An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters.

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

Reprogramming fibroblasts and peripheral blood cells from a C9ORF72 patient: A proof-of-principle study.

As for the majority of neurodegenerative diseases, pathological mechanisms of amyotrophic lateral sclerosis (ALS) have been challenging to study due to the difficult access to alive patients' cells. Induced pluripotent stem cells (iPSCs) offer a useful in vitro system for modelling human diseases. iPSCs can be theoretically obtained by reprogramming any somatic tissue although fibroblasts (FB) remain the most used cells. However, reprogramming peripheral blood cells (PB) may offer significant advantages. In order to investigate whether the choice of starting cells may affect reprogramming and motor neuron (MNs) differentiation potential, we used both FB and PB from a same C9ORF72-mutated ALS patient to obtain iPSCs and compared several hallmarks of the pathology. We found that both iPSCs and MNs derived from the two tissues showed identical properties and features and can therefore be used interchangeably, giving the opportunity to easily obtain iPSCs from a more manageable source of cells, such as PB.

Motor neuron differentiation of iPSCs obtained from peripheral blood of a mutant TARDBP ALS patient.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease, mainly affecting the motor neurons (MNs) and without effective therapy. Drug screening is hampered by the lack of satisfactory experimental and pre-clinical models. Induced pluripotent stem cells (iPSCs) could help to define disease mechanisms and therapeutic strategies as they could be differentiated into MNs, otherwise inaccessible from living humans. In this study, given the seminal role of TDP-43 in ALS pathophysiology, MNs were obtained from peripheral blood mononuclear cells-derived iPSCs of an ALS patient carrying a p.A382T TARDBP mutation and a healthy donor. Venous samples were preferred to fibroblasts for their ease of collection and no requirement for time consuming extended cultures before experimentation. iPSCs were characterized for expression of specific markers, spontaneously differentiated into primary germ layers and, finally, into MNs. No differences were observed between the mutated ALS patient and the control MNs with most of the cells displaying a nuclear localization of the TDP-43 protein. In conclusion, we here demonstrated for the first time that human TARDBP mutated MNs can be successfully obtained exploiting the reprogramming and differentiation ability of peripheral blood cells, an easily accessible source from any patient.

Bcl-2/adenovirus E1B 19-kDa interacting protein (BNip3) has a key role in the mitochondrial dysfunction induced by mutant huntingtin.

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a CAG repeat in the IT15 gene that encodes the protein huntingtin (htt). Evidence shows that mutant htt causes mitochondrial depolarization and fragmentation, but the underlying molecular mechanism has yet to be clarified. Bax/Bak and BNip3 are pro-apoptotic members of the Bcl-2 family protein whose activation triggers mitochondrial depolarization and fragmentation inducing cell death. Evidence suggests that Bax/Bak and BNip3 undergo activation upon mutant htt expression but whether these proteins are required for mitochondrial depolarization and fragmentation induced by mutant htt is unclear. Our results show that BNip3 knock-out cells are protected from mitochondrial damage and cell death induced by mutant htt whereas Bax/Bak knock-out cells are not. Moreover, deletion of BNip3 C-terminal transmembrane domain, required for mitochondrial targeting, suppresses mitochondrial depolarization and fragmentation in a cell culture model of HD. Hence, our results suggest that changes in mitochondrial morphology and transmembrane potential, induced by mutant htt protein, are dependent and linked to BNip3 and not to Bax/Bak activation. These results provide new compelling evidence that underlies the molecular mechanisms by which mutant htt causes mitochondrial dysfunction and cell death, suggesting BNip3 as a potential target for HD therapy.

Counterfactual Thinking Deficit in Huntington's Disease.

BACKGROUND AND OBJECTIVE: Counterfactual thinking (CFT) refers to the generation of mental simulations of alternatives to past events, actions and outcomes. CFT is a pervasive cognitive feature in every-day life and is closely related to decision-making, planning and problem-solving - all of which are cognitive processes linked to unimpaired frontal lobe functioning. Huntington's Disease (HD) is a neurodegenerative disorder characterised by motor, behavioral and cognitive dysfunctions. Because an impairment in frontal and executive functions has been described in HD, we hypothesised that HD patients may have a CFT impairment. METHODS: Tests of spontaneous counterfactual thoughts and counterfactual-derived inferences were administered to 24 symptomatic HD patients and 24 age- and sex-matched healthy subjects. RESULTS: Our results show a significant impairment in the spontaneous generation of CFT and low performance on the Counterfactual Inference Test (CIT) in HD patients. Low performance on the spontaneous CFT test significantly correlates with impaired attention abilities, verbal fluency and frontal lobe efficiency, as measured by Trail Making Test - Part A, Phonemic Verbal Fluency Test and FAB. CONCLUSIONS: Spontaneous CFT and the use of this type of reasoning are impaired in HD patients. This deficit may be related to frontal lobe dysfunction, which is a hallmark of HD. Because CFT has a pervasive role in patients' daily lives regarding their planning, decision making and problem solving skills, cognitive rehabilitation may improve HD patients' ability to analyse current behaviors and future actions.

