Multifaceted interactions mediated by intrinsically disordered regions play key roles in alpha synuclein aggregation

The aggregation of Alpha Synuclein (α-Syn) into fibrils is associated with the pathology of several neurodegenerative diseases. Pathologic aggregates of α-Syn adopt multiple fibril topologies and are known to be transferred between cells via templated seeding. Monomeric α-Syn is an intrinsically disordered protein (IDP) with amphiphilic N-terminal, hydrophobic-central, and negatively charged C-terminal domains. Here, we review recent work elucidating the mechanism of α-Syn aggregation and identify the key and multifaceted roles played by the N- and C-terminal domains in the initiation and growth of aggregates as well as in the templated seeding involved in cell-to-cell propagation. The charge content of the C-terminal domain, which is sensitive to environmental conditions like organelle pH, is a key regulator of intermolecular interactions involved in fibril growth and templated propagation. An appreciation of the complex and multifaceted roles played by the intrinsically disordered terminal domains suggests novel opportunities for the development of potent inhibitors against synucleinopathies.     

Male germ cell specification and differentiation

Understanding the mechanisms by which the germline is induced and maintained should lead to a broader understanding of the means by which pluripotency is acquired and maintained. In this review, two major aspects of male germ cell development are discussed: underlying mechanisms for induction and maintenance of primordial germ cells and the basic signaling pathways that determine spermatogonial cell fate.     

PR结构域蛋白质与原始生殖细胞特化

原始生殖细胞特化在精子和卵子生成过程中发挥着重要的作用,而PR结构域蛋白质(PR-domain protein,PRDM)家族部分成员参与了该过程。PRDM1可抑制体细胞程序化过程中基因的表达,而PRDM1和PRDM14共同参与了潜在的全能性细胞的重新获取和基因组范围内表观遗传学重编程。这三个过程都是原始生殖细胞特化所必需的。此外,原始生殖细胞特化还需要一些其他因素如骨形态发生蛋白4(bone morphogenetic protein4,Bmp4)和RNA结合蛋白Lin28,这些因素通过影响PRDM发挥生理作用。对原始生殖细胞特化的理解有利于生殖细胞发育和相关问题的研究。     

The roles of FGF signaling in germ cell migration in the mouse

Fibroblast growth factor (FGF) signaling is thought to play a role in germ cell behavior. FGF2 has been reported to be a mitogen for primordial germ cells in vitro, whilst combinations of FGF2, steel factor and LIF cause cultured germ cells to transform into permanent lines of pluripotent cells resembling ES cells. However, the actual function of FGF signaling on the migrating germ cells in vivo is unknown. We show, by RT-PCR analysis of cDNA from purified E10.5 germ cells, that germ cells express two FGF receptors: Fgfr1-IIIc and Fgfr2-IIIb. Second, we show that FGF-mediated activation of the MAP kinase pathway occurs in germ cells during their migration, and thus they are potentially direct targets of FGF signaling. Third, we use cultured embryo slices in simple gain-of-function experiments, using FGF ligands, to show that FGF2, a ligand for FGFR1-IIIc, affects motility, whereas FGF7, a ligand for FGFR2-IIIb, affects germ cell numbers. Loss of function, using a specific inhibitor of FGF signaling, causes increased apoptosis and inhibition of cell shape change in the migrating germ cells. Lastly, we confirm in vivo the effects seen in slice cultures in vitro, by examining germ cell positions and numbers in embryos carrying a loss-of-function allele of FGFR2-IIIb. In FGFR2-IIIb(-/-) embryos, germ cell migration is unaffected, but the numbers of germ cells are significantly reduced. These data show that a major role of FGF signaling through FGFR2-IIIb is to control germ cell numbers. The data do not discriminate between direct and indirect effects of FGF signaling on germ cells, and both may be involved.     

IFITM/Mil/fragilis family proteins IFITM1 and IFITM3 play distinct roles in mouse primordial germ cell homing and repulsion

The family of interferon-induced transmembrane protein (Ifitm/mil/fragilis) genes encodes cell surface proteins that may modulate cell adhesion and influence cell differentiation. Mouse Ifitm1 and -3, which are expressed in primordial germ cells (PGCs), are implicated to have roles in germ cell development, but the specific functions have been unclear. Our results show that Ifitm1 activity is required for PGC transit from the mesoderm into the endoderm, and that it appears to act via a repulsive mechanism, such that PGCs avoid Ifitm1-expressing tissues. In contrast, Ifitm3, which is expressed in migratory PGCs, is sufficient to confer autonomous PGC-like homing properties to somatic cells. These guidance activities are mediated by the N-terminal extracellular domain of the specific IFITM, which cannot be substituted by that of another family member. Complex homo- and/or heterotypic intercellular interactions among various IFITMs in PGCs and neighboring cells may underpin coordinated germ cell guidance in mice.     

