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Summary Marking replicating DNA with multiple labels presents the possibility of revealing new features and mechanisms of DNA synthesis and cell division; however, progression beyond double labeling has been hampered by cross-reactivity of label detection and scarcity of appropriate labels. Here, we present a method for triple S-phase labeling of the dividing cells, with a fourth label used to mark cells actively engaged in cell-cycle progression (e.g., using Ki67) or to phenotype the dividing cells or their progeny (e.g., using a GFP-expressing lineage reporter transgene). We apply this method to determine the parameters of neural stem cell division in the adult brain, to birth date up to four cohorts of dividing cells, and to reveal patterns of stem cell division in non-neural tissues., Highlights • Marking replicating DNA is now possible with three thymidine analogs • Triple S-phase labeling allows for multiple birth dating of dividing cells • Triple S-phase labeling increases resolution of cell-proliferation kinetic analysis • Triple S-phase labeling allows for combination of different labeling paradigms, In this article, Enikolopov and colleagues describe a method for triple S-phase labeling of stem cells, with an additional channel used to phenotype the cells or to add the fourth marker of cell division. They demonstrate the method's utility for birth dating multiple stem cell populations and for revealing patterns of stem cell division in the brain, testis, and intestine.

Macrophages are well characterized as immune cells. However, in recent years, a multitude of non-immune functions have emerged many of which play essential roles in a variety of developmental processes (Wynn, Chawla and J. W. Pollard, 2013; DeFalco et al., 2014). In adult animals, macrophages are derived from circulating monocytes originating in the bone marrow, but much of the tissue-resident population arise from erythro-myeloid progenitors (EMPs) in the extra-embryonic yolk sac, appearing around the same time as primitive erythroblasts (Schulz et al., 2012; Kierdorf et al., 2013; McGrath, Frame and Palis, 2015; Gomez Perdiguero et al., 2015; Mass et al., 2016). Of particular interest to our group, macrophages have been shown to act as pro-angiogenic regulators during development (Owen and Mohamadzadeh, 2013; DeFalco et al., 2014; Hsu et al., 2015), but there is still much to learn about these early cells. The goal of the present study was to isolate and expand progenitors of yolk-sacderived Embryonic Macrophages (EMs) in vitro to generate a new platform for mechanistic studies of EM differentiation. To accomplish this goal, we isolated pure (>98%) EGFP(+) populations by flow cytometry from embryonic day 9.5 (E9.5) Csf1r-EGFP(+/tg) mice, then evaluated the angiogenic potential of EMs relative to Bone Marrow-Derived Macrophages (BMDMs). We found that EMs expressed more pro-angiogenic and less pro-inflammatory macrophage markers than BMDMs. EMs also promoted more endothelial cell (EC) cord formation in vitro, as compared to BMDMs in a manner that required direct cell-to-cell contact. Importantly, EMs preferentially matured into microglia when co-cultured with mouse Neural Stem/Progenitor Cells (NSPCs). In conclusion, we have established a protocol to isolate and propagate EMs in vitro, have further defined specialized properties of yolk-sac-derived macrophages, and have identified EM-EC and EM-NSPC interactions as key inducers of EC tube formation and microglial cell maturation, respectively.

Production of new neurons from stem cells is important for cognitive function, and the reduction of neurogenesis in the aging brain may contribute to the accumulation of age-related cognitive deficits. Restriction of calorie intake and prolonged treatment with rapamycin have been shown to extend the lifespan of animals and delay the onset of the age-related decline in tissue and organ function. Using a reporter line in which neural stem and progenitor cells are marked by the expression of green fluorescent protein (GFP), we examined the effect of prolonged exposure to calorie restriction (CR) or rapamycin on hippocampal neural stem and progenitor cell proliferation in aging mice. We showed that CR increased the number of dividing cells in the dentate gyrus of female mice. The majority of these cells corresponded to nestin-GFP-expressing neural stem or progenitor cells; however, this increased proliferative activity of stem and progenitor cells did not result in a significant increase in the number of doublecortin-positive newborn neurons. Our results suggest that restricted calorie intake may increase the number of divisions that neural stem and progenitor cells undergo in the aging brain of females.

In the brain, communication between neural and non-neural cells is crucial for the proper functioning of the central nervous system. Microglia play an important role in the clearance of neural cellular corpses and debris, especially under pathological conditions. It remains, however, unclear how microglia sense the degenerating neurons at a distance in order to migrate to them. In the present study, we explored the interaction between neurons and microglia using an in vitro model of Parkinson's disease (PD). In primary mesencephalic neuronal cultures, 1-methyl-4-phenylpridinium (MPP(+)) induced the selective death of dopaminergic (DAergic) neurons in a dose- and time-dependent manner. Transmigration assay showed that the conditioned medium (CM) from mesencephalic cultures treated with MPP(+) was enough to trigger the attraction of microglia at an early as well as a late phase of neuronal damage. Microglia preferably reacted with the soluble parts separated by ultracentrifugation over the neural debris-containing pellets. This chemoattractive activity was significantly reduced by the removal of the lipidic components in CM, but not by the removal of proteins, DNA or RNA. These results suggest that as yet-unidentified lipid-like components released from dying DAergic neurons are likely to recruit microglia, and thus have a role in neuronal damage.

