SUMMARY Lung alveoli, which are unique to air-breathing organisms, have been challenging to generate from pluripotent stem cells (PSCs) in part because there are limited model systems available to provide the necessary developmental roadmaps for in vitro differentiation. Here we report the generation of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, from human PSCs. Using multicolored fluorescent reporter lines, we track and purify human SFTPC+ alveolar progenitors as they emerge from endodermal precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells form monolayered epithelial “alveolospheres” in 3D cultures without the need for mesenchymal support, exhibit self-renewal capacity, and display additional AEC2 functional capacities. Footprint-free CRISPR-based gene correction of PSCs derived from patients carrying a homozygous surfactant mutation (SFTPB121ins2) restores surfactant processing in AEC2s. Thus, PSC-derived AEC2s provide a platform for disease modeling and future functional regeneration of the distal lung.

Parkinson's disease is caused primarily by degeneration of brain dopaminergic neurons in the substantia nigra and the consequent deficit of dopamine in the striatum. Dopamine replacement therapy with the dopamine precursor L-dopa is the mainstay of current treatment. After several years, however, the patients develop L-dopa-induced dyskinesia, or abnormal involuntary movements, thought to be due to excessive signaling via dopamine receptors. G protein-coupled receptor kinases (GRKs) control desensitization of dopamine receptors. We found that dyskinesia is attenuated by lentivirus-mediated overexpression of GRK6 in the striatum in rodent and primate models of Parkinson's disease. Conversely, reduction of GRK6 concentration by microRNA delivered with lentiviral vector exacerbated dyskinesia in parkinsonian rats. GRK6 suppressed dyskinesia in monkeys without compromising the anti-parkinsonian effects of L-dopa and even prolonged the antiparkinsonian effect of a lower dose of L-dopa. Our finding that increased availability of GRK6 ameliorates dyskinesia and increases duration of the antiparkinsonian action of L-dopa suggests a promising approach for controlling both dyskinesia and motor fluctuations in Parkinson's disease. † To whom correspondence should be addressed. Eugenia.Gurevich@vanderbilt.edu (E.V.G.); Erwan.bezard@u-bordeaux2.fr (E.B.). * These authors contributed equally to this work.Author contributions: E. Bezard and E.V.G. designed and organized the experiments; E.V.G., V.V.G., M.R.A., Y.T.C., and S.K. designed, cloned, and produced viral vectors and viruses; E.V.G., M.R.A., Y.T.C., E. Bychkov, S.K., A.B., and G.P. performed rat behavioral, neurochemical, and histological experiments; E. Bezard, A.B., G.P., Q.L., B.H.B., B.B., I.A., S.D., and E.D. performed monkey behavioral, neurochemical, and histological experiments; E. Bezard and E.V.G. analyzed the data; E. Bezard, V.V.G., and E.V.G. wrote the paper. Competing interests:The authors have declared no competing interests. SUPPLEMENTARY MATERIAL www.sciencetranslationalmedicine.org/cgi/content/full/2/28/28ra28/DC1 Materials and Methods Fig. S1. The GFP-tagged GRK6 is functional and has the subcellular localization of the endogenous GRK6. Fig. S2. Antibodies to GRK6 selectively recognize GRK6A or GRK6B splicing variants. Fig. S3. The lentivirus carrying two chained miRNAs targets both GRK6A and GRK6B splice variants. Fig. S4. Infection of the rat striatum with the miRNA lentivirus induces the GRK6 knockdown. References NIH Public Access

Non‐visual arrestins‐2 and‐3 interact with numerous GPCRs and dozens of non‐receptors partners, such as MAP kinases. Arrestin‐3 promotes JNK3 activation, scaffolding ASK1‐MKK4‐JNK3 cascade. Full activation of JNKs, unlike other MAP kinases, requires two upstream kinases, MKK4 and MKK7, and each preferentially phosphorylating a distinct site, tyrosine (MKK4) and theronine (MKK7). It remains unclear whether arrestin‐3 can promote the activation of JNK3 by MKK7. Using phospho‐Tyr and phospho‐Thr antibodies we found that arrestin‐3 promotes the phosphorylation of both Tyr and Thr within the T‐P‐Y motif in COS‐7 cells. We also found that arrestin‐3 directly binds MKK7 comparably to MKK4. The binding of JNK3 enhanced the association of arrestin‐3 with MKK4, while reduced the binding of MKK7. With pure proteins we show that arrestin‐3 scaffolds MKK7‐JNK3 module at higher concentration than MKK4‐JNK3 module. Arresint‐3 also facilitates JNK3 activation in COS‐7 cells over‐expressing either MKK4 or MKK7. This is the first evidence that arrestin‐3 promotes JNK3 phosphorylation by MKK7. The data suggest that JNK3 binding has opposite effect on arrestin‐3 interactions with MKK4 and MKK7. Support: NIH GM081756, GM077561 and EY011500 (VVG) and GM059802 and Welch Foundation Grant F‐1390 (KND).

Background:The relative contribution of phosphates and active GPCR conformation is unknown. Results: Using WT and mutant arrestins and receptors, we show that phosphates are critical for arrestin binding to some GPCRs but not to others. Conclusion:The role of receptor-attached phosphates in arrestin binding varies widely depending on the arrestin-receptor combination. Significance: Distinct molecular mechanisms mediate arrestin recruitment to different GPCRs.