Cellular and Molecular

From neural crest cells to melanocytes: cellular plasticity during development and beyond Abstract Here, we review melanocyte development and how the embryonic melanoblast, although specified to become a melanocyte, is prone to cellular plasticity and is not fully committed to the melanocyte lineage. Even fully differentiated and pigment-producing melanocytes do not always have a stable phenotype. The gradual lineage restriction of neural crest cells toward the melanocyte lineage is determined by both cell-intrinsic and extracellular signals in which differentiation and pathfinding ability reciprocally influence each other. These signals are leveraged by subtle differences in timing and axial positioning. The most extensively studied migration route is the dorsolateral path between the dermomyotome and the prospective epidermis, restricted to melanoblasts. In addition, the embryonic origin of the skin dermis through which neural crest derivatives migrate may also affect the segregation between melanogenic and neurogenic cells in embryos. It is widely accepted that, irrespective of the model organism studied, the immediate precursor of both melanoblast and neurogenic populations is a glial-melanogenic bipotent progenitor. Upon exposure to different conditions, melanoblasts may differentiate into other neural crest-derived lineages such as neuronal cells and vice versa. Key factors that regulate melanoblast migration and patterning will regulate melanocyte homeostasis during different stages of hair cycling in postnatal hair follicles.

N -acetylglucosaminyltransferases and nucleotide sugar transporters form multi-enzyme–multi-transporter assemblies in golgi membranes in vivo Abstract Branching and processing of N-glycans in the medial-Golgi rely both on the transport of the donor UDP-N-acetylglucosamine (UDP-GlcNAc) to the Golgi lumen by the SLC35A3 nucleotide sugar transporter (NST) as well as on the addition of the GlcNAc residue to terminal mannoses in nascent N-glycans by several linkage-specific N-acetyl-glucosaminyltransferases (MGAT1-MGAT5). Previous data indicate that the MGATs and NSTs both form higher order assemblies in the Golgi membranes. Here, we investigate their specific and mutual interactions using high-throughput FRET- and BiFC-based interaction screens. We show that MGAT1, MGAT2, MGAT3, MGAT4B (but not MGAT5) and Golgi alpha-mannosidase IIX (MAN2A2) form several distinct molecular assemblies with each other and that the MAN2A2 acts as a central hub for the interactions. Similar assemblies were also detected between the NSTs SLC35A2, SLC35A3, and SLC35A4. Using in vivo BiFC-based FRET interaction screens, we also identified novel ternary complexes between the MGATs themselves or between the MGATs and the NSTs. These findings suggest that the MGATs and the NSTs self-assemble into multi-enzyme/multi-transporter complexes in the Golgi membranes in vivo to facilitate efficient synthesis of complex N-glycans.

Dgcr8 knockout approaches to understand microRNA functions in vitro and in vivo Abstract Biologic function of the majority of microRNAs (miRNAs) is still unknown. Uncovering the function of miRNAs is hurdled by redundancy among different miRNAs. The deletion of Dgcr8 leads to the deficiency in producing all canonical miRNAs, therefore, overcoming the redundancy issue. Dgcr8 knockout strategy has been instrumental in understanding the function of miRNAs in a variety of cells in vitro and in vivo. In this review, we will first give a brief introduction about miRNAs, miRNA biogenesis pathway and the role of Dgcr8 in miRNA biogenesis. We will then summarize studies performed with Dgcr8 knockout cell models with a focus on embryonic stem cells. After that, we will summarize results from various in vivo Dgcr8 knockout models. Given significant phenotypic differences in various tissues between Dgcr8 and Dicer knockout, we will also briefly review current progresses on understanding miRNA-independent functions of miRNA biogenesis factors. Finally, we will discuss the potential use of a new strategy to stably express miRNAs in Dgcr8 knockout cells. In future, Dgcr8knockout approaches coupled with innovations in miRNA rescue strategy may provide further insights into miRNA functions in vitro and in vivo.

