Most cortical interneurons are generated in the subpallial ganglionic eminences and migrate tangentially to their final destinations in the neocortex. Within the cortex, interneurons follow mainly stereotype routes in the subventricular zone/intermediate zone (SVZ/IZ) and in the marginal zone. It has been suggested that interactions between invading interneurons and locally generated projection neurons are implicated in the temporal and spatial regulation of the invasion process. However, so far experimental evidence for such interactions is lacking.We show here that the chemokine stromal-derived factor 1 (SDF-1; CXCL12) is expressed in the main invasion route for cortical interneurons in the SVZ/IZ. Most SDF-1-positive cells are proliferating and express the homeodomain transcription factors Cux1 and Cux2. Using MASH-1 mutant mice in concert with the interneuron marker DLX, we exclude that interneurons themselves produce the chemokine in an autocrine manner. We conclude that the SDF-1-expressing cell population represents the precursors of projection neurons during their transition and amplification in the SVZ/IZ. Using mice lacking the SDF-1 receptor CXCR4 or Pax6, we demonstrate that SDF-1 expression in the cortical SVZ/IZ is essential for recognition of this pathway by interneurons. These results represent the first evidence for a molecular interaction between precursors of projection neurons and invading interneurons during corticogenesis.

The N-terminal sequence myr-Gly-Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes Ca, Cp, and Cy. By capillary LC-MS and tandem MS, we show that, in approximately one third of the C a and C p enzyme populations from cattle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to Asp 2. This deamidation accounts for the major isoelectric variants of the cAPK C-subunits formerly called CA and Cg. Deamidation also includes characteristic isoaspartate isomeric peptides from C a and Cp. Asn 2 deamidation does not occur during C-subunit preparation and is absent in recombinant myristoylated Ca (rCa) from Escherichia coli. Deamidation appears to be the exclusive pathway for introduction of an acidic residue adjacent to the myristoylated N-terminal glycine, verified by the myristoylation negative phenotype of an rCa(Asn 2 Asp) mutant. This is the first report thus far of a naturally occumng myr-Gly-Asp sequence. Asp 2 seems to be required for the well-characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a conserved site for deamidation in vivo. Comparable myr-Gly-Asn sequences are found in several signaling proteins. This may be especially significant in view of the recent knowledge that negative charges close to myristic acid in some proteins contribute to regulating their cellular localization.

Nociceptin (also called orphanin FQ) is an endogenous heptadecapeptide that activates the opioid receptor-like 1 (ORL 1 ) receptor. Nociceptin system not only affects the nociception and locomotor activity, but also regulates learning and memory in rodents. We have previously reported that long-term potentiation and memory of ORL 1 receptor knockout mice are enhanced compared with those in wild-type mice. Here, we show the neuronal mechanism of nociceptin-induced modulation of learning and memory. Retention of fear-conditioned contextual memory was significantly enhanced in the ORL 1 receptor knockout mice without any changes in cued conditioned freezing. Inversely, in the wild-type mice retention of contextual, but not cued, conditioning freezing behavior was suppressed by exogenous nociceptin when it was administered into the cerebroventricle immediately after the training. ORL 1 receptor knockout mice exhibited a hyperfunction of N-methyl-D-aspartate (NMDA) receptor, as evidenced by an increase in [ 3 H]MK-801 binding, NMDA-evoked 45 Ca 2 þ uptake and activation of Ca 2 þ /calmodulin-dependent protein kinase II (CaMKII) activity and its phosphorylation as compared with those in wild-type mice. The NMDA-induced CaMKII activation in the hippocampal slices of wild-type mice was significantly inhibited by exogenous nociceptin via a pertussis toxin-sensitive pathway. However, the a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor GluR1 subunit at Ser 831 and Ser 845, and NMDA receptor subunit NR2B at Thr 286 were phosphorylated similarly after NMDA receptor stimulation in both type of mice. The expressions of GluR1 and GluR2 also did not change, but the levels of polysialylated form of neuronal cell adhesion molecule (N-CAM) were reduced in the ORL 1 receptor knockout as compared with wild-type mice. These results suggest that nociceptin system negatively modulates learning and memory through the regulation of NMDA receptor function and the expression of N-CAM. Molecular Psychiatry (2003Psychiatry ( ) 8, 752-765. doi:10.1038 Keywords: nociceptin/orphanin FQ; ORL 1 receptor; Knockout mice; hippocampus; NMDA Opioid receptors are negatively coupled to adenylate cyclase through Gi/o proteins and mediate the inhibition of forskolin-induced cAMP accumulation by morphine and endogenous opioid peptides. Opioid receptor agonists have receptor subtypespecific actions on not only the nociceptive thresholds but also synaptic transmission and long-term potentiation (LTP) in the hippocampus. Thus, opioid system modulates certain forms of learning and memory.

