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Transposons are conserved mobile genetic elements and one of the major sources of genetic variability during organisms’ evolution. PiggyBac Transposable Element Derived 5 (Pgbd5), an evolutionarily conserved vertebrate DNA transposase-coding gene, is highly expressed in the central nervous system during development and adult life, suggesting it may play a role in neuronal differentiation and maintenance.Here, we show thatPgbd5controls the expression of pro-neuronal and proliferative genes in a cell autonomous manner, leading to a decreased generation of neurons with altered migration. This activity depends on the induction of endogenous DNA double-strand breaks (DSBs).These results positionPgbd5as a crucial regulator of brain development through its endonuclease activity.One-Sentence SummaryPgbd5regulates cerebral cortex neurogenesis through DNA double-strand breaks modulation.

The risk of honey bee (Apis mellifera L.) exposure to pesticide residues while foraging for nectar and pollen is commonly explored in the context of agroecosystems. However, pesticides are also used in urban and suburban areas for vegetation management, vector control, and the management of ornamental plants in public and private landscapes. The extent to which pesticides pose a health risk to honey bees in these settings remains unclear. We addressed this at a landscape scale by conducting pesticide residue screening analyses on 768 nectar and 862 pollen samples collected monthly over 2 years from honey bee colonies located in urban and suburban areas in eight medium to large cities in California, Florida, Michigan, and Texas (USA). A risk assessment was performed using the US Environmental Protection Agency's BeeREX model whenever an oral toxicity value was available for a compound. Chemical analyses detected 17 pesticides in nectar and 60 in pollen samples during the survey. Approximately 73% of all samples contained no detectable pesticide residues. Although the number of detections varied among the sampled regions, fewer pesticides were detected in nectar than in pollen. Per BeeREX, four insecticides showed a potential acute risk to honey bees: imidacloprid, chlorpyrifos, and esfenvalerate in nectar, and deltamethrin in nectar and pollen. In general, exposure of honey bees to pesticides via nectar and pollen collection was low in urban and suburban areas across the United States, and no seasonal or spatial trends were evident. Our data suggest that honey bees are exposed to fewer pesticides in developed areas than in agricultural ones. Environ Toxicol Chem 2022;41:991-1003. © 2022 SETAC.

Many neurodevelopmental conditions, including autism, affect males more than females. Genomic mechanisms enhancing risk in males may contribute to this sex-bias. The ubiquitin protein ligase E3A gene (Ube3a) exerts pleiotropic effects on cellular homeostasis via control of protein turnover and by acting as transcriptional coactivator with steroid hormone receptors. Overdosage ofUbe3avia duplication or triplication of chromosomal region 15q11-13 causes 1-2% of autistic cases. Here, we test the hypothesis that increased dosage ofUbe3amay influence autism-relevant phenotypes in a sex-biased manner. We report robust sex-biasing effects on brain connectomics and repetitive behaviors in mice with extra copies of Ube3a. These effects were associated with a profound transcriptional dysregulation of several known autism-associated genes (e.g., FMR1, SCN2A, PTEN, MEF2C, SHANK3, TSC2) as well as differentially-expressed genes identified in human 15q duplication and in autistic patients. Notably, increased Ube3a dosage also affects multiple sex-relevant mechanisms, including genes on the X chromosome, genes influenced by sex steroid hormones, downstream targets of the androgen and estrogen receptors, or genes that are sex-differentially regulated by transcription factors. These results suggest thatUbe3aoverdosage can critically contribute to sex-bias in neurodevelopmental conditions via influence on sex-differential mechanisms.

With recent rapid advancement of methodological tools, mechanistic understanding of biological processes leading to carcinogenesis is expanding. New approach methodologies such as transcriptomics can inform on non-genotoxic mechanisms of chemical carcinogens and can be developed for regulatory applications. The Organisation for the Economic Cooperation and Development (OECD) expert group developing an Integrated Approach to the Testing and Assessment (IATA) of Non-Genotoxic Carcinogens (NGTxC) is reviewing the possible assays to be integrated therein. In this context, we review the application of transcriptomics approaches suitable for pre-screening gene expression changes associated with phenotypic alterations that underlie the carcinogenic processes for subsequent prioritisation of downstream test methods appropriate to specific key events of non-genotoxic carcinogenesis. Using case studies, we evaluate the potential of gene expression analyses especially in relation to breast cancer, to identify the most relevant approaches that could be utilised as (pre-) screening tools, for example Gene Set Enrichment Analysis (GSEA). We also consider how to address the challenges to integrate gene panels and transcriptomic assays into the IATA, highlighting the pivotal omics markers identified for assay measurement in the IATA key events of inflammation, immune response, mitogenic signalling and cell injury.

