Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes

TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative diseases. Although TDP-43 resembles heterogeneous nuclear ribonucleoproteins, its RNA targets and physiological protein partners remain unknown. Here we identify RNA targets of TDP-43 from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). The canonical TDP-43 binding site (TG)(n) is 55.1-fold enriched, and moreover, a variant with adenine in the middle, (TG)(n)TA(TG)(m), is highly abundant among reads in our TDP-43 RIP-seq library. TDP-43 RNA targets can be divided into three different groups: those primarily binding in introns, in exons, and across both introns and exons. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to synaptic function, RNA metabolism, and neuronal development. Furthermore, TDP-43 binds to a number of RNAs encoding for proteins implicated in neurodegeneration, including TDP-43 itself, FUS/TLS, progranulin, Tau, and ataxin 1 and -2. We also identify 25 proteins that co-purify with TDP-43 from rodent brain nuclear extracts. Prominent among them are nuclear proteins involved in pre-mRNA splicing and RNA stability and transport. Also notable are two neuron-enriched proteins, methyl CpG-binding protein 2 and polypyrimidine tract-binding protein 2 (PTBP2). A PTBP2 consensus RNA binding motif is enriched in the TDP-43 RIP-seq library, suggesting that PTBP2 may co-regulate TDP-43 RNA targets. This work thus reveals the protein and RNA components of the TDP-43-containing ribonucleoprotein complexes and provides a framework for understanding how dysregulation of TDP-43 in RNA metabolism contributes to neurodegeneration.     

Differential signaling by adaptor molecules LRP1 and ShcA regulates adipogenesis by the insulin-like growth factor-1 receptor

The low density lipoprotein receptor-related protein (LRP1) is a transmembrane receptor that integrates multiple signaling pathways. Its cytoplasmic domain serves as docking sites for several adaptor proteins such as the Src homology 2/α-collagen (ShcA), which also binds to several tyrosine kinase receptors such as the insulin-like growth factor 1 (IGF-1) receptor. However, the physiological significance of the physical interaction between LRP1 and ShcA, and whether this interaction modifies tyrosine kinase receptor signaling, are still unknown. Here we report that LRP1 forms a complex with the IGF-1 receptor, and that LRP1 is required for ShcA to become sensitive to IGF-1 stimulation. Upon IGF-1 treatment, ShcA is tyrosine phosphorylated and translocates to the plasma membrane only in the presence of LRP1. This leads to the recruitment of the growth factor receptor-bound protein 2 (Grb2) to ShcA, and activation of the Ras/MAP kinase pathway. Conversely, in the absence of ShcA, IGF-1 signaling bifurcates toward the Akt/mammalian target of rapamycin pathway and accelerates adipocyte differentiation when cells are stimulated for adipogenesis. These results establish the LRP1-ShcA complex as an essential component in the IGF-1-regulated pathway for MAP kinase and Akt/mammalian target of rapamycin activation, and may help to understand the IGF-1 signaling shift from clonal expansion to growth-arrested cells and differentiation during adipogenesis.     

Guaianolides and lignans from Centaurea solstitialis subs Schouwii

Aerial parts of Centaurea solstitialis subs schouwii afforded the guaianolides cynaropicrin and aguerin B and the lignans arctigenin and matairesinol. The structure of a third guaianolide previously found also in Centaurea behen was revised.     

Receptor clustering is involved in Reelin signaling

The Reelin signaling cascade plays a crucial role in the correct positioning of neurons during embryonic brain development. Reelin binding to apolipoprotein E receptor 2 (ApoER2) and very-low-density-lipoprotein receptor (VLDLR) leads to phosphorylation of disabled 1 (Dab1), an adaptor protein which associates with the intracellular domains of both receptors. Coreceptors for Reelin have been postulated to be necessary for Dab1 phosphorylation. We show that bivalent agents specifically binding to ApoER2 or VLDLR are sufficient to mimic the Reelin signal. These agents induce Dab1 phosphorylation, activate members of the Src family of nonreceptor tyrosine kinases, modulate protein kinase B/Akt phosphorylation, and increase long-term potentiation in hippocampal slices. Induced dimerization of Dab1 in HEK293 cells leads to its phosphorylation even in the absence of Reelin receptors. The mechanism for and the sites of these phosphorylations are identical to those effected by Reelin in primary neurons. These results suggest that binding of Reelin, which exists as a homodimer in vivo, to ApoER2 and VLDLR induces clustering of ApoER2 and VLDLR. As a consequence, Dab1 becomes dimerized or oligomerized on the cytosolic side of the plasma membrane, constituting the active substrate for the kinase; this process seems to be sufficient to transmit the signal and does not appear to require any coreceptor.     

New melampolides and darutigenol from Sigesbeckia orientalis

Isolation and identification of darutigenol and two new melampolides from Sigesbeckia orientalis, in addition to the previously described orientalide and darutoside, are reported.     

Chromans from helianthella quinquenervis

Investigation of Helianthella quinquenervis afforded, in addition to several known 6-acetylchromenes, 3-prenyl-4-hydroxyphenone and the benzofuran encecalin, cis- and trans-2,2-dimethyl-3,4,7-trihydroxy-6- acetylchroman.