Mice lacking the dopamine transporter (DAT-/-) are characterized by high extracellular dopamine levels and spontaneous hyperlocomotion. We performed a detailed analysis of the behavioural phenotype of DAT-/- mice in order to identify other behavioural impairments associated with the hyperdopaminergic tone of these mutant mice. In particular, we investigated locomotor activity, exploration, and social and maternal behaviours, which are known to be regulated by dopamine. DAT-/- mice were easily aroused by novelty and always responded with hyperlocomotion, which interfered with habituation to the testing environment, exploratory behaviour in an open field and the coping response to forced swimming stress. Social behaviours such as interaction with an unknown congener or aggressiveness were not modified in DAT-/- mice compared with DAT+/- and DAT+/+ mice, although the maternal behaviour of mutant females was severely disturbed. Haloperidol and clozapine reversed the hyperactivity in DAT-/- mice, with a rightward shift of the dose-response curve compared with control animals, suggesting a dopamine-mediated effect. These results emphasize the role of dopamine regulation in locomotion, exploration and maternal behaviours and suggest that mice with a genetic deletion of DAT may represent a useful model to elucidate the altered behavioural processes accompanying pathological conditions associated with hyperdopaminergic function.

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death. Synthesis of lipids critical for Mtb’s cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4’-phosphopantetheine (Ppt) from coenzyme A to diverse acyl carrier proteins (ACPs). We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss of function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor co-crystal structure may aid further development of anti-mycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.

Mice lacking the dopamine transporter (DAT) display biochemical and behavioural dopaminergic hyperactivity despite dramatic alteration in dopamine homeostasis. In order to determine the anatomical and functional integrity of the dopaminergic system, we examined the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis as well as DOPA decarboxylase and vesicular monoamine transporter. TH-positive neurons in the substantia nigra were only slightly decreased (-27.6 +/- 4.5%), which can not account for the dramatic decreases in the levels of TH and dopamine that we previously observed in the striatum. TH mRNA levels were decreased by 25% in the ventral midbrain with no modification in the ratio of TH mRNA levels per cell. However, TH protein levels were decreased by 90% in the striatum and 35% in the ventral midbrain. In the striatum, many dopaminergic projections had no detectable TH, while few projections maintained regular labelling as demonstrated using electron microscopy. DOPA decarboxylase levels were not modified and vesicular transporter levels were decreased by only 28.7% which suggests that the loss of TH labelling in the striatum is not due to loss of TH projections. Interestingly, we also observed sporadic TH-positive cell bodies using immunohistochemistry and in situ hybridization in the striatum of homozygote mice, and to some extent that of wild-type animals, which raises interesting possibilities as to their potential contribution to the dopamine hyperactivity and volume transmission previously reported in these animals. In conjunction with our previous findings, these results highlight the complex regulatory mechanisms controlling TH expression at the level of mRNA, protein, activity and distribution. The paradoxical hyperdopaminergia in the DAT KO mice despite a marked decrease in TH and dopamine levels suggests a parallel to Parkinson's disease implying that blockade of DAT may be beneficial in this condition.

These findings indicate that partial or total DAT gene deletion result in decreased locomotion in response to d-amphetamine and modify behavioral sensitization depending on the proportion of DAT removed, suggesting that inhibition of the DAT is necessary for the development of sensitization to psychostimulant drugs.

Rationale: Monocytes play critical roles in the pathogenesis of arthritis by contributing to the inflammatory response and bone erosion. Among genes involved in regulating monocyte functions, miR-146a negatively regulates the inflammatory response and osteoclast differentiation of monocytes. It is also the only miRNA reported to differentially regulate the cytokine response of the two classical Ly6Chigh and non-classical Ly6Clow monocyte subsets upon bacterial challenge. Although miR-146a is overexpressed in many tissues of arthritic patients, its specific role in monocyte subsets under arthritic conditions remains to be explored.Methods: We analyzed the monocyte subsets during collagen-induced arthritis (CIA) development by flow cytometry. We quantified the expression of miR-146a in classical and non-classical monocytes sorted from healthy and CIA mice, as well as patients with rheumatoid arthritis (RA). We monitored arthritis features in miR-146a-/- mice and assessed in vivo the therapeutic potential of miR-146a mimics delivery to Ly6Chigh monocytes. We performed transcriptomic and pathway enrichment analyses on both monocyte subsets sorted from wild type and miR-146a-/- mice.Results: We showed that the expression of miR-146a is reduced in the Ly6Chigh subset of CIA mice and in the analogous monocyte subset (CD14+CD16-) in humans with RA as compared with healthy controls. The ablation of miR-146a in mice worsened arthritis severity, increased osteoclast differentiation in vitro and bone erosion in vivo. In vivo delivery of miR-146a to Ly6Chigh monocytes, and not to Ly6Clow monocytes, rescues bone erosion in miR-146a-/- arthritic mice and reduces osteoclast differentiation and pathogenic bone erosion in CIA joints of miR-146a+/+ mice, with no effect on inflammation. Silencing of the non-canonical NF-κB family member RelB in miR-146a-/- Ly6Chigh monocytes uncovers a role for miR-146a as a key regulator of the differentiation of Ly6Chigh, and not Ly6Clow, monocytes into osteoclasts under arthritic conditions.Conclusion: Our results show that classical monocytes play a critical role in arthritis bone erosion. They demonstrate the theranostics potential of manipulating miR-146a expression in Ly6Chigh monocytes to prevent joint destruction while sparing inflammation in arthritis.