Epidemiological data have linked cadmium exposure to neurotoxicity and to neurodegenerative diseases (e.g., Alzheimer's and Parkinson's disease), and to increased risk of developing ALS. Even though the brain is not a primary target organ, this metal can bypass the blood brain barrier, thus exerting its toxic effects. The coordination chemistry of cadmium is of strong biological relevance, as it resembles to zinc(II) and calcium(II), two ions crucial for neuronal signaling. A toxicogenomics approach applied to a neuronal human model (SH-SY5Y cells) exposed to cadmium (10 and 20 μM) allowed the identification of early deregulated genes and altered processes, and the discrimination between neuronal-specific and unspecific responses as possible triggers of neurodegeneration. Cadmium confirmed its recognized carcinogenicity even on neuronal cells by activating the p53 signaling pathway and genes involved in tumor initiation and cancer cell proliferation, and by down-regulating genes coding for tumor suppressors and for DNA repair enzymes. Two cadmium-induced stress responses were observed: the activation of different members of the heat shock family, as a mechanism to restore protein folding in response to proteotoxicity, and the activation of metallothioneins (MTs), involved in zinc and copper homeostasis, protection against metal toxicity and oxidative damage. Perturbed function of essential metals is suggested by the mineral absorption pathway, with MTs, HMOX1, ZnT-1, and Ferritin genes highly up-regulated. Cadmium interferes also with Ca2+ regulation as S100A2 is one of the top up-regulated genes, coding for a highly specialized family of regulatory Ca2+-binding proteins. Other neuronal-related functions altered in SH-SY5Y cells by cadmium are microtubules dynamics, microtubules motor-based proteins and neuroprotection by down-regulation of NEK3, KIF15, and GREM2 genes, respectively.

Several harmful modifications in different tissues-organs, leading to relevant diseases (e.g., liver and lung diseases, neurodegeneration) are reported after exposure to cadmium (Cd), a wide environmental contaminant. This arises the question whether any common molecular signatures and/or Cd-induced modifications might represent the building block in initiating or contributing to address the cells towards different pathological conditions. To unravel possible mechanisms of Cd tissue-specificity, we have analyzed transcriptomics data from cell models representative of three major Cd targets: pulmonary (A549), hepatic (HepG2), and neuronal (SH-SY-5Y) cells. Further, we compared common features to identify any non-specific molecular signatures. The functional analysis of dysregulated genes (gene ontology and KEGG) shows GO terms related to metabolic processes significantly enriched only in HepG2 cells. GO terms in common in the three cell models are related to metal ions stress response and detoxification processes. Results from KEGG analysis show that only one specific pathway is dysregulated in a significant way in all cell models: the mineral absorption pathway. Our data clearly indicate how the molecular mimicry of Cd and its ability to cause a general metal ions dyshomeostasis represent the initial common feature leading to different molecular signatures and alterations, possibly responsible for different pathological conditions.

Pollinator populations are declining (Biesmeijer et al., 2006; Brodschneider et al., 2018; Cameron et al., 2011; Goulson, Lye, & Darvill, 2008; Kulhanek et al., 2017; National Research Council, 200...

