J. Neurochem. (2010) 113, 1659–1675. Abstract β‐Carbolines (BCs) are potential endogenous and exogenous neurotoxins that may contribute to the pathogenesis of Parkinson’s disease. However, we recently demonstrated protective and stimulatory effects of 9‐methyl‐BC (9‐me‐BC) in primary dopaminergic culture. In the present study, treatment with 9‐me‐BC unmasked a unique tetrad of effects. First, tyrosine hydroxylase (TH) expression was stimulated in pre‐existing dopa decarboxylase immunoreactive neurons and several TH‐relevant transcription factors (Gata2, Gata3, Creb1, Crebbp) were up‐regulated. Neurite outgrowth of TH immunoreactive (THir) neurons was likewise stimulated. The interaction with tyrosine kinases (protein kinase A and C, epidermal growth factor‐receptor, fibroblast growth factor‐receptor and neural cell adhesion molecule) turned out to be decisive for these observed effects. Second, 9‐me‐BC protected in acute toxicity models THir neurons against lipopolysaccharide and 2,9‐dime‐BC+ toxicity. Third, in a chronic toxicity model when cells were treated with 9‐me‐BC after chronic rotenone administration, a pronounced regeneration of THir neurons was observed. Fourth, 9‐me‐BC inhibited the proliferation of microglia induced by toxin treatment and installed an anti‐inflammatory environment by decreasing the expression of inflammatory cytokines and receptors. Finally, 9‐me‐BC lowered the content of α‐synuclein protein in the cultures. The presented results warrant the exploration of 9‐me‐BC as a novel potential anti‐parkinsonian medication, as 9‐me‐BC interferes with several known pathogenic factors in Parkinson’s disease as outlined above. Further investigations are currently under way.

Notch signaling has a pivotal role in numerous cell-fate decisions, and its aberrant activity leads to developmental disorders and cancer. To identify molecules that influence Notch signaling, we screened nearly 17,000 compounds using automated microscopy to monitor the trafficking and processing of a ligand-independent Notch-enhanced GFP (eGFP) reporter. Characterization of hits in vitro by biochemical and cellular assays and in vivo using zebrafish led to five validated compounds, four of which induced accumulation of the reporter at the plasma membrane by inhibiting γ-secretase. One compound, the dihydropyridine FLI-06, disrupted the Golgi apparatus in a manner distinct from that of brefeldin A and golgicide A. FLI-06 inhibited general secretion at a step before exit from the endoplasmic reticulum (ER), which was accompanied by a tubule-to-sheet morphological transition of the ER, rendering FLI-06 the first small molecule acting at such an early stage in secretory traffic. These data highlight the power of phenotypic screening to enable investigations of central cellular signaling pathways.

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Determining the individual roles of the two dopamine D1-like receptors (D1R and D5R) on sodium transport in the human renal proximal tubule has been complicated by their structural and functional similarity. Here we used a novel D5R-selective antagonist (LE-PM436) and D1R or D5R-specific gene silencing to determine second messenger coupling pathways and heterologous receptor interaction between the two receptors. D1R and D5R co-localized in renal proximal tubule cells and physically interact, as determined by co-immunoprecipitation and FRET microscopy. Stimulation of renal proximal tubule cells with fenoldopam (D1R/D5R agonist) led to both adenylyl cyclase and phospholipase C (PLC) activation using real-time FRET biosensors ICUE3 and CYPHR, respectively. Fenoldopam increased cAMP accumulation and PLC activity and inhibited both NHE3 and NaKATPase activities. LE-PM436 and D5R siRNA blocked the fenoldopam-stimulated PLC pathway but not cAMP accumulation, while D1R siRNA blocked both fenoldopam-stimulated cAMP accumulation and PLC signaling. Either D1R or D5R siRNA, or LE-PM436 blocked the fenoldopam dependent inhibition of sodium transport. Further studies using the cAMP-selective D1R/D5R agonist SKF83822 and PLC-selective D1R/D5R agonist SKF83959 confirmed the cooperative influence of the two pathways on sodium transport. Thus, D1R and D5R interact in the inhibition of NHE3 and NaKATPase activity, the D1R primarily by cAMP, while the D1R/D5R heteromer modulates the D1R effect through a PLC pathway.

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