The glucocorticoid receptor is a ubiquitous transcription factor mediating adaptation to environmental challenges and stress. Selective Nr3c1 (the glucocorticoid receptor gene) ablation in mouse dopaminoceptive neurons expressing dopamine receptor 1a, but not in dopamine-releasing neurons, markedly decreased the motivation of mice to self-administer cocaine, dopamine cell firing and the control exerted by dopaminoceptive neurons on dopamine cell firing activity. In contrast, anxiety was unaffected, indicating that glucocorticoid receptors modify a number of behavioral disorders through different neuronal populations.
BackgroundCharcot-Marie-Tooth type 1A disease (CMT1A) is a rare orphan inherited neuropathy caused by an autosomal dominant duplication of a gene encoding for the structural myelin protein PMP22, which induces abnormal Schwann cell differentiation and dysmyelination, eventually leading to axonal suffering then loss and muscle wasting. We favour the idea that diseases can be more efficiently treated when targeting multiple disease-relevant pathways. In CMT1A patients, we therefore tested the potential of PXT3003, a low-dose combination of three already approved compounds (baclofen, naltrexone and sorbitol). Our study conceptually builds on preclinical experiments highlighting a pleiotropic mechanism of action that includes downregulation of PMP22. The primary objective was to assess safety and tolerability of PXT3003. The secondary objective aimed at an exploratory analysis of efficacy of PXT3003 in CMT1A, to be used for designing next clinical development stages (Phase 2b/3).Methods80 adult patients with mild-to-moderate CMT1A received in double-blind for 1 year Placebo or one of the three increasing doses of PXT3003 tested, in four equal groups. Safety and tolerability were assessed with the incidence of related adverse events. Efficacy was assessed using the Charcot-Marie-Tooth Neuropathy Score (CMTNS) and the Overall Neuropathy Limitations Scale (ONLS) as main endpoints, as well as various clinical and electrophysiological outcomes.ResultsThis trial confirmed the safety and tolerability of PXT3003. The highest dose (HD) showed consistent evidence of improvement beyond stabilization. CMTNS and ONLS, with a significant improvement of respectively of 8% (0.4% - 16.2%) and 12.1% (2% - 23.2%) in the HD group versus the pool of all other groups, appear to be the most sensitive clinical endpoints to treatment despite their quasi-stability over one year under Placebo. Patients who did not deteriorate over one year were significantly more frequent in the HD group.ConclusionsThese results confirm that PXT3003 deserves further investigation in adults and could greatly benefit CMT1A-diagnosed children, usually less affected than adults.Trial registrationEudraCT Number: 2010-023097-40. ClinicalTrials.gov Identifier: NCT01401257. The Committee for Orphan Medicinal Products issued in February 2014 a positive opinion on the application for orphan designation for PXT3003 (EMA/OD/193/13).Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-014-0199-0) contains supplementary material, which is available to authorized users.
The dopamine transporter is an essential component of the dopaminergic synapse. It is located in the presynaptic neurons and regulates extracellular dopamine levels. We generated a transgenic mouse line expressing the Cre recombinase under the control of the regulatory elements of the dopamine transporter gene, for investigations of gene function in dopaminergic neurons. The codon‐improved Cre recombinase (iCre) gene was inserted into the dopamine transporter gene on a bacterial artificial chromosome. The pattern of expression of the bacterial artificial chromosome–dopamine transporter–iCre transgene was similar to that of the endogenous dopamine transporter gene, as shown by immunohistochemistry. Recombinase activity was further studied in mice carrying both the bacterial artificial chromosome–dopamine transporter–iCre transgene and a construct expressing the β‐galactosidase gene after Cre‐mediated recombination. In situ studies showed that β‐galactosidase (5‐bromo‐4‐chloroindol‐3‐yl β‐d‐galactoside staining) and the dopamine transporter (immunofluorescence) had identical distributions in the ventral midbrain. We used this animal model to study the distribution of dopamine transporter gene expression in hypothalamic nuclei in detail. The expression profile of tyrosine hydroxylase (an enzyme required for dopamine synthesis) was broader than that of β‐galactosidase in A12 to A15. Thus, only a fraction of neurons synthesizing dopamine expressed the dopamine transporter gene. The bacterial artificial chromosome–dopamine transporter–iCre transgenic line is a unique tool for targeting Cre/loxP‐mediated DNA recombination to dopamine neurons for studies of gene function or for labeling living cells, following the crossing of these mice with transgenic Cre reporter lines producing fluorescent proteins.
Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited sensory and motor peripheral neuropathy. It is caused by PMP22 overexpression which leads to defects of peripheral myelination, loss of long axons, and progressive impairment then disability. There is no treatment available despite observations that monotherapeutic interventions slow progression in rodent models. We thus hypothesized that a polytherapeutic approach using several drugs, previously approved for other diseases, could be beneficial by simultaneously targeting PMP22 and pathways important for myelination and axonal integrity. A combination of drugs for CMT1A polytherapy was chosen from a group of authorised drugs for unrelated diseases using a systems biology approach, followed by pharmacological safety considerations. Testing and proof of synergism of these drugs were performed in a co-culture model of DRG neurons and Schwann cells derived from a Pmp22 transgenic rat model of CMT1A. Their ability to lower Pmp22 mRNA in Schwann cells relative to house-keeping genes or to a second myelin transcript (Mpz) was assessed in a clonal cell line expressing these genes. Finally in vivo efficacy of the combination was tested in two models: CMT1A transgenic rats, and mice that recover from a nerve crush injury, a model to assess neuroprotection and regeneration. Combination of (RS)-baclofen, naltrexone hydrochloride and D-sorbitol, termed PXT3003, improved myelination in the Pmp22 transgenic co-culture cellular model, and moderately down-regulated Pmp22 mRNA expression in Schwannoma cells. In both in vitro systems, the combination of drugs was revealed to possess synergistic effects, which provided the rationale for in vivo clinical testing of rodent models. In Pmp22 transgenic CMT1A rats, PXT3003 down-regulated the Pmp22 to Mpz mRNA ratio, improved myelination of small fibres, increased nerve conduction and ameliorated the clinical phenotype. PXT3003 also improved axonal regeneration and remyelination in the murine nerve crush model. Based on these observations in preclinical models, a clinical trial of PTX3003 in CMT1A, a neglected orphan disease, is warranted. If the efficacy of PTX3003 is confirmed, rational polytherapy based on novel combinations of existing non-toxic drugs with pleiotropic effects may represent a promising approach for rapid drug development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-014-0201-x) contains supplementary material, which is available to authorized users.