Growing concern suggests that some chemicals exert (developmental) neurotoxicity (DNT and NT) and are linked to the increase in incidence of autism, attention deficit and hyperactivity disorders. The high cost of routine tests for DNT and NT assessment make it difficult to test the high numbers of existing chemicals. Thus, more cost effective neurodevelopmental models are needed. The use of induced pluripotent stem cells (iPSC) in combination with the emerging human 3D tissue culture platforms, present a novel tool to predict and study human toxicity. By combining these technologies, we generated multicellular brain spheroids (BrainSpheres) from human iPSC. The model has previously shown to be reproducible and recapitulates several neurodevelopmental features. Our results indicate, rotenone's toxic potency varies depending on the differentiation status of the cells, showing higher reactive oxygen species (ROS) and higher mitochondrial dysfunction during early than later differentiation stages. Immuno-fluorescence morphology analysis after rotenone exposure indicated dopaminergic-neuron selective toxicity at non-cytotoxic concentrations (1 μM), while astrocytes and other neuronal cell types were affected at (general) cytotoxic concentrations (25 μM). Omics analysis showed changes in key pathways necessary for brain development, indicating rotenone as a developmental neurotoxicant and show a possible link between previously shown effects on neurite outgrowth and presently observed effects on Ca2+ reabsorption, synaptogenesis and PPAR pathway disruption. In conclusion, our BrainSpheres model has shown to be a reproducible and novel tool to study neurotoxicity and developmental neurotoxicity. Results presented here support the idea that rotenone can potentially be a developmental neurotoxicant.

Background Extracellular nucleic acids circulate in plasma. They are expected to be present in manufactured blood products eligible for transfusion, but little is known about their biological activity on human cells. The aim of this study is to investigate whether cell-free nucleic acids (CFNAs) are present and biologically active in red blood cell units (RBCUs), fresh frozen plasmas, and platelet concentrates. Study design and methods CFNAs were extracted from RBCUs, fresh frozen plasma, and platelet concentrates. Their nature and structure were analyzed by regular methods of nucleic acid detection/quantification. A normalized polymerase chain reaction combining amplification of a CFNA marker (Alu 115) and amplification of an internal nonhuman DNA control spiked in all samples (phiX 174) was developed to study CFNA release after RBCU storage. The impact of CFNAs on gene regulation was tested by microarray after coculture with peripheral blood mononuclear cells and macrophages. Results Extracellular double-stranded DNA was present in all blood products, with higher amounts found in cellular suspensions (RBCUs and platelet concentrates). Storage up to 40 days did not influence release from RBCUs, and CFNA amount varied considerably from one unit to another. Microarray experiments showed that exposition of macrophages to CFNA increased the expression of genes involved in the innate immune response including chemokines, chemokine receptors, and receptors of the innate response. Conclusion CFNAs are present in blood products. Immunoregulatory properties of CFNA are shown in vitro, providing new insights on biologically active components of blood products besides those for intended therapeutic use.

BACKGROUND Although the risk of transmitting infectious agents by blood transfusion is dramatically reduced after donor selection, leukoreduction, and laboratory testing, some could still be present in donor's blood. A description of metagenomes in blood products eligible for transfusion represents relevant information to evaluate the risk of pathogen transmission by transfusion. STUDY DESIGN AND METHODS Detection of viruses, bacteria, and fungi genomes was made by high-throughput sequencing (HTS) of 600 manufactured blood products eligible for transfusion: 300 red blood cell (RBC) and 300 fresh-frozen plasma (FFP) units. RESULTS Anelloviruses and human pegivirus, frequent in the blood of healthy individuals, were found. Human papillomavirus type 27 and Merkel cell polyomavirus, present on the skin, were also detected. Unexpectedly, astrovirus MLB2 was identified and characterized in a FFP unit. The presence of astrovirus MLB2 was confirmed in donor's blood and corresponded to an asymptomatic acute viremia. Sequences of bacteria and fungi were also detected; they are likely the result of environmental contamination. CONCLUSION This study demonstrates that HTS is a promising tool for detecting common and less frequent infectious pathogens in blood products

