, miR-181a and the brain-specific miR-124. These miRNAs target many genes involved in cocaine addiction. Precursor and mature miRNA quantification by qRT-PCR showed that miR-124 and let-7d are significantly downregulated, whereas miR-181a is induced in the mesolimbic dopaminergic system under chronic cocaine administration. Results were confirmed by in situ hybridization, Northern blots, FISH analysis and RNase protection assay. Using lentiviral-mediated miRNA expression, we show a significant downregulation of BDNF and D3R both at mRNA and protein levels by miR-124 and let-7d, respectively. Our data suggest that miR-124, let-7d and miR-181a may be involved in a complex feedback loop with cocaine-responsive plasticity genes, highlighting the possibility that some miRNAs are key regulators of the reward circuit and may be implicated in addiction.

Role of accumbens BDNF and TrkB in cocaine-induced psychomotor sensitization, conditioned-place preference, and reinstatement in rats.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is involved in the survival and function of midbrain DA neurons. BDNF action is mediated by the TrkB receptor-tyrosine kinase, and both BDNF and TrkB transcripts are widely expressed in the rat mesolimbic pathway, including the nucleus accumbens (NAc) and the ventral tegmentum area (VTA). OBJECTIVE: BDNF was previously shown to be involved in cocaine reward and relapse, as assessed in rat models. The goal of this study is to explore the role of BDNF and TrkB in the rat nucleus accumbens (NAc) in cocaine-induced psychomotor sensitization and in conditioned-place preference acquisition, expression, and reinstatement. MATERIALS AND METHODS: In vivo genetic manipulations of BDNF and TrkB were performed using a lentiviral gene delivery approach to over-express these genes in the NAc and siRNA-based technology to locally knockdown gene expression. Behavioral experiments consisted of locomotor activity monitoring or cocaine-induced conditioned-place preference (CPP). RESULTS: BDNF and/or its receptor TrkB in the NAc enhance drug-induced locomotor activity and induce sensitization in rats. Furthermore, LV-BDNF- and LV-TrkB-treated rats display enhanced cocaine-induced CPP, delayed CPP-extinction upon repeated measurements, and increased CPP reinstatement. In contrast, expression of TrkT1 (truncated form of TrkB, acting as a dominant negative) inhibits these behavioral changes. This inhibition is also observed when rats are fed doxycycline (to block lentivirus-mediated gene expression) or when injected with siRNAs-expressing lentiviruses against TrkB. In addition, we investigate the establishment, maintenance, extinction, and reinstatement of cocaine-induced CPP. We show that BDNF and TrkB-induced CPP takes place during the learning period (conditioning), whereas extinction leads to the loss of CPP. Extinction is delayed when rats are injected LV-BDNF or LV-TrkB, and in turn, priming injections of 2 mg/kg of cocaine reinstates it. CONCLUSIONS: These results demonstrate the crucial function of BDNF-through its receptor TrkB-in the enhancement of locomotor activity, sensitization, conditioned-place preference, CPP-reinstatement, and rewarding effects of cocaine in the mesolimbic dopaminergic pathway.