The roles of ERAS during cell lineage specification of mouse early embryonic development

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β-catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development.     

Local neurons play key roles in the mammalian olfactory bulb

Over the past few decades, research exploring how the brain perceives, discriminates, and recognizes odorant molecules has received a growing interest. Today, olfaction is no longer considered a matter of poetry. Chemical senses entered the biological era when an increasing number of scientists started to elucidate the early stages of the olfactory pathway. A combination of genetic, biochemical, cellular, electrophysiological and behavioral methods has provided a picture of how odor information is processed in the olfactory system as it moves from the periphery to higher areas of the brain. Our group is exploring the physiology of the main olfactory bulb, the first processing relay in the mammalian brain. From different electrophysiological approaches, we are attempting to understand the cellular rules that contribute to the synaptic transmission and plasticity at this central relay. How olfactory sensory inputs, originating from the olfactory epithelium located in the nasal cavity, are encoded in the main olfactory bulb remains a crucial question for understanding odor processing. More importantly, the persistence of a high level of neurogenesis continuously supplying the adult olfactory bulb with newborn local neurons provides an attractive model to investigate how basic olfactory functions are maintained when a large proportion of local neurons are continuously renewed. For this purpose, we summarize the current ideas concerning the molecular mechanisms and organizational strategies used by the olfactory system to encode and process information in the main olfactory bulb. We discuss the degree of sensitivity of the bulbar neuronal network activity to the persistence of this high level of neurogenesis that is modulated by sensory experience. Finally, it is worth mentioning that analyzing the molecular mechanisms and organizational strategies used by the olfactory system to transduce, encode, and process odorant information in the olfactory bulb should aid in understanding the general neural mechanisms involved in both sensory perception and memory. Due to space constraints, this review focuses exclusively on the olfactory systems of vertebrates and primarily those of mammals.     

Tooth germ epithelial-mesenchymal interactions in tissue culture

The usefulness of the tooth germ in culture arises from the fact that it exemplifies those fundamental attributes of development, cell proliferation, cytodifferentiation, and morphogenesis, which we expect to find in the development of any metazoan organism. In culture, as in the organism, such development takes place in 3 dimensions. This study was undertaken to determine if it is possible to uncouple, by using 2 dimensions, cytodifferentiation from morphogenesis. Under the conditions used, cytodifferentiation in culture was not apparent (at the light microscope level). However, the following interesting observations were made: Cell populations arising from the same types of explants (enamel organ/enamel organ or dental papilla/dental papilla) readily flow together. Cell populations arising from dissimilar types of explants (enamel organ/dental papilla) form sharp boundaries at their interfaces. Additionally, cell populations arising from intact tooth germs differ from those arising from either enamel organs or dental papillae.     

Somatic and germ cell interactions during histogenetic aggregation of mouse fetal testes.

In the present study we examined the capacity of somatic and germ cells dissociated from fetal mouse testes at various stages to reform seminiferous cords in culture. We found that after 12 h in culture, seminiferous cords became segregated from stromal cells. Although Sertoli cells were incorporated into seminiferous cords at all stages studied, the germ cells dramatically changed their histogenetic behavior with age. Most germ cells which had been dissociated at 12.5 days postcoitum (dpc) were incorporated into the seminiferous cords, whereas at 14.5 dpc or later the majority remained among the stromal cells or as clusters on the surface of the aggregates. We considered three possible causes for this change in behavior of germ cells: (i) Failure to deposit some extracellular matrix components in the aggregates. (ii) Decrease in adhesiveness of prospermatogonia to either extracellular matrix components or Sertoli cells. (iii) A change in adhesiveness of Sertoli cells to germ cells with age. We found that laminin and fibronectin were similarly deposited in aggregates at 12.5 and 15.5 dpc. When prospermatogonia at 15.5 dpc labeled with colloidal gold were reaggregated with somatic cells at 12.5 dpc, 50% were incorporated into seminiferous cords. Moreover, [3H]thymidine-labeled Sertoli cells at 15.5 dpc formed heterochronic seminiferous cords with Sertoli cells at 12.5 dpc. These results suggest that mouse Sertoli cells change their surface property which is essential for binding to germ cells when they enter the mitotic resting stage (T-prospermatogonia).     

