Ceramides and other bioactive sphingolipid backbones in health and disease: Lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy

Sphingolipids are comprised of a backbone sphingoid base that may be phosphorylated, acylated, glycosylated, bridged to various headgroups through phosphodiester linkages, or otherwise modified. Organisms usually contain large numbers of sphingolipid subspecies and knowledge about the types and amounts is imperative because they influence membrane structure, interactions with the extracellular matrix and neighboring cells, vesicular traffic and the formation of specialized structures such as phagosomes and autophagosomes, as well as participate in intracellular and extracellular signaling. Fortunately, “sphingolipidomic” analysis is becoming feasible (at least for important subsets such as all of the backbone “signaling” subspecies: ceramides, ceramide 1-phosphates, sphingoid bases, sphingoid base 1-phosphates, inter alia) using mass spectrometry, and these profiles are revealing many surprises, such as that under certain conditions cells contain significant amounts of “unusual” species: N-mono-, di-, and tri-methyl-sphingoid bases (including N,N-dimethylsphingosine); 3-ketodihydroceramides; N-acetyl-sphingoid bases (C2-ceramides); and dihydroceramides, in the latter case, in very high proportions when cells are treated with the anticancer drug fenretinide (4-hydroxyphenylretinamide). The elevation of DHceramides by fenretinide is befuddling because the 4,5-trans-double bond of ceramide has been thought to be required for biological activity; however, DHceramides induce autophagy and may be important in the regulation of this important cellular process. The complexity of the sphingolipidome is hard to imagine, but one hopes that, when partnered with other systems biology approaches, the causes and consequences of the complexity will explain how these intriguing compounds are involved in almost every aspect of cell behavior and the malfunctions of many diseases.     

Membrane microdomains and insulin resistance

A new concept, that “metabolic disorders, such as type 2 diabetes, are membrane microdomain disorders caused by aberrant expression of gangliosides”, has arisen. By examining this working hypothesis, we demonstrate the molecular pathogenesis of type 2 diabetes and insulin resistance focusing on the interaction between insulin receptor and gangliosides in microdomains and propose the new therapeutic strategy “membrane microdomain ortho-signaling therapy”.     

Ampelozyziphus amazonicus Ducke (Rhamnaceae), a medicinal plant used to prevent malaria in the Amazon Region, hampers the development of Plasmodium berghei sporozoites

Most medicinal plants used against malaria in endemic areas aim to treat the acute symptoms of the disease such as high temperature fevers with periodicity and chills. In some endemic areas of the Brazilian Amazon region one medicinal plant seems to be an exception: Ampelozyziphus amazonicus, locally named “Indian beer” or “Saracura-mira”, used to prevent the disease when taken daily as a cold suspension of powdered dried roots. In previous work we found no activity of the plant extracts against malaria blood parasites in experimentally infected animals (mice and chickens) or in cultures of Plasmodium falciparum. However, in infections induced by sporozoites, chickens treated with plant extracts were partially protected against Plasmodium gallinaceum and showed reduced numbers of exoerythrocytic forms in the brain. We now present stronger evidence that the ethanolic extract of “Indian beer” roots hampers in vitro and in vivo development of Plasmodium berghei sporozoites, a rodent malaria parasite. Some mice treated with high doses of the plant extract did not become infected after sporozoite inoculation, whereas others had a delayed prepatent period and lower parasitemia. Our data validates the use of “Indian beer” as a remedy for malaria prophylaxis in the Amazon, where the plant exists and the disease represents an important problem which is difficult to control. Studies aiming to identify the active compounds responsible for the herein described causal prophylactic activity are needed and may lead to a new antimalarial prophylactic.     

Using induced pluripotent stem cell neuronal models to study neurodegenerative diseases

Current application of human induced pluripotent stem cells (hiPSCs) technology in patient-specific models of neurodegenerative disorders recapitulate some of key phenotypes of diseases, representing disease-specific cellular modeling and providing a unique platform for therapeutics development. We review recent efforts toward advancing hiPSCs-derived neuronal cell types and highlight their potential use for the development of more complex in vitro models of neurodegenerative diseases by focusing on Alzheimer's disease, Parkinson's disease, Huntington's disease and Amyotrophic lateral sclerosis. We present evidence from previous works on the important phenotypic changes of various neuronal types in these neurological diseases. We also summarize efforts on conducting low- and high-throughput screening experiments with hiPSCs toward developing potential therapeutics for treatment of neurodegenerative diseases. Lastly, we discuss the limitations of hiPSCs culture system in studying neurodegenerative diseases and alternative strategies to overcome these hurdles.     