Establishment and depletion of the ovarian reserve: physiology and impact of environmental chemicals Abstract The reproductive life span in women starts at puberty and ends at menopause, following the exhaustion of the follicle stockpile termed the ovarian reserve. Increasing data from experimental animal models and epidemiological studies indicate that exposure to a number of ubiquitously distributed reproductively toxic environmental chemicals (RTECs) can contribute to earlier menopause and even premature ovarian failure. However, the causative relationship between environmental chemical exposure and earlier menopause in women remains poorly understood. The present work, is an attempt to review the current evidence regarding the effects of RTECs on the main ovarian activities in mammals, focusing on how such compounds can affect the ovarian reserve at any stages of ovarian development. We found that in rodents, strong evidence exists that in utero, neonatal, prepubescent and even adult exposure to RTECs leads to impaired functioning of the ovary and a shortening of the reproductive lifespan. Regarding human, data from cross-sectional surveys suggest that human exposure to certain environmental chemicals can compromise a woman's reproductive health and in some cases, correlate with earlier menopause. In conclusion, evidences exist that exposure to RTECs can compromise a woman's reproductive health. However, human exposures may date back to the developmental stage, while the adverse effects are usually diagnosed decades later, thus making it difficult to determine the association between RTECs exposure and human reproductive health. Therefore, epidemiological surveys and more experimental investigation on humans, or alternatively primates, are needed to determine the direct and indirect effects caused by RTECs exposure on the ovary function, and to characterize their action mechanisms.

Exosomes: from carcinogenesis and metastasis to diagnosis and treatment of gastric cancer Abstract Exosomes represent an important group of extracellular vesicles with a defined size between 40 and 150 nm and cup-shaped construction which have a pivotal role in elimination of intracellular debris and intercellular signaling networks. A line of evidence revealed the impact of different types of exosomes in initiation, progression, and metastasis of gastric cancer (GC). These bioactive vesicles mediate tumor and stromal communication network through modulation of cell signaling for carcinogenesis and pre-metastatic niche formation in distant organs. Exosomes contain various cargos including DNAs (mitochondrial and genomic), proteins, transposable elements, and RNAs (coding and noncoding) with different compositions related to functional status of origin cells. In this review, we summarize the main roles of key exosomal cargos in induction of exosome-mediated signaling in cancer cells. Body fluids are employed frequently as the source of exosomes released by tumor cells with a potential role in early diagnosis of GC and chemoresistance. These vesicles as non-toxic and non-immunogenic carriers are also found to be applied for novel drug delivery systems.

Non-obesogenic doses of fatty acids modulate the functionality of the circadian clock in the liver Abstract Saturated fatty acids, such as palmitate, lead to circadian disruption in cell culture. Moreover, information regarding the effects of unsaturated fatty acids on circadian parameters is scarce. We aimed at studying the effects of low doses of saturated as well as unsaturated fatty acids on circadian metabolism in vivo and at deciphering the mechanism by which fatty acids convey their effect. Mice were fed non-obesogenic doses of palm or olive oil and hepatocytes were treated with palmitate and oleate. Mice fed non-obesogenic doses of palm oil showed increased signaling towards fatty acid synthesis, while olive oil increased signaling towards fatty acid oxidation. Low doses of palmitate and oleate were sufficient to alter circadian rhythms, due to changes in the expression and/or activity of key metabolic proteins. Palmitate, but not oleate, counteracted the reduction in lipid accumulation and BMAL1-induced expression of mitochondrial genes involved in fatty acid oxidation. Palmitate was also found to interfere with the transcriptional activity of CLOCK:BMAL1 by preventing BMAL1 deacetylation and activation. In addition, palmitate, but not oleate, reduced PER2-mediated transcriptional activation and increased REV-ERBα-mediated transcriptional inhibition of Bmal1. The inhibition of PER2-mediated transcriptional activation by palmitate was achieved by interfering with PER2 nuclear translocation. Indeed, PER2 reduced fat accumulation in hepatocytes and this reduction was prevented by palmitate. Herein, we show that the detrimental metabolic alteration seen with high doses of palmitate manifests itself early on even with non-obesogenic levels. This is achieved by modulating BMAL1 at several levels abrogating its activity and expression.

Establishment and maintenance of blood–lymph separation Abstract Hippocratic Corpus, a collection of Greek medical literature, described the functional anatomy of the lymphatic system in the fifth century B.C. Subsequent studies in cadavers and surgical patients firmly established that lymphatic vessels drain extravasated interstitial fluid, also known as lymph, into the venous system at the bilateral lymphovenous junctions. Recent advances revealed that lymphovenous valves and platelet-mediated hemostasis at the lymphovenous junctions maintain life-long separation of the blood and lymphatic vascular systems. Here, we review murine models that exhibit failure of blood–lymph separation to highlight the novel mechanisms and molecular targets for the modulation of lymphatic disorders. Specifically, we focus on the transcription factors, cofactors, and signaling pathways that regulate lymphovenous valve development and platelet-mediated lymphovenous hemostasis, which cooperate to maintain blood–lymph separation.

