GAP-43，Netrin-1，Collapsin-1和Neuropilin-1在出生后小鼠小脑中的动态表达

GAP-43,netrin-1,collapsin-1和neuropilin-1被认为在成网络分布的神经联系中发挥重要的作用．在年幼的啮齿类动物中,小脑包含5种不同的集中分布层：白质、内颗粒细胞层(IGL)、浦肯野氏细胞层(PCL)、分子层(ML)和外颗粒细胞层(EGL)．与浦肯野氏神经元在出生前产生这一点不同的是,EGL中的细胞在出生后产生,它们接受从前脑olivary核团发出的攀援纤维的主要神经投射,以及从内颗粒细胞发出的平行纤维的神经投射．这些神经投射主要在出生后的前3个星期内建立,同时还有浦肯野氏细胞的发育和成熟．而GAP-43,netrin-1,collapsin-1和neuropilin-1在出生后小脑发育的潜在作用仍然不清楚．为了更加清楚地探讨上述问题,检验了GAP-43,netrin-1,collapsin-1和neuropilin-1的mRNA与蛋白质在出生后5,10,20天和成年小鼠小脑中的表达情况．研究结果显示,这4种分子在小鼠出生后的小脑中有不同的时间和空间表达形式,这些结果与出生后发育和成年期间的轴突发生、延伸以及突触形成都有关联．通过免疫组织化学双标染色,发现小鼠出生后10天的小脑中,GAP-43阳性的浦肯野氏细胞也显示netrin-1或collapsin-1阳性,并且collapsin-1阳性的细胞也对 netrin-1 阳性．上述研究结果证明这4种分子可能参与了小脑的出生后发育．     

Simultaneous synthesis,translation, and storage of mRNA including histone mRNA in sea urchin eggs

The kinetics of accumulation of RNA labeled with uridine and the time course of change in the specific activity of the UTP pool were used to estimate the rate constants for synthesis and decay of RNA synthesized in unfertilized eggs of the sea urchin Lytechinus pictus. The rate of synthesis per haploid genome is similar to that in embryos. Most of the RNA is turning over with a half-life of about 5 hr, and an average of 11 pg of newly synthesized RNA accumulates at steady state. About 3.7% of the RNA in the polysomes of the egg is newly synthesized and this RNA has the heterogeneous size distribution expected for mRNA. Thus most, probably all, of the mRNA translated in the egg is also synthesized in the egg. Little, if any, of the RNA synthesized in the egg enters polysomes following fertilization. Thus the egg synthesizes a population of mRNA which is unstable and translated, but it also contains a more stable, untranslated population of previously synthesized, stored mRNA, which is translated only after fertilization. Since the two populations of mRNA code for the same abundant proteins (Brandhorst, B. P. (1976). Develop. Biol., 52, 310–317), there is a temporal separation in the metabolism and function of coexisting mRNA molecules of identical coding sequence. Among the mRNAs synthesized and translated in the egg are histone mRNAs having the same electrophoretic mobilities and rates of synthesis per genome as those synthesized in rapidly cleaving embryos. Thus the synthesis, entry into the cytoplasm, and translation of histone mRNA are not restricted to the S phase of the cell cycle or the period of cell division.     

Differences in the cytoplasmic distribution of newly synthesized poly (A) in serum-stimulated and resting cultures of BALB/c 3T3 cells.

Density-inhibited, serum-stimulated, and SV40 virus-transformed BALB/c 3T3 cultures were compared with respect to the rates of accumulation of cytoplasmic RNA molecules and with respect to the distribution of newly synthesized messenger RNA (mRNA) between polyribosomes and the post-ribosomal cell fraction. mRNA was isolated and quantitated by virtue of its association with radioactive polyadenylate (poly(A))-synthesized during a 90 min exposure of the cultures to ³H-adenosine. The rate of accumulation of cytoplasmic poly(A) rose slowly after serum stimulation and reached a value of 1.8 times that of resting cultures at 12 h after serum stimulation, which was also the time of onset of DNA synthesis. A change in the cytoplasmic distribution of newly synthesized poly(A) occurred more rapidly than the change in the rate of its synthesis, however. Resting cultures contained 37% of newly synthesized cytoplasmic poly(A)-containing RNA large enough to be mRNA in the post-ribosomal cell fraction, whereas virtually all of this material was found in polyribosomes at 3, 6 and 12 h after stimulation and in transformed cultures. The relatively infrequently translated mRNA of resting cultures was shown to be functional by cycloheximide treatment. (All BALB/c 3T3 cultures, resting or stimulated, contained about 20% of newly synthesized cytoplasmic poly(A) as nearly pure poly(A) in molecules of 4–6 Svedbergs in size, presumably too small to be mRNA.) We conclude that serum stimulation of density-inhibited cultures resulted in a more efficient use of the protein-synthesizing ability of the cell, and that the change in efficiency preceded increases in numbers of ribosomes and mRNA molecules.     