Summary Production of new neurons in the adult hippocampus decreases with age; this decline may underlie age-related cognitive impairment. Here we show that continuous depletion of the neural stem cell pool, as a consequence of their division, may contribute to the age-related decrease in hippocampal neurogenesis. Our results indicate that adult hippocampal stem cells, upon exiting their quiescent state, rapidly undergo a series of asymmetric divisions to produce dividing progeny destined to become neurons and subsequently convert into mature astrocytes. Thus, the decrease in the number of neural stem cells is a division-coupled process and is directly related to their production of new neurons. We present a scheme of the neurogenesis cascade in the adult hippocampus that includes a proposed "disposable stem cell" model and accounts for the disappearance of hippocampal neural stem cells, the appearance of new astrocytes, and the age-related decline in the production of new neurons.

The preimplantation development of mammalian embryo after fertilization encompasses a series of events including cleavage, compaction, and differentiation into blastocyst. These events are likely to be associated with substantial changes in embryonic gene expression. In the present study, we explored the expression patterns and function of epithin, a mouse type II transmembrane serine protease, during preimplantation embryo development. RT-PCR analysis showed that epithin mRNAs were detectable during the cleavage stages from a 1-cell zygote to the blastocyst. Immunocytochemical studies revealed that epithin protein was expressed at blastomere contacts of the compacted 8-cell and later embryonic stages. Epithin colocalized with E-cadherin at the membrane contacts of the compacted morula-stage embryo as revealed by double-staining immunocytochemistry and confocal microscopy, respectively. Post-transcriptional epithin gene silencing by RNA interference (RNAi) resulted in the blockade of 8-cell in vitro-stage embryo compaction and subsequent embryonic deaths after several rounds of cell division. These results strongly suggest that epithin plays an important role in the compaction processes that elicit the signal for the differentiation into trophectoderm and inner cell mass.

Chronic exposure to the pesticide rotenone induces a selective degeneration of nigrostriatal dopaminergic neurons and reproduces the features of Parkinson's disease in experimental animals. This action is thought to be relevant to its inhibition of the mitochondrial complex I, but the precise mechanism of this suppression in selective neuronal death is still elusive. Here we investigate the mechanism of dopaminergic neuronal death mediated by rotenone in primary rat mesencephalic neurons. Low concentrations of rotenone (5-10 nM) induce the selective death of dopaminergic neurons without significant toxic effects on other mesencephalic cells. This cell death was coincident with apoptotic events including capsase-3 activation, DNA fragmentation, and mitochondrial membrane depolarization. Pretreatment with coenzyme Q10, the electron transporter in the mitochondrial respiratory chain, remarkably reduced apoptosis as well as the mitochondrial depolarization induced by rotenone, but other free radical scavengers such as N-acetylcysteine, glutathione, and vitamin C did not. Furthermore, the selective neurotoxicity of rotenone was mimicked by the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), a cyanide analog that effectively collapses a mitochondrial membrane potential. These data suggest that mitochondrial depolarization may play a crucial role in rotenone-induced selective apoptosis in rat primary dopaminergic neurons.

Since estrogen exerts wide ranging effects within the central nervous system, it is important to investigate the sites and actions of this gonadal steroid hormone at extra-hypothalamic locations. In the present report, the effects of estrogen upon catecholaminergic function within the olfactory bulb were examined. To assess the role of estrogen at this site, ovariectomized mice received either no further hormonal treatment or were treated with estrogen, the anti-estrogen, tamoxifen, or a combination of estrogen and tamoxifen as administered in a 21-day release pellet. At 14 days post-hormonal treatment, the olfactory bulbs were assayed for mRNA levels of tyrosine hydroxylase, dopamine transporter and norepinephrine transporter using competitive-PCR. Tyrosine hydroxylase mRNA levels in either estrogen or estrogen+tamoxifen treated females were significantly decreased compared with non-hormonally treated controls. In addition, tyrosine hydroxylase mRNA levels of tamoxifen-treated mice were significantly greater than that of estrogen-treated mice. Dopamine transporter mRNA levels of tamoxifen-treated females were significantly greater than that of non-hormonally treated controls and estrogen treated mice. The combination of estrogen+tamoxifen significantly increased dopamine transporter mRNA levels compared to that of estrogen treated mice. No overall statistically significant differences in norepinephrine transporter mRNA levels were obtained among the four treatment groups. The data demonstrate that estrogen can exert significant modulatory effects upon olfactory bulb catecholaminergic function. Therefore, events which alter estrogen levels (menstrual/estrogen cycle, pregnancy/lactation, menopause, tamoxifen treatment) can modulate olfactory bulb catecholaminergic functions which may be involved with the detection and processing of olfactory stimuli.