In vitro methods of male germ cell specification and differentiation

Germ line specification is an early cell fate decision essential for the transmission of totipotency over generations. Two types of germ line stem cells populate the male gonads in mammals. Primordial germ cells (PGCs) are the germ line founders only present during prenatal life. Spermatogonial stem cells (SSCs) appear a few days after birth and divide asymmetrically to give rise to one stem cell and one spermatogonia that initiates differentiation to produce spermatozoa. Germ cell specification and differentiation involve specific environmental stimuli and a sequential order of maturing phases required for gamete function. Spatio-temporal controls similarly dictate the erasure of somatic methylation marks and the subsequent acquisition of sex-specific marks at imprinted genes in gametes. We review here the recent advancements in male germ cell derivation from ES cells and discuss the limits of these in vitro methods in providing a kinetics and a microenvironment suitable for the programming of a proper gametic and parental identity.     

Role of cell interactions in ascidian muscle and pigment cell specification

Summary Muscle and brain pigment cell specification was studied by disrupting cell adhesion, cell dissociation, and reaggregation in embryos of the ascidianStyela clava. Treatment of embryos with Ca²⁺-free sea water between the 2-cell and gastrula stages disrupted blastomere adhesion but did not prevent acetylcholinesterase or muscle actin expression in presumptive muscle cells. Similar treatments initiated between the 2- and 32-cell stages caused more ectoderm cells to express tyrosinase and develop pigment granules than expected from the cell lineage. Whereas 2 pigment cells become the otolith and ocellus sensory organs in normal embryos, up to 33 pigment cells could differentiate in embryos after disruption of cell adhesion. Replacement of Ca²⁺-free sea water with normal sea water restored cell adhesion and usually resulted in development of embryos containing the conventional number of pigment cells. Dissociation of embryos into single cells between the 2- and 64-cell stages and culture of these cells beyond the fate restricted stage had no effect on the accumulation of muscle actin mRNA and muscle actin synthesis, but blocked pigment cell differentiation. Reaggregation of the dissociated cells did not enhance the number of cells that developed muscle features, but rescued pigment cell development. The results indicate that ascidian muscle cell specification occurs by an autonomous mechanism, whereas pigment cell specification occurs by a conditional mechanism involving cell interactions. In addition, the results suggest that negative cell interactions may restrict the potential for pigment cell development in the ectoderm of cleaving ascidian embryos.     

Akt and its downstream targets play key roles in mediating dormancy in land snails

Estivation, a state of aerobic dormancy, facilitates survival during adverse environmental conditions and is characterized at the molecular level by regulatory protein phosphorylation. The Akt (protein kinase B) signaling pathway regulates diverse responses in cells and the present study analyzes its role in the estivating desert snail Otala lactea. Kinetic analysis (maximal velocity, substrate affinities) determined that Akt was activated in tissues of estivating snails and Western blotting and in vitro incubations promoting changes to Akt phosphorylation state both confirmed that higher amounts of active (phosphorylated Ser473) Akt were present during estivation. Akt protein stability was also enhanced during estivation as assessed from urea denaturation studies. Multiple downstream targets of Akt were differentially regulated during estivation. Estivating animals showed elevated levels of phosphorylated FOXO3a (Ser253) and BAD (Ser136), no change in mTOR (Ser2481 and Ser2448), and reduced amounts of phosphorylated glycogen synthase kinase-3 (GSK-3) beta subunit (Ser9). Kinetic analysis of GSK-3 showed 1.5-1.7 fold higher activities in estivating snails coupled with increased GSK-3 substrate affinities in hepatopancreas. The data suggest an active role for Akt signaling during estivation emphasizing anti-apoptotic actions but uncoupling growth/proliferation actions to help achieve life extension on a limited energy budget.     