Deficits in cerebellar granule cell development and social interactions in CD47 knockout mice

CD47 is involved in neurite differentiation in cultured neurons, but the function of CD47 in brain development is largely unknown. We determined that CD47 mRNA was robustly expressed in the developing cerebellum, especially in granule cells. CD47 protein was mainly expressed in the inner layer of the external granule layer (EGL), molecular layer, and internal granule layer (IGL), where granule cells individually become postmitotic and migrate, leading to neurite fasciculation. At postnatal day 8 (P8), CD47 knockout mice exhibited an increased number of proliferating granule cells in the EGL, whereas the CD47 agonist peptide 4N1K increased the number of postmitotic cells in primary granule cells. Knocking out the CD47 gene and anti‐CD47 antibody impaired the radial migration of granule cells from the EGL to the IGL individually in mice and slice cultures. In primary granule cells, knocking out CD47 reduced the number of axonal collaterals and dendritic branches; by contrast, overexpressing CD47 or 4N1K treatment increased the axonal length and numbers of axonal collaterals and dendritic branches. Furthermore, the length of the fissure between Lobules VI and VII was decreased in CD47 knockout mice at P21 and at 14 wk after birth. Lastly, CD47 knockout mice exhibited increased social interaction at P21 and depressive‐like behaviors at 10 wk after birth. Our study revealed that the cell adhesion molecule CD47 participates in multiple phases of granule cell development, including proliferation, migration, and neurite differentiation implying that aberrations of CD47 are risk factors that cause abnormalities in cerebellar development and atypical behaviors.© 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 463–484, 2015     

PACSIN1, a Tau-interacting Protein,Regulates Axonal Elongation and Branching by Facilitating Microtubule Instability

Tau is a major member of the neuronal microtubule-associated proteins. It promotes tubulin assembly and stabilizes axonal microtubules. Previous studies have demonstrated that Tau forms cross-bridges between microtubules, with some particles located on cross-bridges, suggesting that some proteins interact with Tau and might be involved in regulating Tau-related microtubule dynamics. This study reports that PACSIN1 interacts with Tau in axon. PACSIN1 blockade results in impaired axonal elongation and a higher number of primary axonal branches in mouse dorsal root ganglia neurons, which is induced by increasing the binding ability of Tau to microtubules. In PACSIN1-blocked dorsal root ganglia neurons, a greater amount of Tau is inclined to accumulate in the central domain of growth cones, and it promotes the stability of the microtubule network. Taken together, these results suggest that PACSIN1 is an important Tau binding partner in regulating microtubule dynamics and forming axonal plasticity.     

Neuronal ribonucleoprotein granules: Dynamic sensors of localized signals

Membrane‐less organelles, because of their capacity to dynamically, selectively and reversibly concentrate molecules, are very well adapted for local information processing and rapid response to environmental fluctuations. These features are particularly important in the context of neuronal cells, where synapse‐specific activation, or localized extracellular cues, induce signaling events restricted to specialized axonal or dendritic subcompartments. Neuronal ribonucleoprotein (RNP) particles, or granules, are nonmembrane bound macromolecular condensates that concentrate specific sets of mRNAs and regulatory proteins, promoting their long‐distance transport to axons or dendrites. Neuronal RNP granules also have a dual function in regulating the translation of associated mRNAs: while preventing mRNA translation at rest, they fuel local protein synthesis upon activation. As revealed by recent work, rapid and reversible switches between these two functional modes are triggered by modifications of the networks of interactions underlying RNP granule assembly. Such flexible properties also come with a cost, as neuronal RNP granules are prone to transition into pathological aggregates in response to mutations, aging, or cellular stresses, further emphasizing the need to better understand the mechanistic principles governing their dynamic assembly and regulation in living systems.     