Honey bee (Apis mellifera) colony maintenance depends on foraging workers to obtain food resources from flowering plants year round. Because diverse floral diets have a positive impact on honey bee health, identifying the plants preferred by foragers provides valuable information to manage bee-friendly habitats. Recent studies have utilized palynology to better understand honey bee nectar foraging preferences. Futhermore, the International Honey Commission has established standards for analyzing honey samples. However, standards for studying the plant taxonomic composition of honey bee pollen pellets have not been established. The goal of this project was to determine the minimum number of pollen grains that need to be counted to obtain an accurate floral taxonomic representation in a pollen pellet sample. To do this, pollen samples were collected from pollen traps placed outside honey bee hives, and a pollen subsample foraged by each colony was acetolyzed and identified to the lowest taxonomic level possible. Cohorts of 100 pollen grains obtained from homogenized pollen samples from three different colonies were counted successively 5 times for a total count of 500 pollen grains per colony. This was repeated for each of the replicates from the three separate colonies. We found no statistically significant differences in the number or proportion of floral taxa found between the 200 and 500 pollen grain counts in two out of the three colonies sampled. Species diversity index analysis suggested that the higher number of floral taxa found in some 500-grain counts were attributed to a relatively low presence of minor pollen types. Thus, a 200 pollen grain count seems sufficient to assess the predominant, secondary and important minor plant taxa present in a pollen sample, while a 500-grain count may be needed to elucidate a more specific taxonomic assessment of additional minor taxa floral types to determine a sample's geographic origin.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the voluntary motor nervous system. Genetic factors play a major role in the familial form, represented by 10% of cases. Whereas, in the remaining 90% sporadic cases a multifactorial origin is supposed, in which environmental factors may be involved. Particularly, the involvement of metal toxicity is proposed, based on epidemiologic data and on the increasing number of case reports of ALS-like syndromes associated to metal exposure. Among metals, cadmium is of particular concern for its heavy anthropogenic release (~30,000 tons/year) and for its chemical properties. Cd exposure can primarily occur through food, drinking water, air particles, cosmetics and cigarette smoking. Once assumed, even at very low concentrations, Cd is accumulated with a long biological half-life (> 26 years). Cd accumulation contributes to oxidative stress and alteration of divalent ions homeostasis, primarily Zn2+ and Ca2+, both crucial for neuronal signaling. Studies of neurodegenerative diseases mainly rely on the use of animal models and on stem cells. Already ten years ago in a Nature Report the validity of neurodegenerative disease animal models was questioned, and direction towards the reduction of transgenic models is mandatory. Human neuroblastoma SH-SY5Y is a dopaminergic cell line that reproduces biochemical and morphological properties of neurons, thus being often used as an in vitro model for human neurons in neurotoxicity or neuroprotection studies in experimental disease research. We have applied a toxicogenomic approach to evidence deregulated pathways in SH-SY5Y cells exposed to cadmium to unravel neuronal specific and non-specific responses to the toxic metal. A whole genome analysis by cDNA microarray was performed in SH-SY5Y cells exposed to non-cytotoxic concentrations of cadmium (10 and 20 mM for 48 h). Very interestingly, microarray analysis evidenced that the top up-regulated genes were enriched in pathways related to the mineral absorption biological process in which metallothioneins (MT), and heme oxygenase-1 (HMOX-1) are among the highest up-regulated genes. MT are low molecular weight proteins that bind essential metal ions and sequester toxic metals, thus representing an important broad mechanisms of metal homeostasis. MT expression was visualized in SH-SY5Y cells by Western blot analysis revealing a dose-dependent trend of the protein, validating microarray data. The increased HMOX-1 expression has just been related [1] to the progression of several diseases including neurodegeneration. Another remarkable result concerns NEU4 that was found in our cell samples as the top down-regulated gene upon cadmium treatment. This gene encodes for a neuraminidase specifically expressed in brain that regulates neuronal function by catabolizing brain gangliosides. Sialidase NEU4 has been shown to be localized in the outer mitochondrial membrane and involved in different cellular processes, such as apoptosis, neuronal differentiation and tumorigenesis [2]. A dramatic decrease of NEU4 expression level was previously observed prior to apoptotic neurodegeneration. Overall our results evidence specific neuronal responses to cadmium and suggest the possible mechanisms triggered by this toxic metal in neurodegeneration. The authors acknowledge the European Commission, the Mistral University Research Center, and UniMIB for funding (2016- ATE-0411 to CU). References [1] Waza, A. A., Hamid, Z., Ali, S. et al. (2018). Inflamm Res 67, 579-588. doi:10.1007/s00011-018-1151-x [2] Bigi, A., Tringali, C., Forcella, M. et al. (2013). Glycobiology 23, 1499-1509. doi:10.1093/glycob/cwt078