Pancreatectomy (Px) is known to cause islet hypertrophy and is a putative method to mimic hyperglycemia representing type II diabetes mellitus. Therefore, finding new genes related to pancreatectomy will help to understand the molecular mechanism of hypertrophy and hyperglycemia, and may provide new diagnostic markers of type II diabetes. To this end, mRNA differential display was used to isolate genes that show transcriptional changes in pancreas of rat after 90% partial pancreatectomy. Forty-nine candidate pancreas regeneration-associated transcripts were isolated. cDNA sequencing and subsequent database analysis revealed that 15 transcripts showed no significant sequence similarity to previously reported genes, whereas 34 transcripts showed significant similarity with genes deposited in the GenBank. The differential mRNA expression of 49 transcripts was confirmed using screening of slot blots and Northern blot analysis was performed to several genes. It was noteworthy that the Wnt-1 inducible signaling pathway protein-1 (WISP-1), Ras-associated protein 1B (Rap1B), vascular cell adhesion molecule-1 (VCAM-1), and huntingtin interacting protein genes (HIP) were observed to be over-expressed during pancreas regeneration. Several genes' expression was modified by pancreatectomy. Profiling of gene expression in response to pancreatectomy may lead to new insights into hypertrophy and hyperglycemia representing type II diabetes, as well as into the identification of novel diagnostic markers of type II diabetes.

Following seizures, heat shock protein (HSP)-70 is induced in various brain regions. Since zinc that can induce HSP-70 in various cell systems is enriched in certain glutamatergic terminals and translocates to postsynaptic neurons with seizures, we examined the possibility that HSP-70 induction in the epileptic brain is mediated by synaptic zinc. Adult rats were injected intraperitoneally with kainate to induce seizures. Seizures were halted 3 h after the kainate administration by the injection of phenytoin. Staining of brain sections with zinc-specific fluorescent dye TFL at 24 h after the kainate injection revealed a one-to-one correlation between dense TFL fluorescence and acidophilic neuronal degeneration in the hippocampus. Subsequent staining with anti-HSP-70 antibody, however, revealed that more numerous neurons than degenerating neurons exhibited HSP-70 immunoreactivity. Most of the HSP-70(+) neurons were not stained with acid fuchsin but exhibited mild zinc fluorescence in the cytoplasm. Intraventricular injection of CaEDTA attenuated neuronal death as well as the HSP-70 induction in a dose-dependent manner. Supporting the specificity of zinc rather than calcium as the inducer of HSP-70 in neurons, exposure to zinc but not to a calcium ionophore or excitotoxins increased expression of HSP-70 mRNA and protein in cultured cortical neurons. The present results suggest that not only selective neuronal death, but also HSP-70 induction in neurons after seizures, is mediated by the translocation of endogenous synaptic zinc.

Degeneration of the midbrain dopaminergic neurons during Parkinson's disease (PD) may affect remote regions of the brain that are innervated by the projections of these neurons. The dentate gyrus (DG), a site of continuous production of new neurons in the adult hippocampus, receives dopaminergic inputs from the neurons of the substantia nigra (SN). Thus, depletion of the SN neurons during disease or in experimental settings may directly affect adult hippocampal neurogenesis. We show that experimental ablation of dopaminergic neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydopyridine (MPTP) mouse model of PD results in a transient increase in cell division in the subgranular zone (SGZ) of the DG. This increase is evident for the amplifying neural progenitors and for their postmitotic progeny; our results also indicate that MPTP treatment affects division of the normally quiescent stem cells in the SGZ. We also show that l-DOPA, used in the clinical treatment of PD, while attenuating the MPTP-induced death of dopaminergic neurons, does not alter the effect of MPTP on cell division in the DG. Our results suggest that a decrease in dopaminergic signaling in the hippocampus leads to a transient activation of stem and progenitor cells in the DG.

Human recombinant tissue plasminogen activator (tPA) may benefit ischemic stroke patients by dissolving clots. However, independent of thrombolysis, tPA may also have deleterious effects on neurons by promoting excitotoxicity. Zinc neurotoxicity has been shown to be an additional key mechanism in brain injuries. Hence, if tPA affects zinc neurotoxicity, this may provide additional insights into its effect on neuronal death. Independent of its proteolytic action, tPA markedly attenuated zinc-induced cell death in cortical culture, and, when injected into cerebrospinal fluid, also reduced kainate seizure–induced hippocampal neuronal death in adult rats.

