Background & AimsInteractions between C-C chemokine receptor types 2 (CCR2) and 5 (CCR5) and their ligands, including CCL2 and CCL5, mediate fibrogenesis by promoting monocyte/macrophage recruitment and tissue infiltration, as well as hepatic stellate cell activation. Cenicriviroc (CVC) is an oral, dual CCR2/CCR5 antagonist with nanomolar potency against both receptors. CVC’s anti-inflammatory and antifibrotic effects were evaluated in a range of preclinical models of inflammation and fibrosis.MethodsMonocyte/macrophage recruitment was assessed in vivo in a mouse model of thioglycollate-induced peritonitis. CCL2-induced chemotaxis was evaluated ex vivo on mouse monocytes. CVC’s antifibrotic effects were evaluated in a thioacetamide-induced rat model of liver fibrosis and mouse models of diet-induced non-alcoholic steatohepatitis (NASH) and renal fibrosis. Study assessments included body and liver/kidney weight, liver function test, liver/kidney morphology and collagen deposition, fibrogenic gene and protein expression, and pharmacokinetic analyses.ResultsCVC significantly reduced monocyte/macrophage recruitment in vivo at doses ≥20 mg/kg/day (p < 0.05). At these doses, CVC showed antifibrotic effects, with significant reductions in collagen deposition (p < 0.05), and collagen type 1 protein and mRNA expression across the three animal models of fibrosis. In the NASH model, CVC significantly reduced the non-alcoholic fatty liver disease activity score (p < 0.05 vs. controls). CVC treatment had no notable effect on body or liver/kidney weight.ConclusionsCVC displayed potent anti-inflammatory and antifibrotic activity in a range of animal fibrosis models, supporting human testing for fibrotic diseases. Further experimental studies are needed to clarify the underlying mechanisms of CVC’s antifibrotic effects. A Phase 2b study in adults with NASH and liver fibrosis is fully enrolled (CENTAUR Study 652-2-203; NCT02217475).

Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinson's disease (PD). However, a major stumbling block has been the lack of a reliable source of donor DA neurons. Here we show that a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD.

The plasma membrane dopamine transporter (DAT) takes extracellular dopamine back up into dopaminergic neurons. Although the number of DATs at the cell surface is regulated by endocytosis and recycling, the molecular mechanisms that control this endocytic trafficking of DAT are not defined. To map the sequence motifs that are involved in constitutive DAT endocytosis, mutagenesis of human DAT tagged with yellow fluorescent protein (YFP) and an extracellular HA epitope was performed. Removal of the entire N terminus of DAT resulted in accumulation of the resulting DAT mutant (YFP-HA-⌬N-DAT) in early and recycling endosomes in HeLa and PAE cells, and in primary rat mesencephalic-striatal neuronal cocultures. This endosomal accumulation was due to rapid constitutive internalization of YFP-HA-⌬N-DAT by the clathrin-dependent pathway. Small deletions and multialanine substitutions in the N terminus revealed two molecular determinants within the membrane proximal residues 60 -65 that are important for preventing rapid internalization of DAT. First, mutations of Arg60 or Trp63, leading to disruption of the "outward facing" DAT conformation, correlated with an increased pool of mobile DATs in the plasma membrane and accelerated constitutive internalization of the DAT mutants. Second, mutation of Lys65 also correlated with elevated endocytosis. While none of these mutations alone recapitulated the marked endocytic phenotype of YFP-HA-⌬N-DAT, simultaneous elimination of both the outward conformation of DAT and Lys65 resulted in DAT mutants that were rapidly internalized. Thus, our studies reveal a new link between DAT endocytosis and conformation-dependent uptake activity that represents a novel mode for regulating DAT function.

The plasma membrane dopamine (DA) transporter (DAT) is essential for reuptake of extracellular DA. DAT function in heterologous cells is regulated by subcellular targeting, endocytosis, and intracellular trafficking, but the mechanisms regulating neuronal DAT remain poorly understood. Hence, we generated a knock-in mouse expressing a hemagglutinin (HA)-epitope-tagged DAT to study endogenous transporter trafficking. Introduction of the HA tag into the second extracellular loop of mouse DAT did not perturb its expression level, distribution pattern, or substrate uptake kinetics. Live-cell fluorescence microscopy imaging using fluorescently labeled HA-specific antibody and a quantitative HA-antibody endocytosis assay demonstrated that in axons HA-DAT was primarily located in the plasma membrane and internalized mostly in growth cones and varicosities, where synaptic vesicle markers were also concentrated. Formation of varicosities was frequently preceded or accompanied by highly dynamic filopodia-like membrane protrusions. Remarkably, HA-DAT often concentrated at the tips of these filopodia. This pool of HA-DATs exhibited low lateral membrane mobility. Thus, DAT-containing filopodia may be involved in synaptogenesis in developing DA neurons. Treatment of neurons with amphetamine increased mobility of filopodial HA-DAT and accelerated HA-DAT endocytosis in axons, suggesting that chronic amphetamine may interfere with DA synapse development. Interestingly, phorbol esters did not accelerate endocytosis of axonal DAT.

Dopamine (DA), d‐amphetamine (AMPH) and methamphetamine (METH) are DA transporter (DAT) substrates that also rapidly and transiently reduce cell surface expression of DATs in dorsal striatum (dSTR) and cells expressing cloned DATs. However, it is not clear if DATs are regulated similarly by substrates in nucleus accumbens (NAc). Here, we investigated whether brief in vitro exposure to DAT substrates differentially regulates DATs in synaptosomes prepared from dSTR or NAc of male Sprague Dawley rats. Synaptosomes were pre‐incubated with DA, AMPH, METH (3–30 μM) or buffer for 5–30 min at 37oC and washed twice at 4oC; specific uptake of 0.5 nM [3H]DA, defined with 100 μM cocaine, was measured for 3 min at 37oC. As expected, [3H]DA uptake was 54% lower in NAc than in dSTR, and substrate pre‐incubation significantly reduced [3H]DA uptake in dSTR in a time‐ and concentration‐dependent manner. Although in NAc [3H]DA uptake was also reduced by pre‐incubation with DA, AMPH or METH, statistical significance was not reached with any pre‐incubation time or concentration. Kinetic analyses after a 15 min pre‐incubation with 10 μM AMPH confirmed a lower maximal DAT velocity in dSTR, but not in NAc. Our results suggest that brief substrate exposure more efficaciously down‐regulates DATs in dSTR; however, surface biotinylation experiments are needed to confirm these observations. Supported by DA004216, DA014204, AA007464, DA015050.