Unexpected lateral-lithiation-induced alkylative ring opening of tetrahydrofurans in deep eutectic solvents: synthesis of functionalised primary alcohols.

o-Tolyl-substituted tetrahydrofurans undergo highly regioselective ring opening with the concomitant formation of new C-C bonds as the result of a lateral lithiation reaction. This reaction provides a new method for the synthesis of functionalised primary alcohols and can be run directly in protic eutectic mixtures as benign reaction media at 0 °C and under air, competitively with protonolysis.

Parkin regulates kainate receptors by interacting with the GluK2 subunit.

Although loss-of-function mutations in the PARK2 gene, the gene that encodes the protein parkin, cause autosomal recessive juvenile parkinsonism, the responsible molecular mechanisms remain unclear. Evidence suggests that a loss of parkin dysregulates excitatory synapses. Here we show that parkin interacts with the kainate receptor (KAR) GluK2 subunit and regulates KAR function. Loss of parkin function in primary cultured neurons causes GluK2 protein to accumulate in the plasma membrane, potentiates KAR currents and increases KAR-dependent excitotoxicity. Expression in the mouse brain of a parkin mutant causing autosomal recessive juvenile parkinsonism results in GluK2 protein accumulation and excitotoxicity. These findings show that parkin regulates KAR function in vitro and in vivo, and suggest that KAR upregulation may have a pathogenetic role in parkin-related autosomal recessive juvenile parkinsonism.

Regioselective desymmetrization of diaryltetrahydrofurans via directed ortho-lithiation: an unexpected help from green chemistry.

An efficient functionalization of diaryltetrahydrofurans via a regioselective THF-directed ortho-lithiation is first described. This reaction can be successfully carried out in cyclopentyl methyl ether as a "greener" alternative to Et2O, with better results in terms of yield and selectivity and, surprisingly, also in protic eutectic mixtures competitively with protonolysis.

Preparation of polysubstituted isochromanes by addition of ortho-lithiated aryloxiranes to enaminones.

The reaction of ortho-lithiated aryloxiranes with various enaminones straightforwardly affords new functionalized isochromanes as mixtures of two epimeric stereoisomers in reasonable to very good yields (50-90%). The two diastereomers, which show a high structural variability, can be easily separated by column chromatography.

Short- and long- term effects of cigarette smoke exposure on glutathione homeostasis in human bronchial epithelial cells.

BACKGROUND: Cigarette smoke extract (CSE), a model for studying the effects of tobacco smoke in vivo and in vitro, induces cell oxidative stress and affects the antioxidative glutathione system. We evaluated the impact of CSE on airway epithelial cells and the possible cytoprotective effect of the mucolitic drug S-carboximethilcysteine lysine salt (S-CMC-Lys). METHODS: Reduced glutathione (GSH) and reactive oxygen species (ROS) intracellular levels were evaluated by fluorimetry in human bronchial epithelial cells (16-HBE) and the expression and activity of enzymes of the GSH metabolic pathway were investigated by RT-PCR, Western blot and colorimetric assays. RESULTS: CSE significantly increased cell mortality in a time and dose dependent manner, via an apoptosis-independent pathway. Short-term (3 hours) CSE exposure induced an increase in ROS levels and a GSH intracellular concentration drop. In parallel, the expression of glutathione peroxidases 2 and 3, glutathione reductase and glutamate-cysteine-ligase was increased. S-CMC-Lys was effective in counteracting these effects. CONCLUSION: CSE affects ROS levels, GSH concentration and GSH enzymes pathway. These effects can be to some extent reversed by S-CMC-Lys, that could represent a therapeutic tool to counteract CSE induced oxidative cellular injuries.

Hematopoietic stem/progenitor cells express functional mitochondrial energy-dependent cystic fibrosis transmembrane conductance regulator.

Bone marrow-derived hematopoietic stem/progenitor cells (HSPCs) encompass a wide array of cell subsets with different capacities of engraftment and injured tissue-regenerating potential. The characterization/isolation of the stem cell subpopulations represents a major challenge to improve the efficacy of transplantation protocols used in regenerative medicine. Cystic fibrosis (CF) is one of the diseases whose hope of cure relies on the successful application of cell-based gene therapy. This study was aimed at characterizing murine HSPCs on the basis of their bioenergetic competence and CF transmembrane conductance regulator (CFTR) expression. Positively immunoselected Sca-1(+) HSPCs encompassed 2 populations distinguished by their different size, Sca-1 expression and mitochondrial content. The smaller were the cells, the higher was Sca-1 expression and the lower was the intracellular density of functional mitochondria. Reverse transcription-polymerase chain reaction and western blotting revealed that HSPCs expressed CFTR mRNA and protein, which was also functional, as assessed by spectrofluorimetric and patch-clamp techniques. Inhibition of mitochondrial oxidative phosphorylation by oligomycin resulted in a 70% decrease of both the intracelluar adenosine triphosphate content and CFTR-mediated channel activity. Finally, HSPCs with lower Sca-1 expression and higher mitochondrial content displayed higher CFTR levels. Our findings identify 2 subpopulations in HSPCs and unveil a so-far unappreciated relationship between bioenergetic metabolism and CFTR in HSPC biology.

Hypertension-linked mutation of α-adducin increases CFTR surface expression and activity in HEK and cultured rat distal convoluted tubule cells.

The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats), an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT) or G460W mutated α-adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus) hypertensive model carrying the F316Y adducin mutation), compared to MNS (Milan Normotensive Strain) rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching) and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT cells the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasmamembrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.