Lrp5 and Lrp6 play compensatory roles in mouse intestinal development

Low-density lipoprotein receptor-related proteins 5 and 6 (Lrp5 and Lrp6) are co-receptors of Wnt ligands and play important roles in Wnt/β-catenin signal transduction. Mice homozygous for a germline deletion of Lrp6 die at birth with several associated defects, while Lrp5-deficient mice are viable. Here, we conditionally deleted Lrp5 and/or Lrp6 in the mouse gut ((gut-/-)) by crossing mice carrying floxed alleles of Lrp5 and Lrp6 to a strain expressing Cre recombinase from the villin promoter (villin-Cre). The changes in morphology, differentiation, and Wnt signal transduction were validated using immunohistochemistry and other staining. Consistent with observations in mice carrying a homozygous germline deletion in Lrp5, intestinal development in Lrp5(gut-/-) mice was normal. In addition, mice homozygous for villin-Cre-induced deletion of Lrp6 (Lrp6(gut-/-)) were viable with apparently normal intestinal differentiation and function. However, mice homozygous for villin-Cre inactivated alleles of both genes (Lrp5(gut-/-) ; Lrp6(gut-/-)) died within 1 day of birth. Analysis of embryonic Lrp5(gut-/-); Lrp6(gut-/-) intestinal epithelium showed a progressive loss of cells, an absence of proliferation, and a premature differentiation of crypt stem/precursor cells; no notable change in differentiation was observed in the embryos lacking either gene alone. Further immunohistochemical studies showed that expression of the Wnt/β-catenin target, cyclin D1, was specifically reduced in the intestinal epithelium of Lrp5(gut-/-); Lrp6(gut-/-) embryos. Our data demonstrate that Lrp5 and Lrp6 play redundant roles in intestinal epithelium development, and that Lrp5/6 might regulate intestinal stem/precursor cell maintenance by regulating Wnt/β-catenin signaling.     

Stage-specific expression of the mitochondrial germ cell epitope PG2 during postnatal differentiation of rabbit germ cells

Structural and biochemical differentiation of germ cell mitochondria is supposed to determine the fate and integrity of mitochondria in the early embryo. Immunofluorescent labeling of the primordial germ cell epitope 2 (PG2), which is associated with the outer mitochondrial membrane and is germ cell specific from the time of germ cell segregation during gastrulation, was used to elucidate biochemical characteristics of mitochondrial differentiation leading to a functional gamete. The PG2 epitope is found in both mitotic and meiotic male and female postnatal germ cells, but PG2 expression ceases transiently in initial stages of meiosis, i.e., in the female during early stages of follicle formation and in the male during prespermatogenesis and initial phases of spermatogenesis. Because the PG2 epitope is detectable in germ cells at the time when structurally immature mitochondria are present, we speculate that PG2 immunoreactivity closely mirrors the progress of mitochondrial differentiation during gametogenesis.     

Primordial germ cell specification: the importance of being 'blimped'

In mouse embryos, the expression of Blimp1 has recently revealed a population of allocated primordial germ cell precursors 24 hours earlier than previously thought. Those 'blimped' precursors have been shown to give rise, by mitotic division, to germ cells only and no other cell lineages. Here, we try to understand the events that lead to Blimp1 expression in the primordial germ cell precursors and speculate on what can be the role of Blimp1 during primordial germ cell specification and gastrulation in the mouse. Finally, we discuss the possible involvement of Blimp1 in the two know modes of germ line segregation (epigenesis and preformation).     

Stage-specific damage to synaptonemal complexes and metaphase chromosomes induced by X rays in male mouse germ cells

Synaptonemal complexes reveal mutagen-induced effects in germ cell meiotic chromosomes. This study was aimed at characterizing relationships between damage to synaptonemal complexes and metaphase I chromosomes following radiation exposure at various stages of spermatogenesis. Male mice were irradiated with doses of 0, 2, or 4 Gy, and spermatocytes were harvested at times consistent with earlier exposures as spermatogonial stem cells, preleptotene cells (premeiotic DNA synthesis), or meiotic prophase cells. After stem-cell exposure, twice as many rearrangements were observed in synaptonemal complexes as in metaphase I chromosomes. Irradiation during premeiotic DNA synthesis resulted in dose-related increases in synaptonemal complex breakage and rearrangements (including novel forms) and in metaphase chromosomal aberrations. Following prophase exposure, various types and levels of damage to synaptonemal complexes and metaphase chromosomes were observed. Irradiation of zygotene cells led to high frequencies of chromosome multivalents in metaphase I without a correspondingly high level of damage in preceding prophase synaptonemal complexes. Thus irradiation of premeiotic and meiotic cells results in variable relationships between damage to synaptonemal complexes and metaphase chromosomes. Interpretations of these relationships are based upon what is known about both radiation clastogenesis and the structural/temporal relationships between synaptonemal complexes at prophase and chromosomes at metaphase I of meiosis.