Cordycepin. An inhibitor of newly synthesized globin messenger RNA.

The effect of cordycepin (3'-deoxyadenosine) on newly synthesized globin mRNA in cultured mouse fetal liver erythroid cells is investigated. At cordycepin concentrations that do not inhibit amino acid incorporation into acid-precipitable material, the quantity of pulse-labeled (radioactive) globin mRNA nucleotide sequences is reduced by 90%, as compared to adenosine-treated controls. The reduction of radioactivity in globin-specific RNA sequences is greater than the inhibition of total RNA synthesis in experiments in which the labeling times range from 6 to 60 min. Control experiments demonstrate that cordycepin does not reduce the recovery of total cell RNA or steady state (unlabeled) globin mRNA. The hybridization assay used to detect radioactive globin mRNA sequences is independent of the cellular location or the number of 3'-terminal adenylate residues in the mRNA-containing molecules. These data thus indicate that cordycepin inhibits newly synthesized mRNA as effectively as it inhibits ribosomal and transfer RNA synthesis.     

An analysis of the rate of metallothionein mRNA poly(A)-shortening using RNA blot hybridization.

A progressive reduction in the size of rat metallothionein-1 mRNA following induction by copper chloride or dexamethasone was demonstrated on RNA blots, and was shown to be due to shortening of the poly(A)-tail. The rate of poly(A) removal was the same in rat liver and kidney following copper chloride induction, in rat liver following dexamethasone induction, and in mouse liver following copper chloride induction. In mouse liver metallothionein-1 and 2 mRNAs were shortened at the same rate. The reduction of the poly(A) tail was more rapid in the first 5 hours (approximately 20 nucleotides/h) but much slower (approximately 3 nucleotides/h) after the poly(A)-tail had been reduced to about 60 residues. Metallothionein mRNA molecules with poly(A) tail sizes less than 30-40 nucleotides were not observed. Exonuclease digestion of the poly(A)-tail is suggested, at least in the initial rapid phase. It is hypothesized that poly(A)-tails longer than 30 are required for mRNA stability and that much longer poly(A) tails may give newly synthesized mRNA molecules a competitive advantage in protein synthesis.     

Axotomy of developing rat spinal motoneurons: Cell survival,soma size,muscle recovery,and the influence of testosterone

During the period of synapse elimination, motoneurons are impaired in their ability to generate or regenerate axonal branches: following partial denervation of their target muscle, young motoneurons do not sprout to nearby denervated fibers and after axonal injury, they fail to reinnervate the muscle. In the rat levator ani (LA) muscle, which is innervated by motoneurons in the spinal nucleus of the bulbocavernosus (SNB), synapse elemination ends relatively late in development and can be regulated by testosterone. We took advantage of this system to determine if the end of synapse elimination and the development of regenerative capabilities by motoneurons share a common mechanism, or, alternatively, if these two events can be dissociated in time. Axotomy on or before postnatal day 14 (P14) caused the death of SNB motoneurons. By P21, toward the end of synapse elimination in the LA muscle, SNB motoneurons had developed the ability to survive axonal injury. Altering testosterone levels by castration on P7 followed by 4 weeks of either testosterone propionate or control injections did not change the ability of SNB motoneurons to survive axonal injury during development, although these same treatments alter the time course of synapse elimination in the LA muscle. Thus, we dissociated the inability of SNB motoneurons to recover from axonal injury from their developmental elimination of synaptic terminals. We also measured the effect of early axotomy on motoneuronal soma size and on target muscle weight. Axotomy on P14 caused a long-lasting decrease in the soma size of surviving SNB motoneurons, whereas motoneurons axotomized on P28 recovered their normal soma size. Axotomy on or before P7 caused severe atrophy of the target muscles, matching the extensive loss of motoneurons. However, target muscle recovery after axotomy on P14 was as good as recovery after axotomy at later ages, despite greater motoneuronal death after axotomy on P14. This result may reflect an increase in motor unit size, a decrease in polyneuronal innervation by SNB motoneurons that survive axotomy on P14, or a combination of the two. © 1995 John Wiley & Sons, Inc.     