Stem cells and neurodegenerative diseases

Neurodegenerative diseases are characterized by the neurodegenerative changes or apoptosis of neurons involved in networks, which are important to specific physiological functions. With the de-velopment of old-aging society, the incidence of neurodegenerative diseases is on the increase. How-ever, it is difficult to diagnose for most of neurodegenerative diseases. At present, there are too few effective therapies. Advances in stem cell biology have raised the hope and possibility for the therapy of neurodegenerative diseases. Recently, stem cells have been widely attempted to treat neurodegen-erative diseases of animal model. Here we review the progress and prospects of various stem cells, including embryonic stem cells, mesenchymal stem cell and neural stem cells and so on, for the treatments of neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, Hunt-ington’s disease and Amyotrophic lateral sclerosis/Lou Gehrig’s disease.     

以果蝇模型研究人类神经退行性疾病

人类神经退行性疾病是一类以神经元退行性病变导致个体行为异常乃至死亡为主要特征的疾病.果蝇以其独特的分子遗传学优势成为研究人类神经退行性疾病的理想模型.通过对果蝇模型的研究不仅可以揭示神经退行性疾病发生的细胞分子通路,还可以研究对疾病有调控作用的基因及其表达产物,寻找可能的药物作用靶点并进行药物筛选,为防治人类神经退行性疾病提供新的方法和有效的药物.     

A novel fluorescent timer based on bicistronic expression strategy in Caenorhabditis elegans

Fluorescent timers are useful tools for studying the spatial and temporal cellular or molecular events. Based on the trans-splicing mechanism in Caenorhabditis elegans, we constructed a “fluorescent timer” through bicistronic expression of two fluorescent proteins with different maturation times. When used in vivo, this “timer” changes its color over time and therefore can be used to monitor the activity of the targeted promoters in C. elegans. Using this “timer”, we have successfully traced the time-dependent activity of myo-3 promoter which drives expression in body wall muscle and vulval muscle. We found that the myo-3 promoter started to be active about 7 h after egg-laying and sustained its activity in the following hatching process. We have also determined the myo-3 promoter activity during larval development by this “timer”. We anticipate that more new “fluorescent timers” with variable time-resolution could be designed by bicistronic expression of different fluorescent protein pairs.     

Genome wide sequence analysis grants unbiased definition of species boundaries in “Candidatus Phytoplasma”

The phytoplasmas are currently named using the Candidatus category, as the inability to grow them in vitro prevented (i) the performance of tests, such as DNA-DNA hybridization, that are regarded as necessary to establish species boundaries, and (ii) the deposition of type strains in culture collections. The recent accession to complete or nearly complete genome sequence information disclosed the opportunity to apply to the uncultivable phytoplasmas the same taxonomic approaches used for other bacteria. In this work, the genomes of 14 strains, belonging to the 16SrI, 16SrIII, 16SrV and 16SrX groups, including the species “Ca. P. asteris”, “Ca. P. mali”, “Ca. P. pyri”, “Ca. P. pruni”, and “Ca. P. australiense” were analyzed along with Acholeplasma laidlawi, to determine their taxonomic relatedness. Average nucleotide index (ANIm), tetranucleotide signature frequency correlation index (Tetra), and multilocus sequence analysis of 107 shared genes using both phylogenetic inference of concatenated (DNA and amino acid) sequences and consensus networks, were carried out. The results were in large agreement with the previously established 16S rDNA based classification schemes. Moreover, the taxonomic relationships within the 16SrI, 16SrIII and 16SrX groups, that represent clusters of strains whose relatedness could not be determined by 16SrDNA analysis, could be comparatively evaluated with non-subjective criteria. “Ca. P. mali” and “Ca. P. pyri” were found to meet the genome characteristics for the retention into two different, yet strictly related species; representatives of subgroups 16SrI-A and 16SrI-B were also found to meet the standards used in other bacteria to distinguish separate species; the genomes of the strains belonging to 16SrIII were found more closely related, suggesting that their subdivision into Candidatus species should be approached with caution.     

Putative roles of cilia in polycystic kidney disease

The last 10 years has witnessed an explosion in research into roles of cilia in cystic renal disease. Cilia are membrane-enclosed finger-like projections from the cell, usually on the apical surface or facing into a lumen, duct or airway. Ten years ago, the major recognised functions related to classical “9 + 2” cilia in the respiratory and reproductive tracts, where co-ordinated beating clears secretions and assists fertilisation respectively. Primary cilia, which have a “9 + 0” arrangement lacking the central microtubules, were anatomical curiosities but several lines of evidence have implicated them in both true polycystic kidney disease and other cystic renal conditions: ranging from the homology between Caenorhabditis elegans proteins expressed on sensory cilia to mammalian polycystic kidney disease (PKD) 1 and 2 proteins, through the discovery that orpk cystic mice have structurally abnormal cilia to numerous recent studies wherein expression of nearly all cyst-associated proteins has been reported in the cilia or its basal body. Functional studies implicate primary cilia in mechanosensation, photoreception and chemosensation but it is the first of these which appears most important in polycystic kidney disease: in the simplest model, fluid flow across the apical surface of renal cells bends the cilia and induces calcium influx, and this is perturbed in polycystic kidney disease. Downstream effects include changes in cell differentiation and polarity. Pathways such as hedgehog and Wnt signalling may also be regulated by cilia. These data support important roles for cilia in the pathogenesis of cystic kidney diseases but one must not forget that the classic polycystic kidney disease proteins are expressed in several other locations where they may have equally important roles, such as in cell-cell and cell-matrix interactions, whilst it is not just aberrant cilia signalling that can lead to de-differentiation, loss of polarity and other characteristic features of polycystic kidney disease. Understanding how cilia fit into the other aspects of polycystic kidney disease biology is the challenge for the next decade. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

