Stem cell engineering and grafting of mesencephalic dopamine (mesDA) neurons is a promising strategy for brain repair in Parkinson's disease (PD). Refinement of differentiation protocols to optimize this approach will require deeper understanding of mesDA neuron development. Here, we studied this process using transcriptome-wide single-cell RNA sequencing of mouse neural progenitors expressing the mesDA neuron determinant Lmx1a. This approach resolved the differentiation of mesDA and neighboring neuronal lineages and revealed a remarkably close relationship between developing mesDA and subthalamic nucleus (STN) neurons, while also highlighting a distinct transcription factor set that can distinguish between them. While previous hESC mesDA differentiation protocols have relied on markers that are shared between the two lineages, we found that application of these highlighted markers can help to refine current stem cell engineering protocols, increasing the proportion of appropriately patterned mesDA progenitors. Our results, therefore, have important implications for cell replacement therapy in PD.

Protease nexin-2/amyloid 6 protein precursor (PN-2/A#PP) is an abundant, secreted platelet protein which is a potent inhibitor of coagulation Factor XIa. We examined other potential anticoagulant activities of PN-2/Ai8PP. Purified Kunitz protease inhibitor domain of PN-2/A8PP and PN-2/ABPP itself were found to prolong the coagulation time of plasma and pure Factor IXa. The Kunitz protease inhibitor domain also inhibited the ability of Factor IXa to activate Factor X. PN-2/AAPP inhibited Factor IXa with a K1 of 7.9 to 3.9 X 10-1 M in the absence and presence of heparin, respectively. When the second-order rate constant of PN-2/AO8PP's inhibition of Factor IXa (2.7 X 108 M-'min-') was compared to that of antithrombin III (3.8 X 106 M-'min-'), PN-2/AfiPP was at least a 71-fold more potent inhibitor of Factor IXa than antithrombin III. PN-2/AfiPP formed a complex with Factor IXa as detected by gel filtration and ELISA. The finding that PN-2/ AfiPP is a potent inhibitor of Factor IXa could help to explain the spontaneous intracerebral hemorrhages seen in patients with hereditary cerebral hemorrhage with amyloidosis Dutchtype where there is an extensive accumulation of PN-2 /A46PP in their cerebral blood vessels. (J. Clin. Invest. 1993.92:2540-2545

Looking for novel fission yeast genes that cause cell cycle arrest when overexpressed, Woollard et al. (1) cloned spd1 (for S-phase delayed). The encoded 14-kDa protein is cell cycle-regulated, and the levels decline during S-phase. Overexpression of spd1 inhibits G 1 /S progression downstream of Start, but it is not an essential gene. In a later publication, Borgne and Nurse (2) demonstrated that overexpression of Spd1p blocks the onset of both S-phase and mitosis, and they suggested that the protein might act by interfering directly with DNA replication giving the same phenotype as that seen using the ribonucleotide reductase (RNR) 2 inhibitor hydroxyurea.Null mutants of the fission yeast csn1-d and csn2-d signalosome subunits are slow growing and have a prolonged S-phase (3). In a screen for multicopy suppressors of rad3-ts csn1-d lethality to identify the cause of the slow S-phase in signalosome mutants, Liu et al. (4) identified multiple independent clones of suc22 that rescued the growth defect. Suc22 encodes the small subunit of Schizosaccharomyces pombe RNR that together with the large subunit Cdc22p forms the active enzyme complex (5). Because the phenotype of spd1 overexpression resembled the phenotype of csn1-d or csn2-d mutants, Liu et al. combined the deletion of spd1 with either of the signalosome subunit mutants, and in both cases, loss of spd1 restored the normal cell cycle profile. They concluded from these experiments that Spd1p inhibits RNR activity and that the signalosome complex is required to release the inhibition during DNA replication or repair.Using indirect immunofluorescence microscopy, Liu et al. observed nuclear co-localization of Suc22p and Spd1p. Although no direct evidence was presented for interaction between the proteins, such an interaction was assumed in a model for the regulation of fission yeast RNR activity during S-phase and DNA repair. According to this model, Spd1p anchors the Suc22p inside the nucleus, thereby inhibiting RNR activity. To increase the supply of deoxyribonucleotides during DNA replication or repair, Spd1p is destroyed by the signalosome; this leads to a delocalization of the Suc22p from the nucleus to the cytoplasm where it combines with the Cdc22p to form active RNR. This model is cited in a number of publications and reviews (6 -9).In this report, we describe the expression and purification of the Cdc22p, Suc22p, and Spd1p and demonstrate by in vitro assays with highly purified recombinant proteins that Spd1p is a strong inhibitor of fission yeast RNR acting on Cdc22p. Furthermore, biosensor technique using chips with immobilized Spd1p shows that the inhibitor specifically binds to Cdc22p with a dissociation constant of 2.4 M, whereas the affinity to Suc22p is negligible. Our data support a model where Spd1 regulates RNR activity by binding to the Cdc22p and not to the Suc22p. This mode of regulation is similar to the regulation of RNR activity in budding yeast, where binding of the low molecular weight inhibitor Sml1 to the Cdc22p homologue, the Rn...