Introduction The cause of sporadic Alzheimer's disease (AD) remains unclear. Given the growing evidence that protein aggregates can spread in a "prion-like" fashion, we reasoned that a small population of brain cells producing such "prion-like" particles due to a postzygotic acquired mutation would be sufficient to trigger the disease. Deep DNA sequencing technology should in principle allow the detection of such mosaics. Methods To detect the somatic mutations of genes causing AD present in a small number of cells, we developed a targeted deep sequencing approach to scrutinize the genomic loci of APP , PSEN1 , and PSEN2 genes in DNA extracted from the entorhinal cortex, one of the brain regions showing the earliest signs of AD pathology. We also included the analysis of the MAPT gene because mutations may promote tangle formation. We validated candidate mutations with an independent targeted ultradeep amplicon sequencing technique. Results We demonstrate that our approach can detect single-nucleotide mosaic variants with a 1% allele frequency and copy number mosaic variants present in as few as 10% of cells. We screened 72 AD and 58 control brain samples and identified three mosaic variants with low allelic frequency (∼1%): two novel MAPT variants in sporadic AD patients and a known PSEN2 variant in a Braak II control subject. Moreover, we detected both novel and known pathogenic nonmosaic heterozygous variants in PSEN1 and PSEN2 in this cohort of sporadic AD patients. Discussion Our results show that mosaic mutations with low allelic frequencies in AD-relevant genes can be detected in brain-derived DNA, but larger samples need to be investigated before a more definitive conclusion with regard to the pathogenicity of such mosaics can be made.

Non-coding RNAs, such as microRNAs and long non-coding RNAs, represent the next major step in understanding the complexity of gene regulation and expression. In the past decade, tremendous efforts have been put mainly into identifying microRNAs that are changed in Alzheimer's disease, with the goal to provide biomarkers of the disease and to better characterize molecular pathways that are deregulated concomitantly to the formation of Tau and amyloid aggregates. This review underlines the importance of correctly defining, in a deluge of high-throughput data, which microRNAs are abnormally expressed in Alzheimer's disease patients. Despite a clear lack of consensus on the topic, miR-132 is emerging as a neuronal microRNA being gradually down-regulated during disease and showing important roles in the maintenance of brain integrity. Insight into the biological importance of other classes of non-coding RNAs also rapidly increased over the last years and therefore we discuss the possible implication of long non-coding RNAs in Alzheimer's disease.

For honey bees (Apis mellifera), colony maintenance and growth are highly dependent on worker foragers obtaining sufficient resources from flowering plants year round. Despite the importance of floral diversity for proper bee nutrition, urban development has drastically altered resource availability and diversity for these important pollinators. Therefore, understanding the floral resources foraged by bees in urbanized areas is key to identifying and promoting plants that enhance colony health in those environments. In this study, we identified the pollen foraged by bees in four developed areas of the U.S., and explored whether there were spatial or temporal differences in the types of floral sources of pollen used by honey bees in these landscapes. To do this, pollen was collected every month for up to one year from colonies located in developed (urban and suburban) sites in California, Texas, Florida, and Michigan, except during months of pollen dearth or winter. Homogenized pollen samples were acetolyzed and identified microscopically to the lowest taxonomic level possible. Once identified, each pollen type was classified into a frequency category based on its overall relative abundance. Species richness and diversity indices were also calculated and compared across states and seasons. We identified up to 64 pollen types belonging to 39 plant families in one season (California). Species richness was highest in CA and lowest in TX, and was highest during spring in every state. In particular, "predominant" and "secondary" pollen types belonged to the families Arecaceae, Sapindaceae, Anacardiaceae, Apiaceae, Asteraceae, Brassicaceae, Fabaceae, Fagaceae, Lythraceae, Myrtaceae, Rhamnaceae, Rosaceae, Rutaceae, Saliaceae, and Ulmaceae. This study will help broaden our understanding of honey bee foraging ecology and nutrition in urban environments, and will help promote the use of plants that serve the dual purpose of providing aesthetic value and nutritious forage for honey bee colonies placed in developed landscapes.