New Directions for Neurodegenerative Disease Therapy: Using Chemical Compounds to Boost the Formation of Mutant Protein Inclusions

Neurodegenerative disease such as Huntington’s, Parkinson’s, and Alzheimer’sdiseases are marked by neuronal accumulation of toxic misfolded protein. Developingtherapies for these misfolding diseases requires finding chemical compounds that caneither clear toxic misfolded protein, or can protect neurons from their impact. Suchcompounds could not only provide the starting points for potential drugs, but could alsoprovide valuable research tools for untangling the complexities of the disease process.Until now, chemical screens for these diseases have focused on finding compoundsthat prevent aggregation of mutant protein. We recently published a compound, B2,which promotes the formation of large inclusions by mutant Huntingtin and α-synuclein,while rescuing some of the toxic effects of these proteins. As inclusions were longbelieved to be toxic to cells, this contradicts previous therapeutic approaches. At thesame time, the results support growing evidence for the protective effects of inclusions.In this review, we discuss these results, and place them in the context of ongoingtherapeutic discovery efforts for HD and other neurodegenerative diseases.     

PrP N-terminal domain triggers PrP(Sc)-like aggregation of Dpl

Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP^C, for “cellular prion protein”) into an abnormal state (PrP^Sc, for “scrapie prion protein”). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP^C. In contrast to its homologue PrP^C, Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP^Sc-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP^C (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the α-helical monomer forms soluble β-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy.     

Efficient germ-line transformation of the economically important pest species Lucilia cuprina and Lucilia sericata (Diptera, Calliphoridae)

The green blowfly species Lucilia cuprina and Lucilia sericata are economically important pests for the sheep industries of Australia and New Zealand. L. cuprina has long been considered a good target for a genetic pest management program. In addition, L. sericata maggots are used in the cleaning of wounds and necrotic tissue of patients suffering from ulcers that are difficult to treat by other methods. Development of efficient transgenesis methods would greatly facilitate the development of strains ideal for genetic control programs or could potentially improve “maggot therapy”. We have previously reported the germ-line transformation of L. cuprina and the design of a “female killing system” that could potentially be applied to this species. However, the efficiency of transformation obtained was low and transformed lines were difficult to detect due to the low expression of the EGFP marker used. Here we describe an efficient and reliable method for germ-line transformation of L. cuprina using new piggyBac vector and helper plasmids containing the strong promoter from the L. cuprina hsp83 gene to drive expression of the transposase and fluorescent protein marker gene. We also report, for the first time, the germ-line transformation of L. sericata using the new piggyBac vector/helper combination.     

The sense of water in the blowfly Protophormia terraenovae

The gustatory system of the blowfly, Protophormia terraenovae, is a relatively simple biological model for studies on chemosensory input and behavioral output. It appears to have renewed interest as a model for studies on the role of water channels, namely aquaporins or aquaglyceroporins, in water detection. To this end, we investigated the presence of water channels, their role in “water” and “salt” cell responsiveness and the transduction mechanism involved. For the first time our electrophysiological results point to the presence of an aquaglyceroporin in the chemoreceptor membrane of the “water” cell in the blowfly taste chemosensilla whose transduction mechanism ultimately involves an intracellular calcium increase and consequently cell depolarization. This hypothesis is also supported by calcium imaging data following proper stimulation. This mechanism is triggered by “water” cell stimulation with hypotonic solutions and/or solutes such as glycerol which crosses the membrane by way of aquaglyceroporins. Behavioral output indicates that the “sense” of water in blowflies is definitely not dependent on the “water” cell only, but also on the “salt” cell sensitivity. These findings also hypothesize a new role for aquaglyceroporin in spiking cell excitability.     

Darwin teleologist? Design in the Orchids

Focusing on the Orchids, this article aims at disentangling the concepts of teleology, design and natural theology. It refers to several contemporary critics of Darwin (Kölliker, Argyll, Royer, Candolle, Delpino) to challenge Huxley's interpretation that Darwin's system was “a deathblow” to teleology. The Orchids seem rather to be a “flank-movement” (Gray): it departs from the Romantic theories of transmutation and the “imaginary examples” of the Origin; it focuses on empirical data and on teleological structures. Although Darwin refers to natural selection, his readers mock him for his fascination for delicate morphological contrivances and co-adaptations – a sign that he was inescapably lured to finality. Some even suggested that his system was a “theodicy”. In the history of Darwinism, the Orchids reveal “another” quite unexpected and heterodox Darwin: freed from the hypothetical fancies of the Origin, and even suggesting a new kind of physico-theology.