The adrenal glucocorticoid, corticosterone, induces changes in gene expression in both neural and non-neural tissues. The rhythmic release of corticosterone has been shown in rats to be necessary for the rhythmic expression of the clock protein PERIOD2 (PER2) in select regions of the limbic forebrain. The mechanisms mediating the effects of glucocorticoids on changes in gene expression have been linked to the transcriptional activity of the low affinity glucocorticoid receptor, GR. We examined the patterns of PER2 expression in the brains of mice containing an inactivation of GR gene restricted to neural tissues (GR(NesCre) mice). We found that central deletion of the GR gene blunts the daily pattern of PER2 expression in the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and central nucleus of the amygdala (CEA) both of which make up the central extended amygdala, but not in the suprachiasmatic nucleus (SCN), basolateral amygdala (BLA) or dentate gyrus of the hippocampus (DG). These results implicate brain GR receptors in the regulation of PER2 expression in the BNSTov and CEA and are consistent with our previous findings that the rhythmic expression of PER2 in these areas is selectively sensitive to fluctuations in circulating corticosterone.
Alzheimer disease (AD) represents a major medical problem where mono-therapeutic interventions demonstrated only a limited efficacy so far. We explored the possibility of developing a combinational therapy that might prevent the degradation of neuronal and endothelial structures in this disease. We argued that the distorted balance between excitatory (glutamate) and inhibitory (GABA/glycine) systems constitutes a therapeutic target for such intervention. We found that a combination of two approved drugs – acamprosate and baclofen – synergistically protected neurons and endothelial structures in vitro against amyloid-beta (Aβ) oligomers. The neuroprotective effects of these drugs were mediated by modulation of targets in GABA/glycinergic and glutamatergic pathways. In vivo, the combination alleviated cognitive deficits in the acute Aβ25–35 peptide injection model and in the mouse mutant APP transgenic model. Several patterns altered in AD were also synergistically normalised. Our results open up the possibility for a promising therapeutic approach for AD by combining repurposed drugs.
Aims/hypothesis Beta cell development is sensitive to glucocorticoid levels. Although direct effects of glucocorticoids on pancreatic precursors have been shown to control beta cell mass expansion, indirect effects of these hormones on pancreatic development remain unexplored. This issue was addressed in mice lacking the glucocorticoid receptor (GR) in the whole organism. Materials and Methods The pancreatic phenotype of GR null/null mice was studied at fetal ages (embryonic day [E]) E15.5 and E18 by immunohistochemistry and beta cell fraction measurements. To distinguish between direct and indirect effects, mutant E15.5 fetal pancreata were grafted under the kidney capsule of immunodeficient mice and analysed after 1 week. Results E18 GR null/null fetuses had smaller digestive tracts and tiny pancreata. Massive pancreatic disorganisation and apoptosis were observed despite the presence of all cell types. E15.5 GR null/null mutants were indistinguishable from wild-type regarding pancreatic size, tissue structure and organisation, beta cell fraction and production of exocrine transcription factor Ptf1a, neurogenin 3 and Pdx-1. Grafting E15.5 GR null/null pancreata into a GR-expressing environment rescued the increased apoptosis and mature islets were observed, suggesting that GR null/null pancreatic cell death can be attributed to indirect effects of glucocorticoids on this tissue. Heterozygous GR +/null mutants with reduced GR numbers showed no apoptosis but increased beta cell fraction at E18 and the adult age, strengthening the importance of an accurate GR dosage on beta cell mass expansion. Conclusions/interpretation Our results provide evidence for GR involvement in pancreatic tissue organisation and survival through indirect effects. GR does not appear necessary for early phases, but its accurate dosage is critical to modulate beta cell mass expansion at later fetal stages, presumably through direct effects.
Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterised by the loss of dopaminergic nigrostriatal neurons but which involves the loss of additional neurotransmitter pathways. Mono- or polytherapeutic interventions in PD patients have declining efficacy long-term and no influence on disease progression. The systematic analysis of available genetic and functional data as well as the substantial overlap between Alzheimer’s disease (AD) and PD features led us to repurpose and explore the effectiveness of a combination therapy (ABC) with two drugs – acamprosate and baclofen – that was already effective in AD animal models, for the treatment of PD. We showed in vitro that ABC strongly and synergistically protected neuronal cells from oxidative stress in the oxygen and glucose deprivation model, as well as dopaminergic neurons from cell death in the 6-hydroxydopamine (6-OHDA) rat model. Furthermore, we showed that ABC normalised altered motor symptoms in vivo in 6-OHDA-treated rats, acting by protecting dopaminergic cell bodies and their striatal terminals. Interestingly, ABC also restored a normal behaviour pattern in lesioned rats suggesting a symptomatic effect, and did not negatively interact with L-dopa. Our results demonstrate the potential value of combining repurposed drugs as a promising new strategy to treat this debilitating disease.
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