The catalytic (C) subunit of protein kinase A functions both in the cytoplasm and the nucleus. A major charge variant representing about one third of the enzyme in striated muscle results from deamidation in vivo of the Asn2 residue at the conserved NH2-terminal sequence myrGly-Asn-Ala (Jedrzejewski, P.T., A. Girod, A. Tholey, N. König, S. Thullner, V. Kinzel, and D. Bossemeyer. 1998. Protein Sci. 7:457–469). Because of the increase of electronegativity by generation of Asp2, it is reminiscent of a myristoyl-electrostatic switch. To compare the intracellular distribution of the enzymes, both forms of porcine or bovine heart enzyme were microinjected into the cytoplasm of mouse NIH 3T3 cells after conjugation with fluorescein, rhodamine, or in unlabeled form. The nuclear/cytoplasmic fluorescence ratio (N/C) was analyzed in the presence of cAMP (in the case of unlabeled enzyme by antibodies). Under all circumstances, the N/C ratio obtained with the encoded Asn2 form was significantly higher than that with the deamidated, Asp2 form; i.e., the Asn2 form reached a larger nuclear concentration than the Asp2 form. Comparable data were obtained with a human cell line. The differential intracellular distribution of both enzyme forms is also reflected by functional data. It correlates with the degree of phosphorylation of the key serine in CREB family transcription factors in the nucleus. Microinjection of myristoylated recombinant bovine Cα and the Asn2 deletion mutant of it yielded N/C ratios in the same range as encoded native enzymes. Thus, Asn2 seems to serve as a potential site for modulating electronegativity. The data indicate that the NH2-terminal domain of the PKA C-subunit contributes to the intracellular distribution of free enzyme, which can be altered by site-specific in vivo deamidation. The model character for other signaling proteins starting with myrGly-Asn is discussed.

The crystal structure of a complex of the catalytic subunit (type alpha) of cAMP-dependent protein kinase (PKA C alpha) with ARC-type inhibitor (ARC-1034), the presumed lead scaffold of previously reported adenosine-oligo-arginine conjugate-based (ARC-type) inhibitors, was solved. Structural elements important for interaction with the kinase were established with specifically modified derivatives of the lead compound. On the basis of this knowledge, a new generation of inhibitors, conjugates of adenosine-4'-dehydroxymethyl-4'-carboxylic acid moiety and oligo(D-arginine), was developed with inhibitory constants well into the subnanomolar range. The structural determinants of selectivity of the new compounds were established in assays with ROCK-II and PKBgamma.

The sensory organs of the embryonic lateral line system are deposited by migrating primordia that originate in the otic region. Here, we examine the pattern of cell proliferation in the posterior lateral line system. We conclude that three phases of cell proliferation are involved in the generation of this system, separated by two phases of mitotic quiescence. The first phase corresponds to generalized proliferation

As the key mediators of eukaryotic signal transduction, the protein kinases often cause disease, and in particular cancer, when disregulated. Appropriately selective protein kinase inhibitors are sought after as research tools and as therapeutic drugs; several have already proven valuable in clinical use. The AGC subfamily protein kinase C (PKC) was identified early as a cause of cancer, leading to the discovery of a variety of PKC inhibitors. Despite its importance and early discovery, no crystal structure for PKC has yet been reported. Therefore, we have co-crystallized PKC inhibitor bisindolyl maleimide 2 (BIM2) with PKA variants to study its binding interactions.

Rhombencephala from rat embryos were processed as whole-mounts for immunocytochemical detection of monoaminergic cell populations, using antibodies to tyrosine hydroxylase (TH) and serotonin (5-HT). Specific advantages of the whole-mount technique over the classical serial-section method were that even isolated immunoreactive (IR) cells could be detected easily, and three-dimensional relationships could be ascertained without the need for serial reconstruction. Embryos between embryonic days (E) 12 and 16 (the day following nocturnal mating being considered as E1) were used in this study. Both TH and 5-HT immunoreactivities were already detectable at E12, even in the smallest embryos (crown-rump length: 6 mm), but there was a striking difference in the number and regional distribution of these two types of IR cells. TH was expressed in several cell groups located in the rostral rhombencephalon (the presumed anlage of the A4-7 complex) as well as in the caudal rhombencephalon (the presumed anlagen of groups A1-2 and C1-3), whereas 5-HT was expressed in very few cells located near the rostral border of the rhombencephalon (presumed anlage of the B4-9 complex). Although the three-dimensional distribution of the TH-IR cell groups underwent some modifications during the period studied, its general pattern remained relatively stable after E12. This contrasted with the sequential appearance of the 5-HT-IR cell groups and their spatial transformations during this period. Using the rhombencephalic isthmus as a landmark, we found that conspicuous 5-HT-IR fibre bundles penetrated into the mesencephalon from E13 onwards, but that the 5-HT IR cell bodies were exclusively located caudal to the borderline between the mesencephalon and the rhombencephalon (the rhombencephalic isthmus). We therefore suggest the term "rostral rhombencephalic raphe nuclei" for the rostral 5-HT cell groups instead of "mesencephalic raphe nuclei," which is a misnomer. Close spatial association between TH and 5-HT-IR elements was observed mainly in the caudal rhombencephalon, where 5-HT-IR fibres coursed through an area containing numerous TH-IR cell bodies (the presumed anlagen of groups A1-2 and C1-3).