Progenitor cells committed to eye development become specified in the prospective forebrain and develop subsequently into the optic vesicle and the optic cup. The optic vesicle induces formation of the lens placode in surface ectoderm from which the lens develops. Numerous transcription factors are involved in this process, including the eye-field transcription factors. However, many of these transcription factors also regulate the patterning of the anterior neural plate and their specific role in eye development is difficult to discern since eye-committed progenitor cells are poorly defined. By using a specific part of the Lhx2 promoter to regulate Cre recombinase expression in transgenic mice we have been able to define a distinct progenitor cell population in the forebrain solely committed to eye development. Conditional inactivation of Lhx2 in these progenitor cells causes an arrest in eye development at the stage when the optic vesicle induces lens placode formation in the surface ectoderm. The eye-committed progenitor cell population is present in the Lhx2−/− embryonic forebrain suggesting that commitment to eye development is Lhx2-independent. However, re-expression of Lhx2 in Lhx2−/− progenitor cells only promotes development of retinal pigment epithelium cells, indicating that Lhx2 promotes the acquisition of the oligopotent fate of these progenitor cells. This approach also allowed us to identify genes that distinguish Lhx2 function in eye development from that in the forebrain. Thus, we have defined a distinct progenitor cell population in the forebrain committed to eye development and identified genes linked to Lhx2's function in the expansion and patterning of these progenitor cells.

Protease nexin-2/amyloid beta-protein precursor (PN-2/A beta PP) is a Kunitz-type protease inhibitor which has been shown to be a tight-binding inhibitor of enzymes, factors XIa and IXa (FIXa), suggesting a role for this protein in hemostasis. Since coagulant reactions are modulated on biologic surfaces, we investigated how 25:75 (mol/mol) phosphatidylserine/phosphatidylcholine vesicles (PSPC), thrombin-activated platelets, or umbilical vein endothelial cells influence inactivation of FIXa by PN-2/A beta PP. The Km of human or porcine FIXa activation of human factor X in the presence of PSPC, activated platelets, or endothelial cells in the absence or presence of thrombin-activated factor VIII (FVIIIa) was similar, (0.05-0.39 microM). The presence of FVIIIa increased the catalytic efficiency (kcat/Km ratio) of human and porcine factor IXa's activation of factor X 4952-406-fold, respectively. In the presence of PSPC, the Ki of human and porcine FIXa inhibition by PN-2/A beta PP was Ki = 1.9 x 10(-9) M and 5.8 x 10(-9) M, respectively. After the addition of FVIIIa to the reaction, the Ki for both human and porcine FIXa inhibition by PN-2/A beta PP on PSPC increased 13- and 4-fold to Ki = 2.5 x 10(-8) M and 2.4 x 10(-8) M, respectively. These Ki for inhibition of human FIXa on phospholipid vesicles by PN-2/A beta PP were similar when factor X activation was measured by chromogenic or activation peptide release assays. FVIIIa reduced the inhibition of FIXa by PN-2/A beta PP only in the presence of PSPC.(ABSTRACT TRUNCATED AT 250 WORDS)