Objective: We evaluated microRNAs (miRNAs) as potential biomarkers for Alzheimer disease (AD) by analyzing the expression level of miRNAs in CSF of patients with AD dementia and nonaffected control subjects. Methods: Using quantitative PCR, we profiled the expression level of 728 miRNAs in CSF of nonaffected control subjects and patients with clinically ascertained AD dementia, and we further compared the expression level of candidate miRNAs in 37 control subjects and 35 patients with AD dementia. Results: The level of hsa-miR-27a-3p in CSF is reduced in patients with dementia due to AD in 2 different cohorts of subjects (cohort 1: p = 0.008; cohort 2: p = 0.015; 2-tailed unpaired Welch t test). Moreover, low levels of hsa-miR-27a-3p were accompanied by high CSF tau levels and low CSF β-amyloid levels. Conclusions: Our pilot study highlights hsa-miR-27a-3p as a candidate biomarker for AD and provides the groundwork for further confirmation studies in larger cohorts and in other hospitals.

The importance of dietary salt may explain why bees are often observed collecting brackish water, a habit that may expose them to harmful xenobiotics. However, the individual salt preferences of water-collecting bees were not known. We measured the proboscis extension reflex (PER) response of Apis mellifera water foragers to 0-10% w/w solutions of Na, Mg, and K, which provide essential nutrients,. We also tested phosphate, which can deter foraging. Bees exhibited strong preferences: the most PER responses for 1.5-3% Na and 1.5% Mg. However, K and phosphate were largely aversive and elicited PER responses only for the lowest concentrations, suggesting a way to deter bees from visiting contaminated water. We then analyzed the salt content of water sources that bees collected in urban and semi-urban environments. Bees collected water with a wide range of salt concentrations, but most collected water sources had relatively low salt concentrations, with the exception of seawater and swimming pools, which had >0.6% Na. The high levels of PER responsiveness elicited by 1.5-3% Na may explain why bees are willing to collect such salty water. Interestingly, bees exhibited significant high individual variation in salt preferences: individual identity accounted for 32% of PER responses. Salt specialization may therefore occur in water foragers.

MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ~75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3"-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.

The complexity of the nervous system arises in part, from the large diversity of neural cell types that support the architecture of neuronal circuits. Recent studies have highlighted microRNAs as important players in regulating gene expression at the post-transcriptional level and therefore the phenotype of neural cells. A link between microRNAs and neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and Parkinson's disease is becoming increasingly evident. Here, we discuss microRNAs in neurodegeneration, from the fruit fly and mouse utilized as experimental models to dysregulated microRNAs in human neurodegenerative disorders. We propose that studying microRNAs and their mRNA targets in the context of neurodegeneration will significantly contribute to the identification of proteins important for neuronal function and might reveal underlying molecular networks that drive these diseases.

The architecture and functioning of the mammalian nervous system is partly based on the complexity of combinatorial gene expression in the developing brain that results in a tremendous diversity of neural cells. MicroRNAs are small non-coding RNAs that are particularly abundant in the brain and are emerging as influential regulators of neural gene expression. This mini-review summarizes the recently discovered role of microRNAs in the development and maintenance of the nervous system. MicroRNAs are temporally expressed during neural differentiation, spatially regulated and embedded in molecular feedback loops that may contribute to the robustness of the neural networks.

Peripheral myelin protein 22 (PMP22) is a dose-sensitive, disease-associated protein primarily expressed in myelinating Schwann cells. Either reduction or overproduction of PMP22 can result in hereditary neuropathy, suggesting a requirement for correct protein expression for peripheral nerve biology. PMP22 is post-transcriptionally regulated and the 3'untranslated region (3'UTR) of the gene exerts a negative effect on translation. MicroRNAs (miRNAs) are small regulatory molecules that function at a post-transcriptional level by targeting the 3'UTR in a reverse complementary manner. We used cultured Schwann cells to demonstrate that alterations in the miRNA biogenesis pathway affect PMP22 levels, and endogenous PMP22 is subjected to miRNA regulation. GW-body formation, the proposed cytoplasmic site for miRNA-mediated repression, and Dicer expression, an RNase III family ribonuclease involved in miRNA biogenesis, are co-regulated with the differentiation state of Schwann cells. Furthermore, the levels of Dicer inversely correlate with PMP22, while the inhibition of Dicer leads to elevated PMP22. Microarray analysis of actively proliferating and differentiated Schwann cells, in conjunction with bioinformatics programs, identified several candidate PMP22-targeting miRNAs. Here we demonstrate that miR-29a binds and inhibits PMP22 reporter expression through a specific miRNA seed binding region. Over-expression of miR-29a enhances the association of PMP22 RNA with Argonaute 2, a protein involved in miRNA function, and reduces the steady-state levels of PMP22. In contrast, inhibition of endogenous miR-29a relieves the miRNA-mediated repression of PMP22. Correlation analyses of miR-29 and PMP22 in sciatic nerves reveal an inverse relationship, both developmentally and in post-crush injury. These results identify PMP22 as a target of miRNAs and suggest that myelin gene expression by Schwann cells is regulated by miRNAs.