There is a limitation to estimate vitamin B12 intake due to a lack of data on vitamin B12 content in many Korean foods. In this study, vitamin B12 content was determined in some seaweeds, fish, and shellfish and their product that are con - sumed in Korea using a modified microbioassay with L actobacillus delbruecki ATCC 7830. Dried laver and dried sea- soned and toasted laver contained very high levels of vitamin B12 (66.8 and 55.2-71.3 μg/100 g, respectively. Sea lettuce and seaweed fulvescene also contained high vitamin B12 content of 5.47-9.41 and 6.46-7.20 μg/100 g, respectively, where- as sea mustard and sea tangle contained low levels of vitamin B12; vitamin B12 was not detected in seaweed fusifome. Pa- cific saury, trout, sea-bass, or squid contained 12.01, 2.00, 0.49 and 2.33 μg vitamin B12/100 g, respectively. Ochellatus oc- topus, and naked sand lance contained 0.72-1.43 and 3.68 μg vitamin B12/100 g, respectively. Dried Alaska pollack con- tained 0.19-2.64 μg vitamin B12/100 g. Shellfish such as little neck clam and small ark shellfish contained high levels of vitamin B12 of 30.5-40.5 μg/100 g, and mussel and abalone contained 17.71 and 7.82 μg/100 g, respectively. Of unique Ko- rean traditional fermented seafood products, salt-fermented products of squid (2.91 μg/100 g), clams (34.31 μg/100 g), Ala- ska pollack roe (9.98-12.02 μg/100 g), hairtail guts (4.58 μg/100 g) or small shrimp (0.58-1.55 μg/100 g), and fish sauce from anchovies (1.52-1.78 μg/100 mL), sand eel (0.22-0.24 μg/100 mL) or small shrimp (0.19-0.78 μg/100 mL) were ana- lyzed. A few commercial brands of flying fish roe (0.73-1.73 μg/100 g), canned tuna (0.40 μg/100 g), and fried fish paste (0.25-0.69 μg /100 g) were also analyzed. In conclusion, vitamin B12 content in these foods, chosen considering the Ko- rean food culture, should contribute to improve the present vitamin B12 food database. It may be helpful to estimate vita- min B12 intake more correctly than before, and provide additional information for dietary education related to vitamin B12 and meal management. (Korean J Nutr 2012; 45(1): 94 ~ 102)

There is a limitation to estimate vitamin B12 intake due to the lack of data on vitamin B12 content of Korean commer- cial foods. In this study, vitamin B12 content was determined in favorite Korean restaurant foods, convenient or instant foods, fast foods and bakery products through a modified microbioassay using Lactobacillus delbrueckii ATCC 7830. Bulgogi and seafood & green pepper griddle had high vitamin B12 content, 3.50 and 2.96 μg/100 g, respectively. Pork suyook, pork griddle and pollack griddle had 0.48, 0.31 and 0.32 μg/100 g of vitamin B12, respectively. In stew, soft-tofu stew with seafood and doenjang stew with seafood had relatively high vitamin B12 content, 1.93 and 1.44 μg/100 g, re- spectively. Bibimbap and 4 different types of rice porridge, beef & mushroom, chicken & ginseng, seafood or abalone, had 0.36, 0.08, 0.09, 1.64 and 0.13 μg/100 g of vitamin B12, respectively. One serving of haejanggguk, yookejang, chuo- tang and galbitang had 5.97, 2.04, 2.63 and 1.91 μg of vitamin B12, respectively. One serving of samgetang and sulong- tang had 2.89 μg and 6.64 μg of vitamin B12. In noodles, one serving of cram noodle soup, bibim-nangmyeon, and mul- nangmyeon had 18.8, 1.21 and 0.38 μg of vitamin B12, respectively. One regular gimbap and one triangle gimbap contained 1.09-2.53 and 0.54-1.11 μg of vitamin B12, respectively. One cheese-burger, chicken-burger and bulgogi-burger had 0.76, 0.62 and 0.54 μg of vitamin B12, respectively. A plain bagel and a waffle contained 0.13 and 0.17 μg/100 g of vita- min B12, respectively. Ready-made tomato sauce or cream sauce for spaghetti in a retort pouch contained only a trace of vitamin B12. In conclusion, these results should contribute to improving the present food vitamin B12 content database, most of which were cited from foreign data, thereby it could be helpful to estimate the vitamin B12 intake of Koreans more accurately than before. It will also provide new information for dietary education related to vitamin B12 and health. (Korean J Nutr 2012; 45(6): 588 ~ 599)