Therapeutic potential of menstrual blood-derived endometrial stem cells in cardiac diseases Abstract Despite significant developments in medical and surgical strategies, cardiac diseases remain the leading causes of morbidity and mortality worldwide. Numerous studies involving preclinical and clinical trials have confirmed that stem cell transplantation can help improve cardiac function and regenerate damaged cardiac tissue, and stem cells isolated from bone marrow, heart tissue, adipose tissue and umbilical cord are the primary candidates for transplantation. During the past decade, menstrual blood-derived endometrial stem cells (MenSCs) have gradually become a promising alternative for stem cell-based therapy due to their comprehensive advantages, which include their ability to be periodically and non-invasively collected, their abundant source material, their ability to be regularly donated, their superior proliferative capacity and their ability to be used for autologous transplantation. MenSCs have shown positive therapeutic potential for the treatment of various diseases. Therefore, aside from a brief introduction of the biological characteristics of MenSCs, this review focuses on the progress being made in evaluating the functional improvement of damaged cardiac tissue after MenSC transplantation through preclinical and clinical studies. Based on published reports, we conclude that the paracrine effect, transdifferentiation and immunomodulation by MenSC promote both regeneration of damaged myocardium and improvement of cardiac function.

Presynaptic NMDA receptors control nociceptive transmission at the spinal cord level in neuropathic pain Abstract Chronic neuropathic pain is a debilitating condition that remains challenging to treat. Glutamate N-methyl-d-aspartate receptor (NMDAR) antagonists have been used to treat neuropathic pain, but the exact sites of their actions have been unclear until recently. Although conventionally postsynaptic, NMDARs are also expressed presynaptically, particularly at the central terminals of primary sensory neurons, in the spinal dorsal horn. However, presynaptic NMDARs in the spinal cord are normally quiescent and are not actively involved in physiological nociceptive transmission. In this review, we describe the emerging role of presynaptic NMDARs at the spinal cord level in chronic neuropathic pain and the implications of molecular mechanisms for more effective treatment. Recent studies indicate that presynaptic NMDAR activity at the spinal cord level is increased in several neuropathic pain conditions but not in chronic inflammatory pain. Increased presynaptic NMDAR activity can potentiate glutamate release from primary afferent terminals to spinal dorsal horn neurons, which is crucial for the synaptic plasticity associated with neuropathic pain caused by traumatic nerve injury and chemotherapy-induced peripheral neuropathy. Furthermore, α2δ-1, previously considered a calcium channel subunit, can directly interact with NMDARs through its C-terminus to increase presynaptic NMDAR activity by facilitating synaptic trafficking of α2δ-1–NMDAR complexes in neuropathic pain caused by chemotherapeutic agents and peripheral nerve injury. Targeting α2δ-1–bound NMDARs with gabapentinoids or α2δ-1 C-terminus peptides can attenuate nociceptive drive form primary sensory nerves to dorsal horn neurons in neuropathic pain.

Tight junction proteins at the blood–brain barrier: far more than claudin-5 Abstract At the blood–brain barrier (BBB), claudin (Cldn)-5 is thought to be the dominant tight junction (TJ) protein, with minor contributions from Cldn3 and -12, and occludin. However, the BBB appears ultrastructurally normal in Cldn5 knock-out mice, suggesting that further Cldns and/or TJ-associated marvel proteins (TAMPs) are involved. Microdissected human and murine brain capillaries, quickly frozen to recapitulate the in vivo situation, showed high transcript expression of Cldn5, -11, -12, and -25, and occludin, but also abundant levels of Cldn1 and -27 in man. Protein levels were quantified by a novel epitope dilution assay and confirmed the respective mRNA data. In contrast to the in vivo situation, Cldn5 dominates BBB expression in vitro, since all other TJ proteins are at comparably low levels or are not expressed. Cldn11 was highly abundant in vivo and contributed to paracellular tightness by homophilic oligomerization, but almost disappeared in vitro. Cldn25, also found at high levels, neither tightened the paracellular barrier nor interconnected opposing cells, but contributed to proper TJ strand morphology. Pathological conditions (in vivo ischemia and in vitro hypoxia) down-regulated Cldn1, -3, and -12, and occludin in cerebral capillaries, which was paralleled by up-regulation of Cldn5 after middle cerebral artery occlusion in rats. Cldn1 expression increased after Cldn5 knock-down. In conclusion, this complete Cldn/TAMP profile demonstrates the presence of up to a dozen TJ proteins in brain capillaries. Mouse and human share a similar and complex TJ profile in vivo, but this complexity is widely lost under in vitro conditions.