TPPP/p25, a recently identified tubulin polymerization promoting protein (TPPP), is expressed mainly in myelinating oligodendrocytes of the CNS. Here we show that TPPP/p25 is strongly up-regulated during the differentiation of primary oligodendrocyte cells as well as the CG-4 cell line. The microRNA expression profile of CG-4 cells before and after induction of differentiation was established and revealed differential regulation of a limited subset of microRNAs. miR-206, a microRNA predicted to target TPPP/p25, was not detected in oligodendrocytes. Over-expression of miR-206 led to down-regulation of TPPP/p25 resulting in inhibition of differentiation. Transfection of siRNAs against TPPP/p25 also inhibited cell differentiation and promoted cell proliferation, providing evidence for an important role of TPPP/p25 during oligodendrogenesis. These results support an essential role for TPPP/p25 in oligodendrocyte differentiation likely via rearrangement of the microtubule system during the process elongation prior to the onset of myelination.

Cancer continues to be one of the major health and socio-economic problems worldwide, despite considerable efforts to improve its early diagnosis and treatment. The identification of new constituents as biomarkers for early diagnosis of neoplastic cells and the discovery of new type of drugs with their mechanistic actions are crucial to improve cancer therapy. New drugs have entered the market, thanks to industrial and legislative efforts ensuring continuity of pharmaceutical development. New targets have been identified, but cancer therapy and the anti-cancer drug market still partly depend on anti-mitotic agents. The objective of this paper is to show the effects of KAR-2, a potent anti-mitotic compound, and TPPP/p25, a new unstructured protein, on the structural and functional characteristics of the microtubule system. Understanding the actions of these two potential effectors on the microtubule system could be the clue for early diagnosis and improvement of cancer therapy.

miR-29 is expressed strongly in the brain and alterations in expression have been linked to several neurological disorders. To further explore the function of this miRNA in the brain, we generated miR-29a/b-1 knockout animals. Knockout mice develop a progressive disorder characterized by locomotor impairment and ataxia. The different members of the miR-29 family are strongly expressed in neurons of the olfactory bulb, the hippocampus and in the Purkinje cells of the cerebellum. Morphological analysis showed that Purkinje cells are smaller and display less dendritic arborisation compared to their wildtype littermates. In addition, a decreased number of parallel fibers form synapses on the Purkinje cells. We identified several mRNAs significantly up-regulated in the absence of the miR-29a/b-1 cluster. At the protein level, however, the voltage-gated potassium channel Kcnc3 (Kv3.3) was significantly up-regulated in the cerebella of the miR-29a/b knockout mice. Dysregulation of KCNO expression may contribute to the ataxic phenotype. (C) 2015 Elsevier Inc All rights reserved.

Oligodendrocytes undergo extensive changes as they differentiate from progenitors into myelinating cells. To better understand the molecular mechanisms underlying this transformation, we performed a comparative analysis using gene expression profiling of A2B5+oligodendrocyte progenitors and O4+oligodendrocytes. Cells were sort-purifiedex vivofrom postnatal rat brain using flow cytometry. Using Affymetrix microarrays, 1707 transcripts were identified with a more than twofold increase in expression in O4+oligodendrocytes. Many genes required for oligodendrocyte differentiation were upregulated in O4+oligodendrocytes, including numerous genes encoding myelin proteins. Transcriptional changes included genes required for cell adhesion, actin cytoskeleton regulation, and fatty acid and cholesterol biosynthesis. At the O4+stage, there was an increase in expression of a novel proline-rich transmembrane protein (Prmp). Localized to the plasma membrane, Prmp displays adhesive properties that may be important for linking the extracellular matrix to the actin cytoskeleton. Together, our results highlight the usefulness of this discovery-driven experimental strategy to identify genes relevant to oligodendrocyte differentiation and myelination.