Long-lived Messenger RNA and its Relationship to Protein Synthesis During Germination of Pea (Pisum sativum L.) Seeds

Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Results from in vitro and in vivo protein synthesis experimentsand from studies of polysome formation suggest that much ofthe long-lived mRNA present in the embryo axis does not directprotein synthesis. The increase in the rate of protein synthesisduring germination is thus dependent on recruitment of newlysynthesized mRNA molecules. Pea, Pisum sativum L., germination, mRNA, protein synthesis     

Spatiotemporal expression and functional implication of CXCL14 in the developing mice cerebellum

Cerebellar granule neurons migrate from the external granule cell layer (EGL) to the internal granule cell layer (IGL) during postnatal morphogenesis. This migration process through 4 different layers is a complex mechanism which is highly regulated by many secreted proteins. Although chemokines are well-known peptides that trigger cell migration, but with the exception of CXCL12, which is responsible for prenatal EGL formation, their functions have not been thoroughly studied in granule cell migration. In the present study, we examined cerebellar CXCL14 expression in neonatal and adult mice. CXCL14 mRNA was expressed at high levels in adult mouse cerebellum, but the protein was not detected. Nevertheless, Western blotting analysis revealed transient expression of CXCL14 in the cerebellum in early postnatal days (P1, P8), prior to the completion of granule cell migration. Looking at the distribution of CXCL14 by immunohistochemistry revealed a strong immune reactivity at the level of the Purkinje cell layer and molecular layer which was absent in the adult cerebellum. In functional assays, CXCL14 stimulated transwell migration of cultured granule cells and enhanced the spreading rate of neurons from EGL microexplants. Taken together, these results revealed the transient expression of CXCL14 by Purkinje cells in the developing cerebellum and demonstrate the ability of the chemokine to stimulate granule cell migration, suggesting that it must be involved in the postnatal maturation of the cerebellum.     

Intracellular transport of the major glycoprotein of zymogen granule membranes in the rat pancreas. Demonstration of high turnover at the plasma membrane

The intracellular transport and destination of the major glycoprotein associated with zymogen granule membranes in the pancreas (GP-2) was established. In suspensions of isolated acinar cells from rat pancreas, pulse-chase experiments were performed. The incorporation of the first newly synthesized GP-2 molecules into zymogen granule membranes occurred at about 60 min after beginning of the pulse. We demonstrated by using two different methods that newly made GP-2 reaches the cell surface within the same time span. After 6-8 h chase considerable more newly synthesized GP-2 has reached the cell surface than would be expected on account of secreted newly synthesized zymogens. These observations strongly suggest that at least part of the GP-2 molecules bypass the mature zymogen granule compartment on their way to the plasma membrane. GP-2 is the only protein that appears in discernable quantity in the plasma membrane during 1-4 h after a pulse label. Nevertheless GP-2 comprises only a small percentage of externally 125I-iodinated plasma membrane proteins. We conclude that GP-2 has a high turnover rate at the plasma membrane level. Treatment of the acinar cells with the N-glycosylation inhibitor tunicamycin does not block the intracellular transport of GP-2.     

De novo synthesis of uracil nucleotides in mouse liver and intestine studied using [15N]alanine

The amount of newly synthesized uracil nucleotides in mouse liver and intestine was determined by analysis of 15N incorporation into the uracil nucleotide pool of these tissues after intraperitoneal infusion of 15N-labelled amino acids. The appearance of newly synthesized uracil nucleotides was linear with time, and essentially independent of the rate of infusion of L-[15N]alanine. Varying the amino acid used in the infusion could affect the enrichment in the uracil ring nitrogens, but had no significant effect on the calculated amount of de novo synthesis. These results demonstrate the utility of this method in measuring de novo uracil nucleotide synthesis in mouse liver and intestine in vivo. The method should be a valuable tool in the effort to understand the regulation and pharmacological manipulation of de novo uracil nucleotide synthesis.